CA2361612A1 - Peptides modulating activities of heparin, other glycosaminoglycans or proteoglycans - Google Patents

Abstract

The present invention involves peptides of various sequences and sizes and methods of using said peptides with a strong affinity for glycosaminoglycans and proteoglycans, wherein said peptides interact strongly with heparin, oth er glycosaminoglycans, or proteoglycans (PGs).

Description

WO ON~6~31 PCTNS0Q~3 PEPTIDES MODULATING ACTIVT>~ES OF HEPARIN, OTHER
GLYCOSAMHVOGLYCANS OR P80TIiOGLYCANS
This invention was made in part with govamuent support from the National Institutes of Health Heart, Lung, and Hiood Iastiwte, grant munbera HL 53590 and HL29282 awarded by the National Institwes of Health. The govtrnntcnt has ccrmin rights in the invention.
~O~ ONCE TO RELATED APPLICATIONS
This application claims priority in part under 35 U.S.C. ~119 based upon U.S.
Provisional Patent Application No. 60111$,276, flied February 2, 1999.
FIELD OF TAE INVENTION
The pinvention generally relates to the field of peptide chemistry and to compositions of matter comprising peptides of various aequetxes and sizes and to methods of using said peptides with a strong amity for glycosaminoglycans and pcotooglycans, and more particululy to the various methods of using said peptides of various sequa~cea and sizes as described below, wherein said peptides interact strongly with h~rin, other glycbsaminoglycaos, or p~glyauis (PGs).
BACHGROUND OF THE INVENTION
Glycasaminoglycans (GAG)s modulate enzyme activities (e.g., of at~tithrombin III or heparin cofactor In, regulate call behaviors (e.g., cell adhesion, growth, and differentiation), and control the function of eatracellular matrices (e.g., diffusion of ions through basetttent membranes, and fibrillogenesis and lateral associations of collagens), largely through non-covakmt intera~ions with proteins. (Jackson, R. L., et al., Pltysiol Rev 71;481-539, 1991; Liudahl, U. and M. Hook, Ann Rev Bioehem 47:385-417, 1978). Although many proteins exhibit high affinity interactions with 6eparan sulfate or heparin and other C3AGs, the specificity of such interactions has been defined for only a small number of them. (San Antonio, J. D. and R. V. Iozzo, Eruycl l,~fe Sci In Press, 2000). As lteparan sulfates and heparin are among the most structurally diverse and biologically active of GAGS, their protein-interactive features have been the most thoroughly studied. Fine sttv~u~al fratures of heparan sulfate chains, including defined sequences, rare rnodifieatioas, domain structures, and gross polymer characteristics are each believed to contrilntte to various classes of interactions with different proteins. (San Antonio, J. D., and R. V. Iozzo, Encycl life Sci In Press, 200p). For proteins, domains rich in basic amino acids appear to be ~cessary to facilitate interactions with GAGS; and for a subset of these proteins, potential heparin-binding consensus sequences have been descn-hed. (Cardin, A. D. and H.
J. R.
Weintraub, Arteriosclerosis, 9:21-32, 1989; Jaclrson, R. L., et al., Physiol Rev ?1:481-339, 1991).
Heparin-binding consensus sequences were discovered by Cardin and Weintraub, who surveyed smira acid sequences of known heparin-binding proteins, where they identified two potential consensus sequence motifs for heparin-binding, X-B-B-X-B-X or X-B-B-B-X-X-B-X, where X represents a hydropathic or uncharged amino acid, and B a basic amino acid. (Gardin, A. D. and H. J. R. Weintraub, Arteriosclerosis 9:21-32, 1989). For example, such consensus sequences wore . . . . . i~~ in- proteins incl~ling apolipoprotein B-100, ape E, and vitroe~ectin, to name a few. (See Cardin and Weintraub, 1989, for review). Molecular modeling of these consensus sites predicts the arrangement of amino acids into either a-helices or (3 strands. This allows for the clustering of noncontiguous basic amino acids on o~ side of the helix, thus forming a charged domain to which GAGS could bind. Indeed, for some heparin-binding proteins, disruption of the heparin-binding consensus sequences hinders heparin binding. For example, c6enaical modification of the heparin-binding consensus site in thrombospondin (Lawfer, J. and R. O. Hypes, Cell Biol 103:1635-1648, 1986) or site-directed mutageaesia of a heparin-binding sequence in fibrotuctin (FN) (Barkalow, F. J. B. and J. E. Schwarzbaucr, ,1 Biol Chem 266:7812-7818, 1991) eliminates or diminishes heparin-binding affinity. On the other hand, peptide mimetics of proposed heparin binding consensus sequences often fail to reveal the high aft"tnities demonstrated by the native heparin-binding proteins. (Conrad, H. E, Heparin-Binding Pr~ttins. Academic Press, 1998). Proteins often contain multiple, distal heparin-binding sequences that may come into proximity upon protein folding or t~ltimerization, hetxe enabling binding through cooperativity. It has thus been speculated that the three dimensional arrangement of multipk heparin-binding consensus sites within or between heparin-binding proteins, andlor the presence of novel heparin-binding sites may be responsible far high affinity heparin- or HS-interactions with native proteins. Others have proposed a necessary approximately 20 r~
distance between basic amino acids for heparin binding, regardless of protein tertiary structure. (Margalit, H.; et al., J Biol Chem 268:19228-19231, 1993). Other heparin-binding sequences have been proposed that are variations of those reported by Cardin and Weintraub. The sequence TXXBXXTBXXXTBB, when T is a turn, was identified as a heparin-binding sequence in acidic FGF and bFGF. (Hileman. R. E., et al., BioEssays 20:156-167, 1998). X-ray crystallography revealed that this peptide backbone loops back upon itself in three turns to form a positively charged triangular heparin-binding pocket. The heparin-binding domain of von Willebrand factor resembles the motif XBBXXBBBXXBBX, a palindromic sequence in which the spacing and clustering of basic residues is Important for heparin binding. (Sobel, M., et al., J
Biol them 267:8857-8862, 1992). A third novel seque~e has been demonstrated to be sufficient for weak heparin-binding in, thrombo~spondin: WSXW. (Guo, N. H., et al., J
Biot Chem; 267:19349-19355, 1992). However, for high affuuty binding, this sequence must be flanked by basic residuss. Other proteins including type I
collagen (Sweeney, S. M., et al., PNAS 95:7275-7280, 1998), type VI collagen (Specks, U., et al., EMBO J, :4281-4290, 1992), extracellular-superoxide dismutase (Sandstrom, J., et al., J Biol Chem, 267:18205-18209, 1992), and mast cell chymasas (Matsumoto, R., et al., J Biol Chem, 2T0:19524-19531, 199, bind heparin via highly-basic binding regions which do not conform to any consensus sequence. In fact, is certain proteins, domains rich in basic amino acids have sometimes been shown to be unimportant for heparin binding. For example, the two heparin-binding consensus sequences identified in the FGFs were shown not to mediaoe heparin-binding (along, P., et al., J
Bial we orcrmsoo~oses~
Chem, 270:25805-25811, 1995; Thompeon, L. D., et al., Bioclrem, 33:3831-3840, 1994). Therefore, there are likely other as yet undefined protein characteristics that must confer heparin-binding pote~ial. Of relevance is the recent use of phage display technology to identify such novel heparin-binding sequences. This approach has generated three distinct HSPG-binding antibodies (van Kuppevelt, T. H., et at., J Biol Chem, 273 21:12960-12966, 1998). Signiftcaatly, one of the sequences (GRRLKD) contained a heparin-binding consa~sus seq>,a~e, while the others (SLRMNGCGAHQ
and YYHYKVN) did not. The tatter two Iack significant basic charge, and thus may bind HSl'Gs through non-ionic interactions. All three anti-HS antibodies showed specificity for heparin and HS but not for other GAGS. Additionally, the antibodies all reaaod differently towards HS from various sources, which would suggest a specificity in recognition of discrete HS molecules.
GAG structure may also play a role in determining binding affinity and selectivity for proteins. A classic example is the aatithrombin-binding site on bepariu, t5 which is present on only about one third of heparin chains (Lam, L. H., et al., Biochem Biophys Rcs Common, 69:570-577, 1976), but which has a thousand-fold greater affinity for antithrombin III than the overall heparin structure (Lee, M. K., and A. D.
Larder, Prac. Nat Acad Sci USA, 88:2768-2?72, 1991). Several other sequences or strucxural motifs have been idcntifced in HS GAGS which underlie their binding interactions with basic fibroblast growth factor (bFGF) (Maccarana, M., et al.. J Biol Chem, 268:23898-23905. 1993), lipoprotein lipase (Parthasarathy, N., et al., J
Biol Chcm, 269:22391-22396, 1994), and interieukin-8 (Lindahl, U., et al., J Biol Chem, . . . . . . , 273:24979-24982; 1998): ~ Other ~ aspacts~ of GAG fine structure also contribute w to specific interactions with proteins. Per example, for short basic peptides, heparin displays high affinities for sequences with contiguous clusters of basic amino acids, whereas HS displays high affinities for those sequences in which clusters of basic amino aids are separated by non-basic residues (Frmnm, 1. R., et al., Arch Biochem Biophys 343 (I):92-100, 1997). Such binding preferences may relaoe to the iacressed spacing between sulfates found throughout HS as compared with the snore densely sulfated heparin. Heparin is capable of binding to a wide array of proteins, due to its high degree of flexibility and ability to "fit" itself into proteins.

Bee of the preaeoce of hoparin-binding eeguin many physiologically important proteins, there was a nood for small peptides with high affinities for heparin or for heparin-like molccuks (i.e., PGs, at oth~x GAGs), to are in a variety of applications to modulate the activitios of native GAGS and PGs. Thetefore, in the present invention peptides with high affJnitla for heparin or for PGs have been designed to include heparin-binding orntsensus sequences; however, in doing so it was necessary to take into account previous studies showing that short peptides of native ~oteins do not behave likt the native proteins, due to co~orma~on and size limitations, and a lack of coopetativity in binding to various ligands. Thus, peptides were also designed including multiple conamsua soqaencxs arrausged in tandem:
(X-B-B-X-B-X)n or (X-B-B-B-X-X-B-X)m, where n=1-6, and m=1-5.
The basis for the design of the peptide of the present invention is the inclusion in their structure of multiply copies of sequenees (ixlnding XBBXBX or XBBBXXBX, where X is a hydropathic amino acid and B is a basic amino acid), rqxe~ting consensus sequences for heparin or PG-binding in natural proteins, and, in addition.
may include the prea~oe of a single cysteine residue preferably occupying, but not limited to, a position within a three residue distance of either the G or N-peptide Iermiuus. that promotes peptide dinxr formation and greatly enhances peptide binding interactions with heparin. Any of these peptides may also be constructed of either L- or D-, or combinations of L- and D-amino xid isomer forms, or containing any amino acid in the X position of any peptide. Any of these peptides may also be used as carriers andlor integral components of various pb:rasaceuticals or bioactive agents ~targ~ to. i~act wHh cell atar~a expressing PGs or Mitt molea~les, or to . . .
..
tissues which express PGs as cell surface or extracellular matrix components.
GSureat e~ch~ to dosi~gn peptides which bind to heparin include Wakefield et al. (U.S. Pabeot No. 5.534,619, and U.S. Patent No. 5,919,761) and Harris et al.
(U.S. Patent No. 5,877,153}. The Wakefield peptide sequences, specifically the grouping and spacing of the basic amino acids, are patterned aftea naturally-occurring protamines. The Harris et al. pept~es are a series of single-chain and nw~lti-chain peptides which incorporate argues within a backbone of ayaniaes. The spacings of the arginines are based on the deparia-binding scquettce of antithrombin III.
All the Harris p~tid~s have AE as their N-terminal auanino xid sequence.

WO 01/45831 PCT/U.S001'Ii853 The present invention, however, includes pepbd~ bash an the consensus sequences (XBBXBX) and (XBBBXXBX) detcnnioed by the analysis of a wide range of known heparin-binding proteins by Cardin et al (Cardin, A. D., and H. J. R.
Weintraub, Arteriosclerosis, 9:21-32, 1989). The peptides designated in this application consist of as many as 6 repeating units of these sequences. These sequences are oat found in protamine. In contrast to Wakefield et al., the peptides of the present invention contain repeating moti6 with groups of two add 1 basic residues serrated by a single alanine, or three and one basic residues sepatatod by alaaine-alanine. While single copies of these general sequences are associated with the heparin binding sites in many proteins, peptides derived from these proteins which include single copies of these sequences and their native surrounding amino acids have insignificant binding affinities for heparin. .Furthermore, some proteins comain the Cardin type consensus sequences, but these sequences were shown not w bind heparin, and many other proteins bind heparin yet do not contain such conse~ns sequences. Thus it is r~or intuitive to use these types of sequences as heparin-binding agents.
Furtltermorc, the sequences used by Harris et al. mimic those found in a naturally occurring protein in terms of spacing and grouping of the basic residues, with no internal repeating structures, but the single-chain peptides have relatively weak ability to interact with heparin. Substantial binding is found only when multi-chain structures are fornxd. In contrast, the present invention involves, for the most part, single-chain peptides with repeating Cardin sequences. These peptides have a strong capability for binding to both unfr~tionatad heparin sad low molecular weight heparin.
. . . . . .. ,p~ . gr,~~~ ~. ~ pies in the Wakeffe~' end' ~Hairis patents . ~
, resides in the engineering of alpha-helical structure into the peptides. Some of their peptides have partial alpha-helical structure. In the present invention, peptides arc not alpha-helical in the native state, but assume an alpha-helical conformation when bound to heparin. Thus, the peptides of the present invention may have more flexibility to conform to a variety of heparin sequences encountered is any of the therapeutic heparin formulations.
An additional aspect of the present invasion is the N-terminal -peptide sequences of the prot~glycan serglycin, which contain a single full or partial Cardin site near the N-terminus and a cysteine residue three notion acids from the C-terminus.

~ ~~s~t These peptides dimeriu tltrougti their cysaeme resideca and thus form a strong heparin-binding unit. Another feature of the pit invention is the inclusion of cysteines near the C-termini of s11 the Ca~rdin sift: and the stxglycin pcptldes w further enhance their heparin-binding fbaerions.
The peptides of the present mv~tion have a numb of aces. One ~d of using these peptides is to promote all attachment or adhesion to natural or synthetic surfsuces.
Vascular diseases such as k~osis, restenosis, and aortic anaaysms often result in permanent dama8e to blood veaels; typically, vessels become occluded as a result of vascular insult, causing ~cr~aed blood flow (Robbios, S. L, and R. S.
Cotran, Pathological Basis of Disease. W.B. , Phila~ip~hia. 598-613 pp., 1979). One approach .to treatment of damaged vessels is surgical replacement of the .
diseased sit with an autMogous ac novas native tissue graft (large, J. L, et al., In Principles of Tissue Ehgintsring, R. P. Lariza. et al., editors.
Academic Press, Austin.. 349-364, 199?). This approach is limited in that either healthy vessels must be removed (auLologoua) or a suitable dons vessel must be available (non-autologous). An alternative approach la the use of a synthetic vascular graft in plane of a native tissue graft. In addition to not requiring an appropriate donor vessel or the removal and traosplaatation of a non-disaasad vessel to the diseased area, syatheti~e vascular grafts can be modified to redtue cxnrtplic~ons from immune rejection or to increase pataacy rates and graft success (Muaro. M. S., et al., Tram Am Sec Artif lntsrn Organs, 27:499 503. 153; Leikweg, W. G., and L. J. Groeafield, Sung, . . ,.. .. . . . 81:335-342; ~1~377~: Pelt. I~. D:, et al., J Bionrnd Ilt~
Res, 22:9?7-99?.27=29; .1988):.: . . . , .. .. .
Historically, inert polymers composed of terepbthalate (Dacron) or of expanded polyoaratltmroethyiene (ePTI?6j have been need to ooeatruct prosthetic vsscnlar grafts (large, J. L, H. P., and H. P. Gteiskr, In Pof Tissue Engineering. R. P.
Lama, et al., editors. Academic Press, Austin, 349-364, 1997), but these materials typically invoice an immune respoa~e. Sytkltetic grafts can react with serum proteins and blood cells that can pmmsote thrambna form~ion sad kad to psatdoinrimal hyperplasia. (large, J. L, H. P., and H. P. Greiskr, In Princtpl~s of Tlissue Fatgin~ring, R. P. Lama, et al., editors, Acadeanic Press, Austin, 349-364, 1997).
Vaxular replacement has been limited to large or medaun size arteries when blood WO QOI45831 ~TN~
Dow rates are high, outflow re~mCe is low, and as a consequence, the graft is less likely to become occluded by a thrombus. Conversely, small arteries are more prone to graft failure via thrombosis or hyperplaaia because of lower flow rates and higher outflow resistance. An inappropriate infiltration of smooth muscle cells during the healing process can also result in vessel occhtsion. Control of this immune response and sraooth muscle cell infiltration could occur in a vessel lined with endothelial cells, which secrete factors inldbiting platelet and erythrocyte aggregation (Fantone, J. C., and P. A. Ward, In Pathology, E. Rubin, and J. L. Farbcr, editors. J.B.
Lippincott Co., Phil, 43, 1994), as well as factors that inbibit smooth muscle cell proliferation, but, endothelial cells typically fail to proliferate well on these graft materials. (large, 1. 1., H. P., and H. P. Greisler, In Principles of Tlissue Engineering, R. P.
Lanza, et al., editors, Academic .Press. Austin, 349-364, 1997). Attempts have been made to overcome these limitations by coating the graft with anticoagulants to limit thrombus formation, growth factors to promote endothelial cell proliferation, or proteins with antiproliferative effects on smooth muscle cells. The presence of endothelial cells in the transplanted graft, however, is thought to increase the chance of survival of the graft (Herring, M. B., et al., Surgery. 84:498, 1978). Studies in which prosthetic vascular surfaces were seeded with autologous endothelial cells before transplantation displayed an increase of 30~b in potency rates over three yeas in comparison to non-seeded surfaces (Zilla, P., et al., J Vasc S'hrg, 19:540-548, 1994). The obvious (imitation of pro-seeding, however, is the need to harvest and culture endothelial cells to the appropriate density prior to seeding, as welt as generating vascular graft materials with . , . , . .., surfaee properties optimized for endothelial all auachrnetsl and prolithration. , , . , -Etxiothelial cells carry a negative surface charge (Verges, F. F., et al., Membrane Biochemistry. 9:83, 1990) that can inhibit platelet adherence, and they express a variety of GAGS on their surface that bind the anti-coagulant anti-thrombin III
(Mettens, G., et al., J Biol Clam, 267 (28):20433-20443, 1992). Vargas and co-workers have shown that sulfated GAGS are tire main carriers of surface charge on vascular endothelial cells, primarily as heparan sulfate (HS) and chondroitin sulfate PGs (Verges, F. F., et al., Membrane Biochsmistry. 9:83, 1990). Specific types of PGs on endothelial cell surfaces include the synda~as and giypican (Mcrtens, G., et al., J Biol Chtm, 267 (28):20435-20443, 1992).

WO OII45e3t PCTNSOQ~853 Thus, the surface cheTnistry (i.e., the prodominance of its PG component) of endothelial cells will prove useful as a gas of tethering and maintaining these cells in a transplanted avascular graft. One goal of the preset invention is to deaoover peptides with high affinities for endothelial cell surface PCs. Such peptides are used by covaleatty attaching than to aynvtaic vascul~ grafts, and in the presence of endothelial cells, prrnnote their attachment to the graft surface, thereby increasing the probability of grab succxss.
Anathat nee of the peptides of the proem invemion is for heparin-and PG
binding as modulators of hemostasis via i~rections with endothelial cells and as aati heparin therapy in plasma. These peptides of the present invention function as agents for neutralization of unfraction~ed heparin,1ow molecular weight heparin, or Orgaraa . (Qrg$nnn, mire of.chondroidn sulfatelheparan sutfueJdermatan sulfate) overdose.
Currently, the only FDA-approved L>eparin antidote available is Protamine.
Protamine can cause several serious side ei~Cts in patients, and although Protamitue is effective in humans against unfractioaated heparin, it is not effective against low moleailar weight heparins or against Orgaran. Since Protamine is a natural product that is an undefuxd mixture of amino acids, its content is variable across different preparations, and thus dosage is uncertain, presettcmg problems in its clinical use.
The peptides of the preset invenmon are useful for counteracting the acxians of heparin and other anticoagulam gtycoataninoglycans on thrombin and Factor Xa activity, and may affect other proteins as well. Heparin is used ro~ndy for amicoagulation. The interactions of exogenously administered heparin with the proteins ... . . . . .. of tke: coagulation and. ~ #Ibrinolytic pathways have been summarized ~in detaih (van . . . .
ICuppevelt. T. H., et al., J Bfol tatem. 273 (21):12960-12966, 1998). These ' interactions are cottiplac on many levels. The best-c6aracterizod targets for heparin arc the procoagaluit procaine thrombin and Factor Xa, which are inhibited by AT
llI when heparin binds to AT Ia. However, heparin acts at many sites. In some cases, the effect of heparin is anticoagulant and in other cases procoagulant. Some proteins, e.g. AT III, have heparin-biod~g consensus sites. However, the putative hepe~rin-binding seqwences are different for every lmown protein in these pathways, and the effects may depend on the 3-dimensional relatioahips of lyasic residues resulting from protein folding, rather than a short linear sequonoe, as is known for dte binding of heparin to AT 17I
(Lam, L.

Wo 111'11 . 1PC' H., et al., Biodiem Biopleya Res Connruas, 69aT0-37?, 1976). A tetramerie protein conformation of platelet feeler 4 (PF4) is requitod for long-gain heparin binding (lxe, M. K., and A. D. Larder, Pros lVldt Aced Sci US~I, 88:2?68-2772, 1991;
Maccarana, M., et al., J Biol Chem ?.68:23898-23903, 1993; P'attbmarathy, N., et al., J
Hfol Chem. 219:22391-22396, 1994). Formation of a two-pcocein aootplex (PAI-l/vitronectin) involve= the via heparin binding site (LindabE. U.. et al.. J
Biol C~em, Z'73:24979-24982. 1998; Fromna, 7. R., ~t al., Arch Bioclrem Biophys, (1):92-100, 1997) and r~e~e could be distopted by hepwria. The inactivated AT
III/thrombin complex is released fram the endothelial purface, binds as a complex to vitronectin, a~ then is taken up for catabolism by binding of the vitroncctin heparin binding domain to HSPG an the endothelium (Robbing, S. G_, and R. S. Cotter, . . Pathological Basis of Disease. W.B. Satwndere, Phfldclphia: 598-619 pp., 1979;
Z.arge, 1. L, H. P., and H. P. Qr~aler. In of ?Frsue F.~tgirrearmg. R. P.
Lan7a, R. Larger, and W. L. Clticic, editors. Academic Press, Austin,. 349-364, 199'n.
Heparin is a compbx rsia~re of polyaacc6arldea. Sane of the interactions require long-chain hepatica (AT III for inactivation of thrombin and binding to thrombin, HC D, PF4, and thralnboapoodin) while others depend on or can fttnaion with low moixular weight heparin cbaim(AT III Nor ~ of Factor Xa, vitranectin, TFPI) (van Kuppevelt, T. H., et al., J Bier! (?rem, 273 (21):12960-12966, 1998). To further complicate the situation, specific seqttencea within the heparin chaiag tray be roquired for interactions with the diifcreat ptotdns (van Kuppvwelt, T. H., et ~.. ~. ~ J..,glol. Clttr~c, 2'73 (21):12960-12966; ~199ff~; and all naNtally=ticcurcitig hep~rins . , . . , ..
and heparan anlfates are very diveeae in their carbohydrate strrctnres. The catabolism 2s of me higher ttwleatlar weight hc~ias in the plasma results in a constantly changing of actna! Ixparin chairs that ate available for reaction with the various proteins, and thus the runts of the possible anticoagulation or fibrinolyric reactiora will charge over the hours after the dosage is given. Pimilly, nary other plasma proteins that are not iavwed in tire ~ as ftbrinolytic pmoesses can bitrd heparin, and variation is the eoaxd~ta~ion and nature of these protieins in diifertnt individuals can intluerice the availability of heparin for these two pathways.
Thus specific single peptides or c~ntbimutions of peptides may target specific interactions l0 WO 00/131 PG r/US00~3 between heperins and cell sarface or plasma protein to got the greateu effxtiveoeas and tninitnize adverse reactions.
It is ot~ea n~aary to reverse the effdc~ of 6epan3a when anucoagu>ation has reached a stage a which hemorrhage becomes a threat, notably after the routine use of heparin for anticoagulation during cardiopubmonary bypass, and in patients who develop an eadogenaoa heparin-Woo eo~dc~n id~itos. 11e most commo~r used anti-heparin drug is protamine, a mixture of basic proteins from fish sperm nuclei, thu contains a high concentration of the amino acid arginine, Wheu injected into a person who has been treuod with hqas~, it omoplexea rap~y w the heparin, thereby neutralizing its activity. Protamine also has nutnetous side effects including pulmonary hypotenaion that are difficult to control and provide significant health risks w the . . patient.-_ .Alga, since Protamine is a poorly-defined and potentially vaf'tab>c product, dosage determination can be problematic. Importantly, Protsmine has been shown to be rove for nWaaliaation of low molaular weigh haparins and the mar-h~arh~
1 S glycosaminoglycan amic~agulant Orgaram. Well~defmod heparin-or other GAG-binding peptides could be of considerable utility for reversing overdose of these specific antiooagulaot peeparsrttions. Carao~wed co-workers (Muao, M. S., ~ al., Trues Aar Soc Artif Inttrn Organs, 27:499-303, 1983) have idartified a heparin-binding peptide from an epitbeJisUendothelial call sntfece protein that has same ability to neutralize heparin effects on thrombin genetulon, but optimal eflteets were found only at high peptide concentrations aul low b~aparln and low thrombin concentrations.
Preliminary data is the present invention suggest that the CaMin and saglycin peptides reverse the . . . . .. ..an thratnbin at se~ratal-ilobd lovvrr pootsceritf~ak' and 7-fold . . . , . . .
higher thrombin concentrations than the ptptide described by Carson and co-workers.
We have oleo shown flat several of the peptide: are effective Qeutraliurs of low maleatlar weight heparin (Erwxaparia, Lovenox) and Orgatan in vitro, and of Lovenox in vivo in rata, in accordance with their affinity constaa~ts for low ttrolecular weight heparin in vitro. Thus the peptides described in this application may have important clinical applicatioag, espeaally if they can be mrgetod to specific reactions in the relevaat pathway and to specific classes of heparitms.
Another use for the peptides of the present invention is to block the uptake std clearance of heparin by bloclang uptake receptors on tissue, without binding to the 1l WO 0011st31 PGTNSOOn0Q~s3 circulating heparin itself, and thus prolotigiag the half life in the circuLtion. Such an agent would reduce the frequency of adtniuistration of the drug, aq well as the atnottrtt nadod. This could be especial)y useful fur >rotno-bared therapy with low molecular weight heparin. which is adaninisteted by subcutaneous it4joetion and is becoming the standard for post-hospitalization anticoagulation.
Multiple interactions between the protWns of the coagulation and fibrinolysis pathways and endothelial cell surface PGs gestate a complex surface on which ongoing coagulation and fibrinolysis are normally balanced to create a non-thrombotic state. The heparan sulfate PGs ~ (HSPGs) of the endothelium mediate antithrombotic/anticoagulant function through binding and activation of Antichrombin IQ (AT III) and binding of tissue factor pathway inhibitor (TFPI). AT Ill bound to endothelium heparan sulfate can imuctivata both thrmnbin and Factor Xa. TFf'I
binds to Factor Xa and this ccxnplex then interacts with the Factor VIIaltissue factor complex to inactivate both Factors VIIa and Xa. Ad6exenee of TPPI to the endomelium via the HSl'G protects against proteolysis of the heparin-binding C-terminal domain (Thompson, L. D., et aL, Biochem, 33:3831-3840, 1994); without this domain, activity is lost. Heparin-binding pept~ such as those dacribod in this study could behave similarly to platelet factor 4 (PF4) io that they could bind to the heparan sulfates on the endothelial surface. For example, docking of a peptide onto the heparan sulfate chain in a revastble manner could protect the GAG $~om degradation by platelet heparitinase released by aggregating platelets at the site of a devclaping thrombus, leaving the GAG
able to resume its antithrombotic funcrion in a shorter time frame than would be .~ ~y~. ~ the ntlte! hand,'s peptidc'with a very high affinity fbr t'hC - v .
.
AT III-binding sequences of endothelial heparatt sulfate could block the binding a~
therefore the activity of AT III and provide a nacre favorable surface for clot formation, thus promoting wound healing.
Therefore, in some embodiments, the peptides of the present invention have affinity for 6epariolheparan sulfate on cell surf~es sad can be used as agents to pt~ote healing, either by injection or by topical application. Injection or typical application of the peptides alone also might serve to assist wound-heating by dislodging ATI1I andlor tissue faeoor pathway inhibitor (TFPn $~om their binding sixes and subtly blocking these binding sites on the mdad~eiium of broken blood vessels, we aoresx~t ~'NSOO~~
thereby reducing the anticoagulant activity of the surface and enabling a clot to form.
Alternatively, oo~oranodus i~ctioun ~ applic~on of a mixarre of heparim and a heparin-binding ptptide could generate a molecular campkx, or low affinity heparin sink, that will then transfer the heparin to proteins whh greater heparin-binding affinities.
The peptides of the present invention can be used to bind and neutralize or uxivate, or otherwise modulate the actions of various PGs or GAGS, thereby influencing their growth- or differentiation-modulating activities. For example, )uparin and heparin-like molecules such as cell surface HSPCis are known to inhibit smooth muscle cell proliferation, to potentiate the activities of growth factors like basic or acidic fibroblast growth factor on eadothdisl calls, and to inhibit or pro~te cell . differentiation of smooth muscle cells, chondrocyta, and other cell types.
The peptides . _..
described here cauW be used to modulate the actions of heparin or endogenous heparan sulfate PGs, with significant consequences to cell growth and differentiation.
GAGS exhibit a wide variety of potent activities on cell growth, migration, diffeaentiation, metabolism, and adhesion (Jackson, R. L., et al., Physial Rev, 71:481-539, 1991; San Antonio, J. D., and R. V. lo~zo, F.racylcL~'e Scl, In Press, 1999). One of the earliest reports of an effect of GAGs on cell growth reported that fibroblastic mouse L cells in suspension culture exposed to 50 pglml heparin were growth inhibited by seventy-three percent (Karnovsky, M. J., et al., Mnaxs of the Ncw York Academy of Science, 556:268-281, 1989). Sevaal strong antipmliferative activities of GAGs on a varidy of cell types have been reported since (San An~Da~nio, et al., Comucaive TissWt Res, 37:87-103, 1998). Tli(~effects of heparin dh vascular ~mooth muscle cells (VSMC) have been the most extensively studied owing to the relevance of this topic to vascular disease. Although for some cell types heparin antipmliferative action may involve displacement of HS or heparin-binding growth factors from cdl surface receptors, for VSMC heparin may also be intarnaliz~ and act directly in the cytoplasm and nucleus (Itamovsky, M. J:, ~ al., Armals of t!u New Yont AaMeary of Silence, 556:268-281, 1989). An i~ortant component of vascular diseases including atherosckrosis and restenosis is the pathological growth of vasaalar smooth muscte cells. As GAGS
are strong regulators of VSMC growth they are potentially useful in treating these diseases.
The effect of heparin on VSMC growth trt vtvo was first discovered in experiments WO aW45831 t'CTNSt10JD1ti53 aimed at determining whether Zteparitt tray inhibit the response to injury cascade of accelerated atherosclerosis owing to its autithrombotic activity; a dramatic inhibition of VSMC proliferation by heparin was observed (Karnovsky, M. J., et al., Annals of the New York Academy of Science, 356:268-281, 1989). It was next shown that the growth effect of heparin on VSMC in vtvo is exhibited by either anticoagulant or non-anticoagulant fractions, and that these effects are mimicked by heparin or HS
on VSMC
in vitro (Karnovslry, M. J., et al., Aratals of tlu New York Academy of Science, 556:268-281, 1989). It has been proposed that in the heahhy vascular wall, endothelial-derivod HS maintains VSMC in a quiescent growth state, but that injuries which result in endothelial denudation remove this patacrinc mechanism, resulting in uncontrolled VSMC proliferation and vascular lesion formadoa (Karnovsky, M. J., et al., Annals of the New York Acadenry of Science, 556:268-28 t , 19$9). Thus, the peptides described here are useful in neutraliting the aatiproliferative activities of endogenous or exogenous heparins or heparan sulfates on vascular smooth musck cells or other cell types. For example, the peptides may be used to neutralize endothelial cell~lerived HSPG's during vascular wound lxaiing.
Heparins and heparsn sulfates have been shown to pmmote cartilage development at low concentrations, and to inhibit it at high concentrations (San Antonio, J. D., et al., Devel Biol, 123:17-24, 1987). Thus, the peptides described here may prove useful as modulators of cartilage difyerentiation, especially in instances where cartilage tissue scaffolds are being constructed for autologous tissue transplants, e.g., for use in orthopedic surgical applications.
. . . ~ Tumor matrix stromas may piI2~Y ibpottant roles in potentiating tumor growth . , . . .
and metastasis (Iozzo, R. V., Lab Invest, 73:157-160, 1993). For example, increases in perlecan expression are seen during devetopmeat of colon carcinomas and of malignant melanomas; its HS chains may potetmate growth factor activity and induce angiogenesis surrounding the tumor, thereby enhancing its growth (Nugent, M., and R. V.
Iozzo, Iraernat J Biochem. and Cell Blol, In Press, 1999). Furthermore, the binding selectivity of HS chains for various members of the fibrobLast growth factor family can be influenced by fme swcdn~al features such as tJ~ patteans of 6-O-sulfation and the abu~ance of sulfated domains (Lindaht, U., et al., J Biol Chem, 273:24979-24982, 1998). A pathological role of tumor cell surface PGs has also been suggested.
For la PCTNSOO/Ud~3 example, Chinese hamster ovary edls carrying various aaztations of PG
synthesis were in,~ected imo nude mice and tested for their tunurn~nic abilities. Mutants which expressed low levels of PGs failed to produce tumors, and of those with normal PG
levels but with defects in the synt'6esis of specifuc GAG types, the structure of HS, but not of CS, was most important to their tumorigemci:y (Esko, J. D., a al., Science, 241:1092-1096, 1988). Thus, if the peptidGR desc~od here e~rhibit GAG type specific or GAG sequence specific binding preferences, they may be useful in directly modulating the fitactiom of tumor cxll GAGS or PGs, or as carriers of drugs to be targeted to control the gmwth of, or to kill tumor ctlls expressing unique GAGS or PG
variants.
PGs secreted by normal cdts are proposed to play a key barrier functiast by inhibiting the migration of tumot cells across basctneat membranes. However, tumor cells have been shown to sere the enzyme heparatinase, which degrades the HS
chains within basement membranes, thereby potentially enabling such malignant cells to breach the basement membrane, enter the circulation, and spread throughout the body (Katz, B. Z., et al., Invasion arid Mttastasis, 14:2?6-289, 1994-5). The peptide described here could thus be used as inhibitors of OAG hydrolase-mediated tumor me~taais.
Another key component of tumor growth and survival Is proposed to be the developtrnttt of a blood vessel supply to the tumor (Folkman, l., and M.
Klagsburn, Sciarce, 235:442-44?, 1987). Tuanor aagiogw~is is strongly in6ibitod by the hqa<ria_ binding protein endostatin (O'lteitly, M. S., et al., Call. 88 (7):27?-285.
199?), and in vitro, heparin is required to promote angiogatesis in respamse to growth tacxors (Jackson, C. J., et al., F.zp Cell Res, 215:294-302, 1994). The peptides described here could therefore function as inhibitors of growth-factor dependent angiogenesis in vivo, therefore inht'bitiag tumor growth.
Yet another application of the present invention is the targeting of drugs to call of endothelium or other cell types which express PGs. For example, drugs to be targeted to endothelial cells could be com~exed with the peptides described here, or the peptide seqtxncea could be itmegrated Into the drug, and then the drug could be administered W the systemic ion. The peptide component of the drug would WO 0014~i1 PCTNS0016~853 mediate high affinity interactions with the andothalial cell surface, effectively delivering the drug for action at that site, or poo~itlly psomd~Og the cellular uptake of the drug.
Since endothelial cell surfatx charge is IarRely due to cell surface GAGs and PGs (Verges, F. F., et al., Membrane B~odm~riatry, ~:8, 1990), and the peptides described in this patent extubh high affinity ietstac<ions with eacbthelial cell PGs, then the peptides can be applied as tools oo deliver drugs to endothelial cells in vivo. For example, a drug which is designed to acx on eadothelial cells could be complexod witb the peptides either covalently or non-oovalently, and delivery to the systemic circuistion. The peptide oomponatt of the ooet~lex would facilitate high affinity inceracxions with the endothelial cell autface, thereby bringing the drug in contact with the endothelial cell surface to exert its activity there, or to facilitate its uptake by the ~dothelial tells. Such a use for these peptides is not limited to endot>xlial cells,'smce many cell types in the body expr~ distinct classes or types of PGs.
Furthermore, within each type of cell population, structural variants of GAGS and PGs may be expressed, thereby distinguishing these cell variants on a structural and functional level.
Thus, for example, it has been sho~m that naramd B cells and various transformed (cancerous) B cells express different variants of syndecan-1 which show distinct differ~e~uc~s in the chemistry of their hqmran aulfa~e chains (Saaderson, R.
D., et al., J
Biol Chem, 269:13100-13106, 1994). If some of the peptides described here show binding prefetfor the heparsn svifams expraesal on the cancerous B cells, then such peptides could be used as carriers of drugs targeted to those cells.
Finally, another potetdial target cell for the peptide deacn'bai here era the chondrocytes, which are present in all joint surfaces and which express high amounts of sulfated GAGs in their pericellular spacxs. Previous work has shown that the basic endogenous protein lysoayme accumulates in joints, likely owing to its interactions with cartilage GAGS
(ICeittbner, K., et al., Clin Orthop Relat Ret, 112:316-339, I975). These results suggest that basic peptides such as those dea<xibed here, wb~ injected systemicauy or directly into or near joints, should also concentrate attdlar be retained in cartilagenous regions.
Therefore, systemic application of the peptide d~xibed here, oompiatod witb drugs targeted to chondroeytes for trea~m of, for example, arthritic diseases, could potentially find many uses.

WO ON4S83I PCTNSOOf'OQ.B.~'.i Another application of the peptides of the present invention is their use to modulate the activities of enzymes that act on GAG substrates. For example, GAG
6ydrolases including aomue of the hepfarinasea and >~paadnases contain heparin-binding consensus sequences which they are proposed to use in binding to their GAG
substrates. The peptides described hero could be used to inhibit this binding thtbugh competition, thereby inhibiting the activity of the enzymes.
Several of the enzymes that hydrolyze heparin am! heparan sulfates are used commonly in scientific mvatigatio~na to charaeeterizc the strucdrre ate function of GAGS within tissue and cell preparations. Furtt~rmore, these enzymes are important natural products as they are secreted by specific types of bacteria, are present in the venom of some poisonous snakes, and arc secrerood by normal human cells, and by human tumor cells, where they arc propoaud to promote humor cell metastasis (IEatz, B.
Z., et al., Invasion and Metastasis, 14:276-289, 1994-5; Sasiselcharan, R., et al., Proc Natl Acad Sci USA, 90:3660-3664, 1993). Since these enzymes contain heparin-binding consensus sequences that arc pmpoaed to aoediatt the interactions between the enzyme and their substrates, the peptides here will serve as effective inhibitors of enzyme action for many in vitro and in vivo applications.
Yet another use of the peptides of tire present invention is in the affinity ptuiBcation of bioactive spa of GAGS. For example, soave hep~in-binding proteins have been shown to interact with specific sequences or domain structural features on heparins or heparan sulfates, including antithrombin III, lipoprotein lipase, and laminin. T'hns, the peptides described here may similarly exhibit binding preferences for distinct sequences in GAGa, making them useful as affinity ~natricxs for the purification of specific GAG aequenxs fat a variety of uses.
Heparin-binding proteins have been shown to interact with specific soqueaces or domain structural features on taeparins or heparan sulfates, including ATID (Lam, L. H., et al., Biochem Blopltys Res Conunun, 69:570-577, 1976), lipopmtein lipase (Parthasarathy, N., et al., J Biol them, 269:22391-22396, 1994), and laminin. For example, the demnmvimnt o~n heparin tu~dsary for AT-DI binding was located on only abo~ one third of heparin chains, and is a pentasaccharide sequence con~posad of a 6-O-sulfated glucoeamine in the first position, a 3-O-sulfated central glucosamine, two N-sulfated gJucosamisxs, and a carboxylated iduconic acid (Jackson, ptp p8//~p1 PCTNS~0~3 R. L., et al., PJtysiol Rev. 71:481-539, 1991). To purify this sequence from heparin is an expensive endeavor as it re~uves heparin fiagmemation followed by atllnity chromatography on ATIII oohs. However, if imy of the peptides deacribod here showed binding preference for specific sequences snch as the ATIII binding site, then they could be used as low cost affinity matrices for the large scale purification of bioactive GAG soqueners and fragments. Furthermore, such approaches could pooentiafly be useful in endeavors to sequ~e or tuncxionally characterize GAG
samples of unknown chemistries, if litxari~ of heparin-binding peptides contain peptides with unique binding sdectivi;ies for distinct features of heparin or heparan sulfate chemistry, these could be used as tools Vie, isolate, and quantitete specific GAG soqfrom complex GAG mixtures.
DI~NITIONS
"Modulating" means binding, neutralizing, activating, or modulating.

The present invention genarally relates to peptides of various sequences and sizes with a strong a>Ttnity for glycosaminoglycans sad protooglycxns. The present invc~tion also comprises the methods of using said peptidta of various sequences and sizes, wherein said peptides interact strongly with heparin, other glycosaminoglycans, or ZS proteoglycans (PGs).
of the praettt invention can be used to:
1. Promote cell attachmt~rr or adhesfon to naturN or synthetic s~erfiacts. For example, a use of these peptides may include erutothetialiZaiteon of synthetic vein gt~ft sur,~crs, which is known to increase the chaacea for the long term success of the vein graft. Thus, peptides am be oovakotly linked to syntt~ic or natural polymers used to construct vascular graft scaffolds, where they will interact strongly with endothelial cell surface PGs, thereby promoting endothelial cell attachment and t3tus graft colonization WO 01 PCTIU~0~2853 and success. Peptides cottld also be linked to atissue culture surfaces, to promote rapid and strong attachment of cells exptPGs.
2. Bind heparin and PG to neodulate hemostasls via iraerc~ti~s with endothelial cells and as anti-heparin therapy in plasma. These peptides function as agents for neutralization of unfractioaatod heparin, low mola~lar weight heparin, or Orgaran (Orgtmon, mixwre of chondroitin suifatel~atan snifateldacmatan sulfate) overdose.
3. Block the uptake and clearance of heparin 6y blocking uptake receptors on tissue, without binding to the circulaxing htparirt itae~f; acrd thus prolonging the halll~e in the circulation. Such an agent would reduce the ftwuency of administration of the drug, as well as the amount needed.
4. Counteract the actions of heparin and other anticoagulant glycosaminoglycans on thrombin and Factor Xa actvity.
5. Agents to promote lferrting, by bete~ng heparlnlltepQran sulfate on cell surfaces. The peptides could be administered either by injection or by topical l5 application. In addition, contemporaneous iqjecaon or application of a mixture of heparin and a heparin-binding peptide could generate a molecular complex, or low affinity heparin sink, that will then iraosfar the deparin w prdans with greater heparin-bitxling affinities.
6. Bind and neutralize or activate, or otherwise a the actions of various PGs or GAGS, thereby influencing their growth- or d~''nrentiatton-modulating activities.
For example, heparin and heparin-like rnolaules such as cell surface HSPGs are known to inhibit smooth muscle cell proliferation, to potenriate the activities of growth factors like basic or acidic fibroblast gmwch factor on ead~othelial cells, and to inhibit or promote cell differentiation of smooth muscle cells, chondrocytes, and other cell types.
The peptides described here could be usod to inhibit the actions of heparin or endogenous heparan sulfate PGs, with significant oonscqctences to cell growth and differentiation.

7. Neutralize tlu antiprolsferatlve activities of endogenous or exogenous heparins or Ireparan sues on vascular smooth t cells 8. Modulate cartilage dl,~ferenttatian, espedally in instances where eartlta$e tissue sods are being constnrctte~d far autologous tissue trnnsplmrts, e.g., for use in orthopedic surgical applications.

WO 00/41 PCTN80AI~10t8J3' 9. 1liodu1ate the,f tnaiott of twnor cell GAGS or PGs, or as carriers of dings to be targeted to cotarnl the gmwtb of, or to ~tll tumor cselts slag rmiqtu GAGs or PG variants.

10. Inhibit GAG Itydrolase-rrtedlated twnor mdastasls.
S 11. Inhibdt gro~ltdepeaaleat aurgiogettesu is vivo, tlkreforc inhibitin,~
tumor growth.
12. Target dr~rga m cell awfaces of adOf~tirwe oar otlmr cell tyyxs wJtidr txpras PGs.
13. Modrdate tlrt activities of tAiat act ore GAG sabvstrrttes.
14. Facilitate amity puri,/tcation of bioactive seqreences of GAGS.
BBIEP' DB6CRIPTION Olt THB DRAWINGS
FIG. 1 Caka>slbe of heparin-bh>Idiag sues of pop~des co~i~ heparie-6indiog eoeseqtemaes. R~c~dstioa ooe~cieats (R) for the mi~ation of low Mr '~I-tyramino-6eprin dnrastgh peptides were determined from ACE gel tlocu~opboretograms, and are pbtted against peptide cono~rakiou as detailed in Materials and Methods. Smooth cnrva rep~eM non-linear least aqoms fits to the eguatiaa R~ Rd}1+ (Kel(peptide])°}. Peptides containing single consensus sequences (ARKARA, -~- or AItRKAAKA, -O-) do not bind heparin with s measurable affinity;
in contrast, significant h~arin-binding is seen with pcont~ing heps<in-binding oonaeasus and increases as a thnction of peptide Mr.
(AKICARAyi, -R-, ICS 40 ~tM: (AItIQCAAKA)z, -~, Ku= 6 L~M~ (AKKARA)s ,-a, IG~ 2 ~M; (AItIQCAAKA~, -~, ICS 135 nM and (ARKICAAKA~, -~, IC,as 40 nM.
FIG. 2 Calculado~ of heparin-binding attlnitia of SG peptides. R values for the ~rstion of low Mr '~I-tyramine-hepatjn through pe~ides cono~io~g sequences native to the mouse (YPARRARYQWVRCKP; O -) or human (YPTQRARYQWVRCNP, -~) SGi PG core poroteina were daetmined flrom ACE gel electropLorebogiams as detailed in Materials sad Methods. SG peptides displayed relatively strong affinities for WO 06J~5831 fCTNS00/0Z»3 heparin (ICr= 200 and 900 nM for the nonuse and human peptides, respectively), in cmnparison to peptides of similar size which contain multiple repeats of heparin-binding consensus sequences [e.g. (A1CKARAh Key 2000 nM, (ARIQ~AAKA)z ICS 6000 nNi;
Table I]. Peptide AAARRARAAAARAKA, (-~-) displayed negligible heparin-binding affuiity (K~ 75 pM) indicating the impoatt~nce of the non-basic residues to heparin-binding. YPARRARYQWVRCKP-heparin binding in the presence of (3-ME
(YPARRARYQWVRCKP+ [i-ME, -X-), was daxaaed by over 20-fold (K~ 4 ~.
Replacement of cysteine by alanine in the mouse SG ptpt~e (YPARRARYQWVRAKP, -l7-) former roducod heparin-binding affinity (K~ 36 ~rI).
la FIG. 3 CD spedroeoopy o( (AKKARAIK in the presmoc or aloof low 11~
heparin. CD spectra measurements of (AKKARA)e in the absence of heparin (l: 0,-~-), reveals peaks at 195 um ami 216 inn and a crossover at 210 nut, indicative of an extended charged coil conformation. Upan heparin addition (I : 0.25, -O-; I:
0.50, -X-), the peptide conformation is alterod and at a 1:1 peptide-heparin ratio (-~-) the peptide becomes a-helical with characteristic a-helical at approximately 190, 207, and 222 um. Excess heparin (1: 2, -D-, or 1: 4, -..) disrupts this interaction, and the spectra resembles that of a pmtein in a random coil conformation. Spectra are heparin andlor blank (water) corrected.
FIG. 4 CD spectroscopy of (AKItAkAh in the presence or absence of low 11~
heparin. The CD spectra meastnr~eats of (AKKARA~ in the of heparin (1: 0, -~-), indicates a charged coil conformation (peaks at 195 and 216 am, crossover at 210 um). . However, in contrast to the hepuin-itaducod conformational .change seen for the high affinity heparin-binding peptide (AIGCARA)6, (AKKARA~, which binds heparin weakly, remains a charged coil in the presence of heparin (1: 0.25, -O-; 1:
0.50, -X-;
l: 0.75, -~-; 1: 1, -O-).
FIG. s ACE s~ralysis of the i~nterodioas betwroat peptides contadnieg heps~rin-binding consensus sequences surd AIJVEC PGs. ACE gel images as obtainod by a phosphorimager in whleh (A) EC PGeIGAGs or (B) be~tn was fractionated through w0 OW4~ii PCTN800~953 peptides. la (A), at least two popnlati~a of high affinity PGIGAG, seen as two bands of radiolabeled tnatetial migrating with differed mobtv'ties, is visible at peptide concentrations of < 50 nM. At a peptide concentration of 250 nM (near the ICS

nM), a separation of the PG/GAG species is evident as a brotul smear throughout the lane, and as a sharp band that migrates approxfnnacely half way down the lane, indicating 6doroga~eity in sine, dHrge deity and/or pe~ide binding interactions of the PGIGAG population. PG/GAG samples in which HSPGs have been chemically degradod by nitroas acid (EC PGsINA), also displayed high binding a~rnitY
(1Ca= 300 nM), implying that choadroitiddermataa sulfates which remain in the sample bind the peptide stmngly. In contrast to the heterogeneity seen in (A), (B) shows that heparin migrates as a single broad band of radiolabeled masaaial.
FIG. f Atlflnity nt. (AR>f~AAKA~ fat HI)VFC PGs sand PG ooapon~. 'ILe peptide was analyzed for binding affinity no HUVEC PGs/GAGs by ACE, and the ICd of the peptide-PGIGAG interactions were calculated from binding plots as detallod in Expetirnental Proc~ttres. Similar affinities (-300 eM) were obtained for total PGs, for PG samples devoid of HS GAGs via nitrous acid treatment (NA) or heparatinasc I
dig~ion (H), and f~ P'Gs devoid of CS GAGs via ch~dmitim~aa AHC (ABG~
digestion. Liberation of GAG chains from the core protein by borohydride reduction (BH) of total PGs caused a 3-fold reduc~n in affinity (ICa~ 1200 aM).
FIG. 7 Nentrali~tion of Lorenoot by P~Wa in Vivo: Anti -Fs~ctor Xa Assay.
Nerr~ of Lorenox by Pepddss fx T~ro: .!~ F~terr Xa Away Absorbanee ~ 405 nm ~finea the heparin concentration in the plasma as a funexion of the amount of anti-Factor Xa activity. Heparin ooorplett~es with Autithrocnbin III and the complex inhibits Factor Xa. The amount of Faedor Xa activity is deberminod by the change in A.oa over 1 minute by chromogcnic assay. 'Ihe low point on each curve represents the highest amonrtt of and-Factor Xa activity, a function of the highest cono~tration of heparin obtained in the particular animal. A~ of 1.0 represents about O.SUImt of anti-Factor Xa activity, and A~ of 0.5 represents about 1.0 Ulml anti-Factor Xa activity, based on staudaaditation against Hepanorm low molecular weight heparin standards fur the Stachrom Heparin kit. Administration of the peptide results WO 0114'831 PCTNS00~12a53 in formation of a peptidelheparin complex, thus reducing the amount of ATIII-heparin complex and therefore reducing Factor Xa activity, resulting in reduced breakdown of the dye and return to baseline of the Ams. Rats were injected with Lovenox alone (Panel A) or with Lovenox followed by place minutes after injection of Lovenox (arrow) (Panels B-J). Peptides were adaranisoered at 2 mgI300 gm animal except where noted otherwise. Blood samples (0.1 ml) were obtained for anti-Factor Xa analysis immediately before the iqjection of Lovenox, at 30-second intervals after the injection until IO mutes, then at 1 minute intervals until 15 minutes, and at 5-minute intervals until 30 minutes.
DETAILED DESCRIPTION
Peptides included in the present invention (all single letters represent conventional nomenclature designations for amino acids):
1. (ARKKAAKA)o 2. (AKAAKI~A)e 3. (AKKARA)~
4. (ARAKKA)e 4. YPARRARYQWVRCKP
5. YPTQRARYQWVRCNP
Examples of variations of the above peptide motifs included in this patent:
A. (XBBBXXBX~ or (XBXXBBBX~ where B denotes arginine (R), lysi~ (K) or a combination of the two, X denotes preferably but is not limited to alanine (A) or glycine (G), and n > 2. For example some possible permutations of the sequences covered by these patents will include but not be limited to:
(ARRRAARA~ (ARRKAAKA)~ (AKKRAAKA)~ (ARAARRRA)n (ARAAKRKA)o wo wbat rcrn~soo'aea3 (GRRKGGRG~ (GRKKGGRG). (GI~C(~(iRG). (GRG(iKRRG~ (GRGGKKRG).
B. (XBBXBX~ or (XBXBBX)n where B ie arginine (it), lysine (K) or a combination of the two, X is p~ferably brut not limvood to sLniae (A) cc glycine (G). sled n > 2. For example some possible perk of the sequences covered by these patents will include but not be limped to:
(ARRARA). (ARKAKA). (ARARRAh (ARAKKA~
(GRRGKG). (GKKGRG~ (GRGRKG)n (GKGKRG).
C. Inchudon of a aia~le cysteioe (C) within 3 residues of ait6or the N- or C-laminas as in YPARRARYQWVRCKP or YPTQRARYQWVRCNP, or in peptide sequctxes (XBBBXXBX)., (XBXXBHBX),, (XBBXBJQ., or (XBXBBX)., where n > 2. Por example some possible permutations of the sequencxs covered by these patents will include but not be limited to:
ARRKAARA-ARRKACRA ARCAKKRA-ARAAKIGiA-ARAAKKRA
ARRAKA-ARRAKA-ARRCKA AKGIQiA-AKAKRA
D. For any of the above peptides, this patent will also cover inclusion of the D- isomer forms of amino acids in place of the L-forms, or inclusions of any combinations of D-a L-isomer forms to etreatc ra~em~ts resistant to proteolydc degradation for in vitro and in viva applications.
E. Fs any of the above pept~es, tiffs prmem will also cover inclusion of any other amino acids in any X position.
F. This pwac wB1 also peptides which inoo:poaate muivpk copies of the heparin-binding co~osus sequdtees, but which are not necessarily arranged as concatamera, e.g., two such peptide may be ARKKAARAAAAAAAAARICKAAIZA or ARKKAARAAAAAAAAAAAAAAAAARKKAARA

WO ltQl~5831 PCTNS00Vn2lISi Embodirneruc of this invention The present invention relsttes to a tmcnber of different peptides of various sequetrces and sizes, including pharrmaceuticals or bioactive agents composed of the peptides coalplexed with or incorporated into delivery vehicles such as salts, solutions, solvents, and/or carriers, andlor covalently linked to other bioactive agents.
These approaches are well known in the ad and thus will not be discussed in great detail here.
Fur example, the peptides may be used as phuma~utieal salts of agents including, but not limited to alkali metal salts, organic carboxylic of sulfonic acids, or inorganic acids, etc. Acceptable carriers for the peptides may include any of a variety of diluents, solvents, time release polymers, fillers, or binders, etc, and formulated into d~age forms such as pills or injeaable solt~ions, eoc. This patent also includes any of the peptides derivatized with functional groups andlor linked to other molecules to facilitate their delivery to speeiflc sites of action, to potentiate their activity, or complexed covalently or non-covalendy to other pharnnaccuticals, bioactive agents, or other molecules. Such derivatizations must be aeaomplighed so as to not significantly interfere with the heparin- or PG-interactive properties of the peptides.
Carriers and derivatizations must also be designed or chosen so as not to exert toxic or untoward activities on animals or humans treated with these forraulations. Functional groups which may be covalently linked be the peptides rnay include, but not be linvted to, amines, alcohols, or ethers. Functional groups to be oovalently linked to the peptides to increase their in vivo half lives may include, but not be limited to, polyethylene glycols, small carbohydrates such ere sucrose, or peptides and proteins. The peptides may also be synthesized by recombinant DNA techniques with expression vectors for . . 2~ use in biological.systems, such as bacteria, yeast, insect, or mammalian cells. Methods are well known in the art.
Desixn and synthuis of novel hixh e~nity heparin-~bindinR Deptides and determination of their heparin- and EC PG-bindin~t sties.
Peptide Synthuis- Peptides wen synthesized and purified by the University of Virginia Biomolecular Research Facility (Charlottesville, VA) or by Ci~enosys Biotech~logies (The Woodlands, TX). Peptides were synthesized by standard solid WO o0/Ii631 t'CfIUS00ro2l.S3 phase synthesis using FMOC chemistry. P~cptide amieatlar weight was verified by mass spectroscopy, and purity (> 70%) analyzed by HPLC.
Preparation of Radiolabelerl Heparin- Whole heparin from pig intestinal muoosa (Sigma) was tyramine end-labeled and radiolabeled with Na~l (Amersham, Pharmacia Biotech, Inc., Fiscataway, NJ) to an average specific activity - 1.0 x 10' CPM/pg as describod59. Radiolabeled heparin was &ao<ionated on S~hadex G 100 (Bio-Rad Laboratories, Hercules, CA) and the final ~ 12% of material to elute was retained as the low M~ material of < 6,000 (Jordan, R., D. Baler, and R. Rosenberg, J Biol Chem, 254:2902-2913, 1979; Laurent, T. C., et al., Biochem J, 175:691-701, 1978).
Electrophoretie Analysis of Binding of Heparin and Human Umbilical Vein Endothelial Cell (HUVEC) PGs to Peptides- Binding of radio>abelal heparin and H U V EC PGs to peptides was studied by ACE as detailed elaewherc (McPhcrson, J. M., et al., Collagen Rel Res, 1:65-82, 1988), since the hepuin protein binding affinities revealed by ACE
match reasonably well with those obtained by other well establislxd qualitative techniques for measuring binding intersetiona, e.g.,( McPherson, J. M., et al., Collagen Rel Res, 1:63-82, 1988; San Antonio, J. D., et aL, J Cell Biol, 125:1179-1188, 1994; San Antonio, 1. D., et al., Glycobiot, 4:327-332, 1994; Tsilibary, E. C., et al., J Biol Chew, 263:19112-19118, 1988). BrieBy, peptides were dissolved in 1X
ACE running buffer, 50 mM sodium 3-(N morpholino)-2-hydroxypropanesulfonate (MOPSO, Sigma) I 125 mM sodium acetate, pH 7.0, and serially dihrted in running buffer at 2X concentrations. Peptides were then mixai 1:1 with 296 agarose/l%
CHAPS, 3-[(3-cholamidopropyl~imethylaanmonio]-1-propanesulfonate, (Boehringer Mannheim, Indianapolis, IN), and loadod into wells of a 1 '% agarose gel.
Radiolabeled heparin or HUVEC PGs were then loaded in a slot on the anode side of the gel, a~
electrophoresed through the peptide-eon~ning wells, towards the cathode. Gels were driod and PG mobility was measured with a P6osphorimager (Molecular Dynamics, Su~yvale, CA) by scanning oath protein latx and determining the relative radioacxivity content per 88-lun pixel through the leagth of the lane. Retardation coefficient (R) measurements, binding isotherm curve fittings, and apparent Ke value determinations WO 10/45831 PCTIU'5001~2853 were calculated as detailed previously (Lee, M. K., and A. D. Lender, Proc Nat Aced Sci USA, 88:2768-2772, 1991; San Antonio, J. D., et al, Bioc)rem, 32~.4746-~4755, 1993).
Some pepti~s were also analyzed by ACE for heparin-binding under reducing S conditions. Thus, after serial peptide dilution, p-mercs~ptoethanol (a-ME) was added at ~ to each peptide sample, and these were mixed I : I with 2% agarosell %
CHAPSIS:~ ~-ME, sad added to the AC8 gel sample wells as usual.
Binding analysis of peptides to enzymatiCally or chemically degraded PGs (see below) was carried out by ACE as detailed, excxpt that PG samples inclt>ded 6 M urea to denature any residual enzymes.
Cell Culture- HUVEC were isolated as detailed elsewhere (Gimbrone, M. A., In Progress in Hematobgy and Throio4bosis. Vol. III. B. S. Collen, oditor. W B
Saunders, Philadelphia. 1-28, 1975) and were used up to passage seven. Cells were t5 caltured on 0.2% ge>sdn-coated tisane cultare fla~CS in normal calture media composed of medium l99 (Gibco HItL, Gaithersburg, MD), IO% fetal bovine serum (FBS, Mediatxh Inc.), 80 ltg/tnl endotlulial cell growth supplanent isolated fmm bovine hypothalami as described (Maciag, T., et al., Proc Nat Acad See USA, 76 (11):5674 5678, 1979), 50 teglml heparin (porcine intestinal mucosa, grade lA, Sigma) 1 %
penicillin-streptomycin, and 0.196 fungizone.
Radiolabeling and IsolatFon of Total HUVEC PGs acrd GAGS- Exponentially growing, subcontluent HUVEC were labeled with 35 pCi/ml '~S.NabSO~ (ICN
Pharn>aceuticals, Costa Mesa, CA) in normal culture media minus hepatic for 12h. Culture media and cell layers were harvested separately. After removal of the media, cells were washod with 2.0 ml PBS ph~s Ca2+ -Mgzt. Media and rinses were pooled and brought to 6 M
urea, 10 mM EDTA, 1 mM plunyin~hylaulfo~rl fluoride (PMSF7, 5 mM N-ethylmaleicnide (NEM), 50 mM 6-aminocaproic acid, 5 mM benzamidine, and 1 ~glcnl pepsratin A. Sample were stirred for 15 min at room temperature, .then centrifuged at 10,000 rpm far 30 min to remove insoluble materials:
To the all layer was added 2.0 ml of exttacdon solution, 6M urea, 100 mM
NaCI, 0.296 Triton X-100, 30 mM Tris-HCI, pH 7.0, and protease inhibitors as WO 00145f~i1 P4TNSOOI~853 described above. Cells were scraped off the dishes, and the extracts were pooled and stirred for 5 min at room temperature, and then ~trifuged as described above.
Samples were concentrated on DEAE (DEAE Bio-Gel A Agarose, Bio Rad Laboraoorics) equilibrated with low salt buffer, 0.1 M NaCI, 6 M urea, and 50 mM
Tris-HCI, pH 7Ø Columns were rinsed with IO ml of low salt buffer; flow through was discarded. Bound PGsIGAOs were chafed with 3 ml of high salt buffer, 1.5 M
NaCI, 6 M urea, and 50 mM Tris-HCI, pH 7Ø Eluted samples were dialyzed against distilled water, lyophilized and stored at -20°C until binding analysis.
Enrymatic Digestions of PG~r- The contributions of PG GAG chain components to peptide binding affinities were assessed by aeleetive enzymatic degradation of GAG
chains prior to ACE analysis. ~S-Na~,SO~ labeled HUVEC PGs were digested with chondroitinase AHC, or hepuratinase I (Seikagabu Antnerica, Ijamsville, MD).
PG
samples were resuspended in 100 pt enzyme buffer [chondroitinase buffer: 50 mM
Tris-HCI, 30 mM sodium acetate, pH 8.0, 0.1 mM pepstatin A, 0.5 mglml BSA, 10 mM NEM, 1 mM PMSF, and 5 mM EDTA; hepatatinaae buffer: 50 mM Tris-HCI, 5 mM calcium acetate, pH 7.0, 0.5 mg/ml BSA, atbd 1 mM PMSF]. Samples were digested with 0.05 Ulml chondroianase ABC ~ 37°C for 3h. Fresh enzyme was then added to the same concentration and the incubat'ron continued for an additional lh.
Heparatinase I was added to samples at 0.01 U/ml. Samples were incubated for 3 h at 43°C, and fresh enzyme was then added to the same concentration, and incubation continued for an additional lh. All saaoples were that stored at -20°C
u~il binding analysis.
Chemical Degradation of PGs- The contributions of PG GAG chain ~nponents to peptide binding affinities were further assessed by selective chemical degradation.
Total secreted HUVEC PGs were subjected to nitrous acid degradation as detailed (Shively, J. E., and H. E. Conrad, Btochem, 15:3932-3942, 1976), which selectively degrules HS GAG chains. Binding analysis to peptides was then measured by ACE.

we W'~s°°~a~
~3-elimina~on of PGs- GAG chains were released from PG core proteins by allralime baohydride reduction as de4iled (lozzo, R. V., and W. Mulkr-Glanser, Canc Res 45:5677-5687, 1985).
Giircrtlar dichrolsm spectroscopy- Circular dichmism (CD) spectra were rooorded at 22°C using a JASCO J-3000 spectcopolarimeter interfaced to a 486 PC.
The padr length of du CD alla was D.5 mm, and the CD was expt~saed in terms of ellipticiry (O] in degree~ad~dtnol'. Samples were initially dialyzed tn water to remove residual synthesis cotttamittants, lyophilized, and resttspeoded in water at 1 mglmi and the pH was adjusted to 7Ø Peptide concentrations of 0.1 mg/ml or 0.2 mglml were analyzed. Typically, two scans were averaged for each spectrum. CD spectra of peptides in a-helical oon6amations were roooatied in the prey of trifluorocthanol (TF~, which nerved as a positive control of peptides in a-helical conformations. To determine the effaxs of heparin on peptide conformation, solutions containing ptptide phts heparin were prepared at various peptide: heparin ratios (wlw).
Analysis of Ptptidt Conc~ntratlon- C0A00ntCafionti of peptides used in CD were verified by ID NMR spectroscopy based on an interns! 2,2-d~thylsilapentane-5-sulphonate (DSS) standard. NMR experiments were ron a Hruker AMX 600 NMR spaxrometer equipped with a 5 mm broadband inverse probe, using the XwinNMR 2.1 software package run on a Silicon Graphics INDY work station. 1 D
proton spectra was acquired at 303K using a 4 a relaxation delay and were proaessod with 0.5 Hz exponential line broadening. 230 pl of a 1 mglml peptide solution (as deoaminod by weight) was lyophilized std dissolved in 443 ltl D20, cvutaining 0.123 mM DSS. The degenaaGe arginitte ~~ resooeaces at 3.2 ppm, ascertained by a TOCSY experiment, were integrated and compared to the internal standard.
Results:

~p~ls-l~rin interacriionr- To dean amtll pq~tidea rx)n'bit high affinities for hepsritt and for the CiAtl components of PQa, peptide sequwas modeled after proposed heparin-binding eonsa~ua sequence motifs. Thus, a collection of peptides oootaitaing ace of two cooeena~ eetpanoe taotlb, r,~ither XHBXBX
or S XBBBXXBX, as wall as various modifications of these, were synthesized (Table n. As peptides containing a single heperln~alndlog seqttanoe often show little or no affinity for heparin (Conrad, H. E, Heparin-Binding Prota'ras. Academic Press, Sao Diego, 1998), a strategy used here was to include eanaatsus secpionces In multiple copies within peptides. In initial snldies we adected for ayats tire seqo~ces (AKKARA)~ or (ARKKAAKA~, where n = 1-6. Alanine was itrhrded in the hydropathic positions becatue of its stabilizing activity cu a-he~ces (Petran, D. S., et at., Biorhsm, 31:5010-5016, 1992) cad the basic amino acids were chosen to represent rheas with the highest probability of occurrence in each basic poaiaon in the h~arin binding oonseasus sequences of native heparin-brag proAeins (Candin, A. D. and H. J. R.
Weintc~wb, ArteriosclerosW, 9:21-32, 1989). When single eopiea of either sequence were tested for ivparin billing by ACE, no w~eree de~ec~ad. In coast, pads oonrai~ag two copies of the consensus sequence exhibited weak but detectable affinities for heparin ( < 6 ~M), and peptides of higher molecular weight containing 4-6 copies of a co~aseasus showed a nnarlsed i~aeae is haparln-bi~d3ng aflb~r (10-150 nM) (Fig. 1). The heparin-binding afftn'sty of both the 6-mer ea~d 8-mer tandem-repeat peptides reached a plateau as peptide length approached 30 amino acids [(AKKARA~, K~ 90 nM and (ARICKAAKA~, ICS 40 nM] . Larger peptides I(AKKARA~ and (ARKKAAKAh] displayed similar affinities (IGrn 100 and 50 all respectively, Table I).
To define the saluence and aonformstional fadu~s of the ran-rat peptides which confer flair high affttftty haparitrblnding charaaeristica, peptides cotitaimttg variants of one of the consanaus sequences first tested, (ARKKAAKAr were synthesized. These included those in which alaniaes wan replaced by other hydropadnic residtxs, the spaeings between conseoeus saEttences were altered by removal or addition of alsmine residues, or the poterrdal of the peptides to form stable a-helixs was , , inhibited by including prolinc reaiduea at varlaus positions. It was found that peptide afFmityr for hepaaln was decrased when alaouse was replaced by glycine in all the hydrapathic positions [(ARKKAAIKAh K~ 135 aiNi, (aRKKC3aKCib ICS 200 nM, p <

WO 10J13g31 PCTNSAaIOQ~i 0.01]; less conservative substitutions had varying ei~cts as heparin-binding affinity, i.e. for (LRIQ~LGKR~, ICS 103 nM, avers uaafBeaood; for ~CRKIG.GICy, ICS
740 oM, (p < 0.01) affinity was docressad (Table I).
Two peptides were synthes'zod in which the spacings between adjacent consensus swere shared. lBam increasing (ARKKAAICA-AAAA
ARKKAAKA-AAAA-AR,KKAAICA) or doaeatdttg (ARKKAAKA-RKKAAKA
RKKAAICA) the distaaoe baween consensus aequresulted in decreased bcparin bindlng affinity (K~ 250 and 450 nM respectively). Inclusion of prolines also decreased the heparin-biming atlinity, ~ the dagtee of which was influenced by their position and number. Thus, the heparin-bi:oddg atllnity deareasad to 360 nM
when prolines were present in each tandem repeat in plax of an elanine:
(ARKICPAKA)3;
however, a weaker affinity was obtainod vvhw a sale proline was substitutal in the center of a series of three heparin-binding coatsensus sequenxs (AR1~KAAKA-ARKKPAICA-ARKKAAICA, K~ 730 nM, Table n.
Other peptides synthesized and died include sequencxs native to the moose (YPARRARYQWVRCICP) or human (YPTQRARYQWVRCNI~ scrglycin (SG) ooare proteins, which contain either a single or partial conseaaus sequence, respectively.
These showed significant affinities for heparin (Key 200-900 nM, Table I and Fig. 2), despite their small sizes (about 2000 Da). To ebci~e the basis for the strong heparin-binding features of these peptides, the ability of the basic residues to sustain high affinity binding was tented by studying a pq>t~ide which contained all the basic residues of the mouse seqnence in their native positl~, but in which all other residues were - changed to a>aniaes (AAAR:RARAAAARAIKA). A 3f0-fold decrease in hepuia binding affinity (K~ 72 ~ for this peptide iadic~ed that the member and arrangement of basic residues in the mouse sequence was not sufficient for high affinity binding, and saggests the importance of one or more of the other non-ba$i~c residues (Fig 2). We aezt tested whether the C-terminal cysteine in the miwtse SG peptide may promote peptide dittser formation, thereby influencing heparin-binding affinity. Thus, heparin-binding was tested by ACE under reducing Ana, and it was found that this treatment yielded negligibk heparin-binding. Likewise. when the cys<eine rcsidne was reph>cod by an alaninc in the native mouse SO sequence, (YPARRARYQWVRAKI~, heparin-WO 00/lS~i1 PGTN900/0~853 binding affinity was again negligible; both results are consistent with the potential cross-linking function of the cysteine residues (Fig. 2).
CD- The intrinsic structural properties of the peptides were exptored using CD
spectroscopy. Short peptides of frnown heparin-binding protci~ containing tteparin-binding consensus sequences have previously boon shown to fold into a-helical conformations. In doing so, the basic amino acids lac~te to one face of the helix, and thus are potentially exposed far binding. Peptides which displayed weak (AKKARAy~, moderate (AKKARA)~, aad strong (AKICARA)s and (AKKARA)~, heparin-binding affinities, were analyzed by CD to ~hat'ectetize the'u degree of a-helical contents and pro~asities to form an a-helix. All peptides exhibit very similar spectra with peaks at 195 nm and 216 nm a~ a crossover at 270 not [for example, see Fig. 3, (AKKARA)e, 1:0, -~-, and Fig. 4 (AIC1CARA)x, 1: 0, -~-]. These spears are indicative of an attended charged coil conformation that was previously reported for P~Y(L)_ i5 lysittes and pofy(L)-arginines (GdaQaa, R. A., et al., 8iopolymers, 12:541-558, 1973).
Intrinsic CD of the peptides shows the they do not adopt a-helical conformations. To explore the confonnetional repertoire of the peptides and to record CD spectra for the a-helical conformations, peptides wet~e analyzed by CD in the of the noon-polar solveoi TFE. Non~olar solvems are luwwn to increase the degree of a-helicity of a peptide in solution by enhancing hydrogen bonding and electrostatic irneractions (Adler, A. J., and D. D. Fasman, J Phys Cir~m, 75:1516 1526, 1971). CD of (AKKARA~, at 0.1 mglml ~rataining 0, 10, 20, 30, 40, and TI~E (v/v) was measured. At TPE concentrations >309b, with an apparent maximal effax induced at 4096 TFE, the peptide assumes an a-helical conformation with classic a-helical peaks at 206 and 220 mn and a cross over at 19'7 nm (data not shown).
The CD spectra of (AKKARA)s recorded in the presence of increasing amounts of heparin (Fig. 3) demonstrates that a charge frown a charged coil conformation displayed in the absence of heparin (1: 0) ooeurs upon heparin additioa (1:
0.25, 1:
0.50, 1: !). Heparin induces a similar a-helical conformation at a 1: 1 peptide: heparin ratio that was obi in the pret~oe of >3096 TFE , with classic a-hWical pealrs at 190, 207, and 222 tun. At higher hepuin concentrations (1: 2 or 1: 4) the a-helical WO O~I4~31 PCTN900J02~i form is lost sad the apearum rIes that of a random Coil structure. This ability of excess GAG to disrupt the a-lulical eouformatio~ of a polyptptide in solution has been reported previously (Gebaan, R. A., et al., R~pot~era, 12:541 558, 19?3).
This same heparin effect is not obtained for the weak heparin-binding peptide (AKKARA)z (Fig. 4). In the abaenca of heparia (1: 0), the peptide assumes a similar charged coil conformation as t6u for (AKKARA)6. but fails to display a-helical character in the presence of h~ (1: 0.25, 1: 0.50. 1: 0.075, or 1:1).
Peptide-PG intcradeo~s- The ioaxactitrna bauroen conseosas sequence peptitka and PGs were also examined. Per these expernnents total PCis were isolated from HUVEC cultures, since HUVEC have bees shown to aq~reas a variety of types of HS
and CS PGs, io~eJndi~; for exsoople: syndecans; pertacan; gIYP~ ~ M81Y~
(Mertens, G., et al., J Bio Chem, 269 (28):20433-20443, 1992; Jarvelainen, H.
T., et al., J Hiol !~, X66 (34):232'!~1-23281, 1991). llua, cell laycx~aesociated and secreted'sS-SO~-radiolabeled PGs were purified by extraction with urea, and those PGs retained on DEAF after a 0.1 M NaCi rims were studied for their binding to (ARKKAAKA)< by ACE (Fig. 5A, l~C P'Ga). This pride exlu'bital Bad for secreted HUVEC PGs, although the average affinity was somewhat weaker than that exhibited by the peptick for hepau~in (PG Its 300 nM, heparin 1~ so nM).
similar affmi~a were obtiitard for cell layer associated P(3a (data not s~hawn).
Inapaaion of ACE gels in which aeaeted PGs were 6~actiottated ehrough peptides demonstrated the presence of at least two populations of PG evident as two distinct bands of radiolabekd maarial mig<saing through the peptide lanes wild ditfarenc mobilides (Fig. 5A, EC
PGs). This difference in migration rate could indicate heterogeneity of the PG
in size or charge. In contrast to the heteroge~,ty seen is Fig,. 5A, Fig. 5B slwwa that heparin mig~ as a single band of radio)abaled materW.
Thus, to ascertain which GAG chains, as well as which PG component, (i.e.
core per, GAG chains, or bob, vra~e cespa~bk For Peptide~iodiog, total HUVEC PGs were subjected to various chemical and enzymatic degradations.
Samples were then faced. for tlnir ability to bind to (ARIGCAAKA)~. PGs in which HS
GAGS
were c)~em~ally degraded by nitrous acid or e~ymatically degraded by Iteparatinase I, were able to maintain comparable affin'tty for the peptide as was displayed by the toW

WO 001ISE31 PGTnJS0010ZS53 FG sample ~g SA, EC IPGaINA aoQ Pig. 6). PGs in which CS GAG chains werc digested with choadroitinase AHC were also able to maintain comnparable affinity for the peptide. Release of GAG chains fimn oocea by borohydride reduction resulted in a 3-4 fold diminished affinity (Fig. ~.
Heparin irrssrodions with cavramsrcial nohcatioru For the binding of polylysinas (p~rehased from Sigma Chemicals) to heparin, it was shown that at least for those polymers of high M~ (= 100 Idlodaltons), inclusion of either L- or D- amino acid isooQer forms or combinstlotts of these isomers in the polymers did not influence binding affnoity. Thane data suggest that that inclusion of the D-isomer. amino: acid forms in the pepddes described in this patent application will be useful in in<xessing their longevitiea for various applic~tioos (e.g., in vivo use or uses in vitro with living cells or coil or tissue extracts) where protsolytic processing of the peptides could present a problem.
Summary arid Discussion:
The goal of these exparhndas arcs to design high afEnity heparin- and PG-binding peptides; the strategy we used is to incorporate into their stru~xure copies of sequences proposed to bind heparin in native proteins. Our approach is also based on the fact that truncation of peptide structure without loss of activity cam sometimes be achieved by eot~srraining or manipulating peptide conformation (Starovasaik, M. A., et al., Proe Natl Aced Sci USA, 94:10080-10483, 1997). In the case of apolioproteia E
and apolipoprotein &100, heparin4aurdina sites arc believed to form a-helices upon heparin-binding, and molecular modeling illustrates that basic amino acids in the binding sites align to one side of the helix to form a region of high positive charge through which heparin-binding encore (Cardin, A. D, acrd H. J. R. Weintraub, Arteriosclerosis, 9:21-32, 1989). Thus, in our design of heparin-binding peptides, we also incorporated structural features conducive to stable a-helicity.
In our initial experiments, families of peptides were synthesized that contained single or multiple copies of heparin binding consensus sequences. When their heparin-WO 00V41~31 binding was examined by ACE, p~tides oo~im~g single seqtKaces showod no measurable affinity for hepania. This t~ewlt is as expecoed aiace peptides carrying single heparin-binding seguences foutd is native proteins often fail bo display significant heparin-binding (Conrad, H. E., Heparin-Binding Proteins. Academic Press, San Diego, 1998), but they may contain multiple cottaeneus sequences that come into proximity upon protein folding or multinterization, thet~eby enhancing heparin-binding through cooperativity (Huntittgton, J. A., et al., &ocfum, 35:8495-8503, 1996). 1n contrast, the affinity of pe~idea (AKI~CARA~ or (ARBI~AAKA). ranged imttt weak (ICS 6~0 it,M) when n= 2, to atrong~ (ICS 30-100 nM) when n= 3-6. These latter affmiaes arc in the range of those dlaplayed by >>epan~in-binding proteins such as bFGF
(K~ 10 nM), or type I collagen, (Ku~ 100-200 nM) (San Antonio, J. D., et al., Biochem, 32:4746-47, 1993). However, the fact that the peptidts are roughly 4 times smaller than bFGF and 100 times smtller than type I collagen highlights their significant heparin-binding abilities. The atktnity appearrod to plateau at n > 5, or 36-40 amino acids, augg~ag that pepddea of approxizt~dy 30-32 amigo acids were of sufficient length to occupy all available binding sites on Iow Mr heparin, and that additional amino acid residues beyond this did not contn'bute to heparin binding due to a lack of available ligand. However, this hypothesis can not be testod without knowing the M~ distribution of the heparin used in these expertmmts.
Other expaitnents exa>niwed heparin binding by peptides iocludiag seqne~
native to proteins which catraitt a single or partial t~pauin-binding consensus sequence.
lEteanlts again atggestod the aiti~l Mare of peptide M~ and nttmbetr of oonsenaus ,~~~ ~. h_b~g. ~~ surprisingly, a stroyig >irparid-bindiitg affinity was displayed by a peptide caaeaponding to the tnotrse SCi proteoglycan core protein containing a single consensus sequence, YPARRARYQWVRCKP (K~ 200 nM).
However, the affinity was dimittished over 200-fold by disulfide reduction, or replacanent of tire cysteine with alanine, thus itttplyfng that its strong heparin-binding relies on peptide dimerlzatioa, and that the other traidues flanking the consensus sequence ware of little consequaree. Indeed, others have shown that inclusion of cysteines near peptide termini to promote disulfide bond fonnauon tray iatpmve peptide-ligand binding (Stamv~nik, M. A., et al., Proc Nail Aced Sci USA, 94:10080-10085, 1997); our results suggest this to be a simple strategy to greatly enhance the WO 00I~i61si1 PCTN801~0QgS3 affinity of peptides for heparin. SG, cer~ro~lyam (with PRRLRL) (Stipp, C. S., et al., J Cell Biol, 124:149-160, 1994), aztd parlxan (with TRRFRD) (Murdoch, A.
D., et al., l Blol Gum, 26'7 (12):8544-8557, 1992) are the few PGs drat contain heparin-binding consensus aequet~ea on their sort pr~eins. Interestingly, the SG core protein, which carries tinny hepafin drains, migrates at tarice its ptodicted tnolaxilaJ
weight on PAGE gels under reducing cotdit3ons (Perin, J.-P., et al., Biockem J, 255:1007-1013, 1988); suggesting ditneri~etion. This could result from GAG
chains of o>ye PCi binding to the core protein of another, or core-core associations through disulfide bottling. The potential physiological fanca~on of such PG-PG
interactions remains to be explored.
Additional cons~tsus sequence peptides were designed to determine other aspxts .of peptide striuxtire important to heparin-bitding. Including glycine in place of alaaine in the hydropathic positions weakemd heparin-binding, and peptides in which arginine was included in all basic positi~ disp4yal higher aff~ for heparin than 1 S did those containing argittinea and lysiaes. The later es consistent with work showing a higher affinity interaction of argittino-heparin and argini~ne-HS than lysine-heparin or lysine-HS (Protean, J. R., et al., ~lrclt Bfochsm Biop~hys, 232 (2):279-287, 1995). This suggests that the heparin-binding characxeristics of the peptides developed here may rely on amino acid type and arrangearent in addition to ionic interactions.
Inclusion of prolines within or between cottaenstts sequence motifs weakened affinity for heparin, possibly as a result of alterations in peptide seoaatda:y conformation; this issue was investigated in our CD experiments. Finally, diangittg the spacing between consensus ~tnot;fs weakenod affiaity~ for heparin; however, aaqtiettcx orientation did not appear to influetux binding ability as long as the mss were contiguous sad in one ori~tion.
Molecular modeling of consensus seqitetices in native heparin binding proteins predicts their presence within a-helical regions (Cardin, A. D, and H. J. R.
Weintraub, Arttrlosclerosis, 9:21-32, 1989). Additionally, GAG-directed conformational changes on polypeptides such as poly(L)-lysine and poly(L)-arginine have been idemified (Gelmatt, R. A., et al., Blopolynurs, 12:541 358, 1973; Gelmen, R. A., and 1.
Hlackwell, Arch Biochem Elophys, 159:427-433, 1473; Gelmaa, R. A., and J.
Hlackwell, Btopo~y»urs, 13:139-156, 1974). Aqueous aolutiosis of these poiypeptides at neutral pH were shown by CD to alopt charged coil conformations, and to display a-WO 00llstil PCTNS0QI0~is3 helical oonformati~s in the pre of hepuin. Our results showed that peptides of the type (AKKARA)p have charged coil eonfnrma<ions ~ neutral pH. In the presence of heparin, however, a peptide that showed high affinity for heparin, (AKKARA~, underwent a confortnatiOnal cbapge 0o an a-helix. In the presence of excess heparin, a further conformational change produced a randasn coil structure. In conua~, a peptide which displayed weak heparin-binding, (AKKARA~, failod to undergo any confomnFUional change. Thus, tlm sotmaott oouaform~ion of a peptide and its propensity to change conformation in the preaenct of heparin may be an indication of its ability to bind to heparin strongly. These data sad those from experiments examining the effects of including prolines in ptptides, which an known to disrupt the a-helical conformation, suggest that peptide se~ary a facilitates heparin-binding.
. . . Here vvc also examined the interaction txrvoett the high affinity heparin-binding . .
peptide (ARKKAAICA)< and EC PGs. Results showed that ECs secreted several types of PGsIGAGs which displayed signiflc~ affinities for (ARKKAAKA~ (Ks - 300 nM).
ACE gel images revealed the t~esolution of multiple PGIGAG species after their migration through ttte peptide-codainittg lanes, g heterogeneity in PGIGAG
charge, size, andlor binding affinities. It was found that the CSPGs or HSPGs likely bind the peptide similarly, since affinity was maintained even after treatment of total PGs with nitrous acid, which selectively degrades HS GAGS, heparatinase I or chondzoitinase ABC. The free GAG chains had 3-4 fold lower affinity than the intact PGs. Thus the core proteins of cauin EC PGs either may contribute to binding directly, or act as a tether to bring multiple GAC~ into proximity for cooperative binding: Similar wbservations have been made previously for cartilage PG-type B ' collagen irtteracnons (Toole, B., .l Blot C9tem 251:895-897, 1976) and SG-type I
collagen interactions (Schiek, B. P., et al., J Ctl1 Pltyaeol, 172:87 93, 1997). Our results are inconsistent with carbohydrate sequence selectivity in the binding of these peptides with EC PGs, since similar affinities for peptides were displayed by either total EC PGs or its CSIPG fraction.
Of note is that the heparin-binding peptides designed here incorporate concatatners of heparin binding conaeasas aeqv~, which should rarely, if ever, appear in native proteins. Nonetluleas, the proposed characteristics of heparin-binding motifs in proteins, as set faa~th by Cardin anti Weintraub based on their theoretical WO 0~t~t31 _ PCTNS6GI~l~S3 analysis of putative bq~ria-biodicg dom~dms of oedve pn~a~as (Cardin, A. D.
and H.
J. R. Weintraub, Arteriosclerosis, 9:21-32, 1989), hold true with our model peptides.
Thus, our data suggest that prides ciag tire Cardin std Weiatraub heparin-binding consensus sequetuxs may abow a selective advaeetage in heparin-binding over certain other sequwhich do not fit their txitaria.
In summary, optimally naive heparin binding peptides should include multiple seqotooes of die types: (XBBJ~JQ. and (X~,HXJCHJC~. Sequence member and peptide M~ are the moat critical features; peptides should be of at least approx'unately 30 residua, which could be decreaaod to 15 if cyateiiee is included near either terminus to promote dimerization. Peptides should contain coadiguous seqnencc arrays, without intervening residues bawan sequences. Ala»ipe, which stabilizes a-helical confioTmation, _ should occupy the hydmp~iia residue. positions, and arginine the basic positions. The high affinity P(3- or (3AG-biad~g peptides developed here, or derivatives thereof, could pmve uacfat as eoo~s f~ the promotion of cell-substratum 1 S attachment of PG-expressing cells, in the drgetiog of drugs to PG-expressing cells and PG-rich extraodlular matrices, or as a~go~ata of GAGrnediaced actions, e.g., neutralization of the anticoagulant activity of heparin, as is presented in this patent apptic~tion.
ds the hs and P on tlYa reversal ir>leibitton thrombin activity:
. . . , . . ... ~t ~~~ ya descalptiun of the effxt3v~eness of the peptide ooenpositans for counteracting the effects of nnfractiooated heparin, Lovenox, and 4rgaran on Factor Xa activhy in vitro, and on Loveeeoa ire vlro. Heparin is sdministered to patients either as unfractionated heparin or as heparin fragments. The fragments are prepared in several different ways, and result in a heterogauo~ miuttrt that varies adding to the methods of preparation. The low molecalar weight heparins currently approved for clinical use, dye s~ndard dosage in anti-Xa units wdere known, and their molecaelar weight distributions are:
Lovenox: < 2000 daltoets < 20?6 WO 00IIS~i1 PCTN900I0~53 (3000-6000 2000-8000 daltoms a 6896 a-Xa ILI) > 8000 daltons < 15 96 Fragmia: < 3000 daltons 3-1596 (5000 a-Xa IU) 3000-8000 daltons 65-7896 > 8000 14-2696 l:,ogiparin 600-20,000 daltons (Debark) > 10,000 daltoas 3096 Orgaran Heparan sulfate 84 96 (750 a-Xa IU) l7errnataa sulfate 12 96 (~ondroitin sulfate 4 9b Mean chain length 5500 daltons IS
Heparin inhibits the activity of throa~in and Factor Xs by binding to AT III
and thus enhancing the ability of antithrombin III to bind to these enzymes. The higher-MW >teparins prdumably set as a bridje between ATIII and throrttbin, and the binding of both proteins to the same molecule of heparin appears to be ia>po~uit for thrombin inhibition by ATDI. Thus the low auoloatlar weight heparins do not affect thrombin activity, but still render ATIII capable of inhibiting Factor Xa activity.
Wakefield et al in PN 5,919,761 tested their peptide against two preparations of LMWH, Logiparin ~,. ~venox, and got very different results with prataaiilie;; ptotatiiine produced 6096 reversal of Logiparin bnt only 309b against Lovenoa, and the other peptides tested with both heparins likewise were more effective against Logiparin. There arc insufficient data be judge whether these differences are due to differences in the comentration of active heparin sequences in the preparations, or differences in the molecular weight diatcihutions of the heparin chains, which could result in different razes of removal fmm the circulation and thus different levels of anticoagulation in the animals after the 30-minute waiting period. It is difficult to assess these data also because the actual levels of anti-Factor Xa activity and the aanounts neutralized, i.e. the absolute amounts of anti-factor Xa activity at the time of injectlon of the peptide, and at the subsequent time Wo IOI15631 PCTIUdIO~OSt63 points, as well as the lose of and-i~x Xa activity ~e to natural clearance of heparin in the dogs, are not gives. in the Harris study, the dose of u~racaonated heparin was only about 2596 of the standard doseltg in hums, and thus it is difficult to assess the efficacy of their peptides vvhhmtt lmawing the pwtjust stated for the Wakefield S atodios. Thus we cannot correlate out data with those of tl~ previous patents.
Metlmds.for in vitro cktermirmtJora o,~the s oftxntides on reNSrøal ofFaaor Xa adiv' Solutions of Lovenwc, Orgaraa, or uafractiam~tod heparin (Sigma) were Irrepared in 0.3296 sodium citrate or in normal human play to contain O.SU/ml anti-FXa activity. Caiibrativns we.te made against the atapdatds provided by the S~chroa~
Heparin (Diagnostics Stago) assay ldt. The lteparlolATIII complex was allowed to farm at 37°C Rx 2 urinates, peptide was added, the mixaue was incubamed for an additional 1-5 minutes, stb then Factar Xa was addod, and finally the color reagent, for one mimtte, and the absorbemce was reed m A43am. The iac~x in abeorbance fran the heparin i'red control to the test sample was divided by the difference in A,aa between the heparinizod control and the couQOl without heparin to obtain the 96 reversal.
As shown in the Table II, several of the pepddea which we tested wcre highly effective agaimtt heparin, tow mola;ular weight he~rin, and orgaran in vitro, and the activity was co~od with tin; >>iadiog ato the LMWH of 56000 daltona which had been used for the ACE binding atadies deacribod above (Table I). The exdeption was the human aerglycin peptide, which was highly effective against UFH and Orgarau, but net agai~t Lovenox.
In vitro efftCt of the ys'at/dsa on reverml of a~ib&ion of thrombin adi w~'roaionatad la~nwin Plasma was oba~d tram normal do~nocs. Tlwombin aona~tratia~ (human alpha thrombin, Enzyme Research Laboratories, South Bend, IN) was standardized to produce a time of 20-22 seooads. Hqwtrin was added at 0.5 IU anti-thin WO OW45ai1 1'CTNS0Qh2a53 activitylml. The clotting time for hepwain aloo~e was approximately 3 minutes.
To test the ef~Cts of the peptides in this syatmn, one atdmo~e aRa addition of heparin to the plums, the peptides were added In co~iotb ranging from 1-200 pglml. After one minute, thrombin was added and the Clotting time determined. The eof the peptides were ooncendration dcpaod~att. The clotting limo was rast~ared to normal values in all samples at the dosages shown botow in Table 1D. The ability of the peptides to na~liae heparin inhibition of thrtune was consistean with the b~odmg affinities shown in Table I, and with the effeclivextess against low molecular weight heparin anti-F~tor Xa acxivity in Table ~II.
.. In Viva leas gf Prides in Re~r.~t l9Alea~r ~'Lovxnux on Factor Xa dativi,~y Methods:
Rats (300-400 gm) were ,ed with tetaminelacepromazine amd were camnnhted is the kA jugular vein and night baoiooral vein. Blood satnplen vaxe all 0.1 ml. Blood wu drawn immediately before i~o~tion of Lovenox to establish baseline Factor Xa activity. Lovanox (43 IU anti-FXa activicylkg in 0.1 ml saline, based on suggested dosage for humans) was injaxed through the juguLr c~thexer, followed immediately by 0.2 ml of saline. Blood (0.1 tn!) was collected into sodium citrate frown the femoral vein every 30 aaAads for 3 mdn.The peptide was injoctod at 3 min through the-,fittgu>~ cadtetcr in 0.1 ml of phoaplnbe-bu!!%red saline, foflowod by a 0.2 ml~ saline flush. P~ida were admiaiatered at 2 mg exocpt where noted otherwise. Blood eot~on was immediately resumed way 30 seconds until 10 minutes after the initial Lovenox injection, then at 15, 20, 23 and 30 mdn. The samples were centrifttged to obtain plasma and were assayed far r~ual Lovenox by assay of anti-FXa xtivity by the Stac6rom heparin test idt. Aeos was after a I-minute incubation with the chromogenic Fatxor Xa substrate. The nosy is described in further detail in the Figure lt~td b F'ig. 9, w0 0114~i1 PGT/1TS00J~1~i~3 The aai~ala appared to tolGra~e the adm~oration of the heparin std the peptide without obvious changes in heart rate and t~espiration. No animals died follovring admiration of the pq~daa of ial~t in Ihis appljatia~, a>tho~ one animal died following administruion of Protamine. The results arc shown in Flg. '1.
The auxiossl ooocemntioa of Love~o~c ~ the plasma was 0.3-1.0 Ulml ami-Factor Xa activity for all the animals tested. The maximal plasma heparin conxntration was found by 2 2.5 aninntes after '"agertion. About half the Lovenooc was cleared from the circulation by 23-30 minutes after injocxioa, in an approxiumately Linear fashion for minutes and more slowly t6ereal~er. Panel A shows a repaeaentative clearance c~uve. The three peptides at shown is paae4 B-D ctused no. rernaval of Lovenox above that due to dared cla~soCe ~ the circulation alone (Panel A).
The peptide (ARICKPAKAh (Panel D) appaated to cklay clarance of the heparin from the 1 S circulation.
Ptotamiae and thret of our high affinity heparin-binding peptides [(AKKARA~, (ARKKAAKA)S, and (ARKKAAKA~] nauralined the Loveaox conamratioa by at least 5096-8096 within 2 minutes of iqjxtion of the peptide, the less tightly-binding mono serglycin Cardin-site peptide was aonoawhat lean ef6xtive. and th~exe was no farther clearance of Lovenox in all cxxa for the r~aoaimdrr of the 30-mimite e~cperiment (Panels E-7).
A series of peptides have been generated, wherein said peptides have the ability to ttwase the anod-FXa levels of Lovenox in r~s within a fow mime at Lov~x concentrations to be in patients. The ability of these peptides to reverse the efleas of Lovenox in viva appears b be consistent with their ability to ceverae du effects in vitro. The peptides reverse both anti-thronnbin and anti-Xa activity of unfra~ted heparin ire vttnn, and atu~t-Xa activity, un&actionated heparin.
Loveaox, and Orgaran in vitro.

W~ ~~1 TABLE I

Hepa>tn-binding of psJ~arfn-bf~n~ ou~sauirs se~asas ~ ~Y~ ~ ~B bY ACE, and K~ of peptido-hepuin iuoaace~oaa were ealcul~ed frasn binding piotx as daailod in Expecimemt Procedures.

Each supple was tested for Lepuin-binding three to clever lanes, with an average of four times. Ks represents an average of data oDdmed for all trials staudud deviation (S.D.); -p values < (AKKARAb and ~p valves < 0.01 versus 0.01 versus (AKKARA)s, p values < 0.01 and''p values < O.O~S versus (ARKKAAKAH.

Peptide Sequence IvG Ks, (tdVl? S.D.

XB9IBX r~ab AKKARA 644 Not detedabie (AKKARAb 1270 10,000 + 18,000 (AxKAItAb lags 1,900 21a~

1 (AxKARA>< 2s2a 174 19 s (AKKARA)s 3146 94 41' (AKKARAb 3770 104 32' (ARItAKAb 1979 900 170 ARKKAAICA 843 Not delectable (ARKKAAKAb 1668 6,200 3,000 (ARKKAAKAh 2493 135 54 , (ARKKAAKAb 3318 42 15' .

(ARKKAAKAh 4143 S I 11' 2s (ARI~tRAARAys 2745 72 22'~

(AKAAKKRAh 2493 132 93 XBBB%XBX tasrdme repeats with h,~Opatlek mod~tcatlowla (AKRKKAAKAb 28T8 ?5 41 (GRKKGGK(lb 2325 200 98' (LRKI~LGKRb 2939 105 37 ('fRKICLGKns 2794 73'7 350' wo oo~4sam pcrnrsro (ARKKPAKA~ 2571 360 + 12T

ARKKAAKAARKKPAKAARKKAAKA

2319 730 + 340' AItKKAAKARKKAKARKKAAKA

2351 450 + 93' ARKKAAKAAAAAARKKAAKAAAAAARKKAAKA

3062 254 + 13T

Native or coifed aarglyciu aequsrecea YPARRARYQWVRCKP 1948 187 + 54 YPT(~RARYQWVRCNP 1936 817 + 170 YPARRARYQWVRAKP 1918 37,000 6,700 AAARRARAAAARAKA 1482 72,000 60,000 wo a~r4~~ rrrNeaw TABLE II
Parcent Reversal of anti-Fa~toa~ Xa Activity by Pepti~s in Vitro Lovenox (Rhone Poulenc Ru6ra)~ Or~nt~ (Or~oon), and tmfractionated hepmin (Sigma Cheenieal Co.) vvete added to pleana to obtain 0.5 Ufml anti-Factor Xa activity as detennirnd by the Stachrom Heparin sassy. The ATIITJheparin complex was allowed to form, the peptide was added at the indicated cone~riana, and the residual Factor Xa activity was measured by a clu~omoge~nic assay acco~ to the directions suppliod with the kit. The per cent reversal of 6op~it~lilGe activity w~aa determined. F,ach numbs represents the average of one-three duptica6e ar ttiplicaie determinations, in which _ ACS were ,~ 2%. Data were not aMained for areas ~ blank in the Table.
I5 Peptide ~ in plasma Peptide Type of H~in 0.6 mg/ml0.4m~m1 0.12 mgfml (AItKARAh Lovenax 61.5%

Orgaran 37.0%

Heparin 67.0%

(AKI~ARA)4 Lovenox 73%

Orb 75%

Heparin 99%

. (AKICARA~Lovenox 8Z.5% 86.5% 86%

Olgaran 776 81.5% 74.5%

Heparin 93% 97.3% 99%

(ARICICAAKA)aLovenox 73%

Orga<an 70.5%

H~ 87.5%

(ARKI~AAICA~Lovemox 81.3% 71.0~1~ 68.5%

Orgaran 83.0% 75.0 75.0%

Heparin 94.5% 93.0% 64.5%

(AICAAKICRA)3Lovenox 93.5%

Orgaran 72.0%

Hep~in 94.0%

WO 00J/S~il !'C1N80O1~113 (ARKKPAKA~ Lovenox 100% 62.5% 62.0'Yo Orgetan 66.5% b7.5% 45.0%
Heparin 99.a/o 50.0% 90.0%
(A,RRRAARA)3 Lovenox 67.5%

Otgaran 67.0%

Heparin 57.5%

ARKICAA
AARKKP
K

IC AAKA
A 83.0/s AARKK
Lovenox Orb 67:a/o Heparin 57.5%

YPARRYQWVRCKP (marine aerglycin) Lovenox 25.0%

Orgaran 16.5%

Heparin 77.0%

YPARRYQWVRAKP (marinesergiycin, cysteine replaced with alanine) Lovenox a/o Orgaran 2.a/e Heparin ZS.alo YPTQRARYQWVRCNP (human serglycin) Lovanox a/o Orgaran 5%

He~atin 9a/

4b Reversal of Anti-Thrombin Et~OCta of Unfractionated Heparin by Peptides in Vitro Plasma waa obtained from aorn4al donors. Tluombin coation was standardized to plod» a clotting time of 20-22 seoonde. Hep~n was addod at O.SUImI.17u ct~g time for heparin alone was appraxima~aly 3 miatttes. C>oe minute after addition of heparin to the plasma, the pepaida woes added in conca~ationa rnngiag finrn uglml. After one minute, thrombin was added aad the clotting time determined.
The Wing time was narmalizod at the peptide coacea>ttration; shown below.
. Peptide ~ peptidelml abed plasma noedod to nosnnaliae thrombin time (AICKARA~ 5 (AICKARA~ 10 (AKKARA~ > 30 (ARKKAAKA)s 21 (AICAAI~KRA~ >5O

(mouse serglycin)

Claims (75)

WE CLAIM:

1. A synthetic peptide with a high affinity for glycosaminoglycans and proteoglycans, wherein the sequence of amino acids is one of the group of (XBBBXXBX)n or (XBXXBBBX)n, wherein:
B is one of the group of arginine, lysine, or a combination of lysine and arginine;
X is alanine or glycine; and n is at least 2.

2. A synthetic peptide with a high affinity for glycosaminoglycans and proteoglycans, wherein the sequence of amino acids is one of the group of (XBBXBX)n or (XBXBBX)n, wherein:
B is one of the group of arginine, lysine, or a combination of lysine and arginine;
X is alanine or glycine; and n is at least 2.

3. A synthetic peptide with a high affinity for glycosaminoglycans and proteoglycans, wherein the sequence of amino acids is one of the group of (XBBBXXBX)n, (XBXXBBBX)n, (XBBXBX)n, or (XBXBBX)n, wherein:
B is one of the group of arginine, lysine, or a combination of lysine and arginine;
X is alanine or glycine;
n is at least 2; and a single cysteine residue is within three residues of either an N- or C-terminus.

4. The synthetic peptide of Claim 1, wherein the peptide comprises one of the group of D-isomer amino acids, L-isomer amino acids, or a combination of D- and L-isomer amino acids.

5. The synthetic peptide of Claim 2, wherein the peptide comprises one of the group of D-isomer amino acids, L-isomer amino acids, or a combination of D- and L-isomer amino acids.

6. The synthetic peptide of Claim 3, wherein the peptide comprises one of the group of D-isomer amino acids, L-isomer amino acids, or a combination of D- and L-isomer amino acids.

7. A synthetic peptide with a high affinity for glycosaminoglycans and proteoglycans, wherein the sequence of amino acids is one of the group of (XBBBXXBX)n or (XBXXBBBX)n, wherein:
B is one of the group of arginine, lysine, or a combination of lysine and arginine;
X is any amino acid; and n is at least 2.

8. A synthetic peptide with a high affinity for glycosaminoglycans and proteoglycans, wherein the sequence of amino acids is one of the group of (XBBXBX)n or (XBXBBX)n, wherein:
B is one of the group of arginine, lysine, or a combination of lysine and arginine;
X is any amino acid; and n is at least 2.

9. A synthetic peptide with a high affinity for glycosaminoglycans and proteoglycans, wherein the sequence of amino acids is one of the group of (XBBBXXBX)n, (XBXXBBBX)n, (XBBXBX)n, or (XBXBBX)n, wherein:

B is one of the group of arginine, lysine, or a combination of lysine and arginine;
X is any amino acid;
n is at least 2; and a single cysteine residue is within three residues of either an N- or C-terminus.

10. A method of modulating heparin or other glycosaminoglycans with anticoagulant activity in a mammal, wherein a therapeutically effective amount of the peptide of Claim 1 or Claim 4 is administered to said mammal.

11. A method of promoting cell attachment or adhesion to natural or synthetic surfaces in a mammal, wherein an effective amount of the peptide of Claim 1 or Claim 4 is covalently linked to a natural or synthetic polymer used to construct a synthetic vein graft surface and wherein said peptide interacts strongly with endothelial cell surface proteoglycans to promote cell attachment and graft endothelialization in vivo in said mammal or in vitro prior to surgical implantation of a vein graft in said mammal.

12. A method of modulating tumor cell metastasis a growth in a mammal, wherein a therapeutically effective amount of the peptide of Claim 1 or Claim 4 is administered to said mammal.

13. A method of modulating cartilage differentiation in mammal, wherein a therapeutically effective amount of the peptide of Claim 1 or Claim 4 is administered to said mammal.

14. A method of targeting drugs in a mammal to cell surfaces of endothelium or other cell types expressing proteoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 1 or Claim 4 is administered to said mammal.

15. A method of modulating enzymes that act on glycosaminoglycan substrates in a mammal, wherein a therapeutically effective amount of the peptide of Claim 1 or Claim 4 is administered to said mammal.

16. A method of affinity purification of bioactive sequences of a glycosaminoglycan, wherein an effective amount of the peptide of Claim 1 or Claim 4 interacts with at least one sequence or structural domain of said glycosaminoglycan.

17. A method of modifying endothelial cell pro-coagulant or anti-coagulant functions mediated through glycosaminoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 1 or Claim 4 is administered to said mammal.

18. A method of modulating wound healing in a mammal, wherein a therapeutically effective amount of the peptide of Claim 1 or Claim 4 is administered to said mammal.

19. A method of modulating heparin or other glycosaminoglycans with anticoagulant activity in a mammal, wherein a therapeutically effective amount of the peptide of Claim 2 or Claim 5 is administered to said mammal.

20. A method of promoting cell attachment or adhesion to natural or synthetic surfaces in a mammal, wherein an effective amount of the peptide of Claim 2 or Claim 5 is covalently linked to a natural or synthetic polymer used to construct a synthetic vein graft surface and wherein said peptide interacts strongly with endothelial cell surface proteoglycans to promote cell attachment and graft endothelialization in vivo in said mammal or in vitro prior to surgical implantation of a vein graft in said mammal.

21. A method of modulating tumor cell metastasis a growth in a mammal, wherein a therapeutically effective amount of the peptide of Claim 2 or Claim 5 is administered to said mammal.

22. A method of promoting cartilage differentiation in mammal, wherein a therapeutically effective amount of the peptide of Claim 2 or Claim 5 is administered to said mammal.

23. A method of targeting drugs in a mammal to cell surfaces of endothelium or other cell types expressing proteoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 2 or Claim 5 is administered to said mammal.

24. A method of modulating enzymes that act on glycosaminoglycan substrates in a mammal, wherein a therapeutically effective amount of the peptide of Claim 2 or Claim 5 is administered to said mammal.

25. A method of affinity purification of bioactive sequences of a glycosaminoglycan, wherein an effective amount of the peptide of Claim 2 or Claim 5 in interacts with at least one sequence or structural domain of said glycosaminoglycan.

26. A method of modifying endothelial cell pro-coagulant or anti-coagulant functions mediated through glycosaminoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 2 or Claim 5 is administered to said mammal.

27. A method of modulating wound healing in a mammal, wherein a therapeutically effective amount of the peptide of Claim 2 or Claim 5 is administered to said mammal.

28. A method of modulating heparin or other glycosaminoglycans with anticoagulant activity in a mammal, wherein a therapeutically effective amount of the peptide of Claim 3 or Claim 6 is administered to said mammal.

29. A method of promoting cell attachment or adhesion to natural or synthetic surfaces in a mammal, wherein an effective amount of the peptide of Claim 3 or Claim 6 is covalently linked to a natural or synthetic polymer used to construct a synthetic vein graft surface and wherein said peptide interacts strongly with endothelial cell surface proteoglycans to promote cell attachment and graft endothelialization in vivo in said mammal or in vitro prior to surgical implantation of a vein graft in said mammal.

30. A method of modulating tumor cell metastasis a growth in a mammal, wherein a therapeutically effective amount of the peptide of Claim 3 or Claim 6 is administered to said mammal.

31. A method of promoting cartilage differentiation in mammal, wherein a therapeutically effective amount of the peptide of Claim 3 or Claim 6 is administered to said mammal.

32. A method of targeting drugs in a mammal to cell surfaces of endothelium or other cell types expressing proteoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 3 or Claim 6 is administered to said mammal.

33. A method of modulating enzymes that act on glycosaminoglycan substrates in a mammal, wherein a therapeutically effective amount of the peptide of Claim 3 or Claim 6 is administered to said mammal.

34. A method of affinity purification of bioactive sequences of a glycosaminoglycan, wherein an effective amount of the peptide of Claim 3 or Claim 6 in interacts with at least one sequence or structural domain of said glycosaminoglycan.

35. A method of modifying endothelial cell pro-coagulant or anti-coagulant functions mediated through glycosaminoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 3 or Claim 6 is administered to said mammal.

36. A method of modulating wound healing in a mammal, wherein a therapeutically effective amount of the peptide of Claim 3 or Claim 6 is administered to said mammal.

37. A method of modulating heparin or other glycosaminoglycans with anticoagulant activity in a mammal, wherein a therapeutically effective amount of the peptide of Claim 7 is administered to said mammal.

38. A method of promoting cell attachment or adhesion to natural or synthetic surfaces in a mammal, wherein an effective amount of the peptide of Claim 7 is covalently linked to a natural or synthetic polymer used to construct a synthetic vein graft surface and wherein said peptide interacts strongly with endothelial cell surface proteoglycans to promote cell attachment and graft endothelialization in vivo in said mammal or in vitro prior to surgical implantation of a vein graft in said mammal.

39. A method of modulating tumor cell metastasis a growth in a mammal, wherein a therapeutically effective amount of the peptide of Claim 7 is administered to said mammal.

40. A method of promoting cartilage differentiation in mammal, wherein a therapeutically effective amount of the peptide of Claim 7 is administered to said mammal.

41. A method of targeting drugs in a mammal to cell surfaces of endothelium or other cell types expressing proteoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 7 is administered to said mammal.

42. A method of modulating enzymes that act on glycosaminoglycan substrates in a mammal, wherein a therapeutically effective amount of the peptide of Claim 7 is administered to said mammal.

43. A method of affinity purification of bioactive sequences of a glycosaminoglycan, wherein an effective amount of the peptide of Claim 7 in interacts with at least one sequence or structural domain of said glycosaminoglycan.

44. A method of modifying endothelial cell pro-coagulant or anti-coagulant functions mediated through glycosaminoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 7 is administered to said mammal.

45. A method of modulating wound healing in a mammal, wherein a therapeutically effective amount of the peptide of Claim 7 is administered to said mammal.

46. A method of modulating heparin or other glycosaminoglycans with anticoagulant activity in a mammal, wherein a therapeutically effective amount of the peptide of Claim 8 is administered to said mammal.

47. A method of promoting cell attachment or adhesion to natural or synthetic surfaces in a mammal, wherein an effective amount of the peptide of Claim 8 is covalently linked to a natural or synthetic polymer used to construct a synthetic vein graft surface and wherein said peptide interacts strongly with endothelial cell surface proteoglycans to promote cell attachment and graft endothelialization in vivo in said mammal or in vitro prior to surgical implantation of a vein graft in said mammal.

48. A method of modulating tumor cell metastasis a growth in a mammal, wherein a therapeutically effective amount of the peptide of Claim 8 is administered to said mammal.

49. A method of promoting cartilage differentiation in mammal, wherein a therapeutically effective amount of the peptide of Claim 8 is administered to said mammal.

50. A method of targeting drugs in a mammal to cell surfaces of endothelium or other cell types expressing proteoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 8 is administered to said mammal.

51. A method of modulating enzymes that act on glycosaminoglycan substrates in a mammal, wherein a therapeutically effective amount of the peptide of Claim 8 is administered to said mammal.

52. A method of affinity purification of bioactive sequences of a glycosaminoglycan, wherein an effective amount of the peptide of Claim 8 in interacts with at least one sequence or structural domain of said glycosaminoglycan.

53. A method of modifying endothelial cell pro-coagulant or anti-coagulant functions mediated through glycosaminoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 8 is administered to said mammal.

54. A method of modulating wound healing in a mammal, wherein a therapeutically effective amount of the peptide of Claim 8 is administered to said mammal.

55. A method of modulating heparin or other glycosaminoglycans with anticoagulant activity in a mammal, wherein a therapeutically effective amount of the peptide of Claim 9 is administered to said mammal.

56. A method of promoting cell attachment or adhesion to natural or synthetic surfaces in a mammal, wherein an effective amount of the peptide of Claim 9 is covalently linked to a natural or synthetic polymer used to construct a synthetic vein graft surface and wherein said peptide interacts strongly with endothelial cell surface proteoglycans to promote cell attachment and graft endothelialization in vivo in said mammal or in vitro prior to surgical implantation of a vein graft in said mammal.

57. A method of modulating tumor cell metastasis a growth in a mammal, wherein a therapeutically effective amount of the peptide of Claim 9 is administered to said mammal.

58. A method of promoting cartilage differentiation in mammal, wherein a therapeutically effective amount of the peptide of Claim 9 is administered to said mammal.

59. A method of targeting drugs in a mammal to cell surfaces of endothelium or other cell types expressing proteoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 9 is administered to said mammal.

60. A method of modulating enzymes that act on glycosaminoglycan substrates in a mammal, wherein a therapeutically effective amount of the peptide of Claim 9 is administered to said mammal.

61. A method of affinity purification of bioactive sequences of a glycosaminoglycan, wherein an effective amount of the peptide of Claim 9 in interacts with at least one sequence or structural domain of said glycosaminoglycan.

62. A method of modifying endothelial cell pro-coagulant or anti-coagulant functions mediated through glycosaminoglycans in a mammal, wherein a therapeutically effective amount of the peptide of Claim 9 is administered to said mammal.

63. A method of modulating wound healing in a mammal, wherein a therapeutically effective amount of the peptide of Claim 9 is administered to said mammal.

64. The synthetic murine serglycin peptide having the sequence YPARRARYQWVRCKP.

65. The synthetic human serglycin peptide having the sequence YPTQRARYQWVRCNP.

66. The synthetic peptide of Claim 64, wherein the peptide comprises one of the group of D-isomer amino acids, L-isomer amino acids, or a combination of D- and L-isomer amino acids.

67. The synthetic peptide of Claim 65, wherein the peptide comprises one of the group of D-isomer amino acids, L-isomer amino acids, or a combination of D- and L-isomer amino acids.

68. A synthetic nonconcatameric peptide with a high affinity for glycosaminoglycans and proteoglycans, wherein Cardin sites are separated by at least one of any amino acid and wherein the sequence of said synthetic peptide is at least two of the group of (XBBBXXBX)n, (XBXXBBBX)n, (XBBXBX)n, or (XBXBBX)n, wherein:
B is one of the group of arginine, lysine, or a combination of lysine and arginine;
X is alanine or glycine; and n is at least 1 for each of the two groups.

69. A synthetic nonconcatameric peptide with a high affinity for glycosaminoglycans and proteoglycans, wherein Cardin sites are separated by at least one of any amino acid and wherein the sequence of said synthetic peptide is at least two of the group of (XBBBXXBX)n, (XBXXBBBX)n, (XBBXBX)n, or (XBXBBX)n, wherein:
B is one of the group of arginine, lysine, or a combination of lysine and arginine;
X is alanine or glycine;
n is at least 1 for each of the two groups; and a single cysteine residue is within three residues of either an N- or C-terminus, either within a Cardin sequence or extended beyond the Cardin sequence.

70. A method of blocking tissue uptake and clearance of heparin or other glycosaminoglycans in a mammal to increase heparin half-life in circulation, wherein a therapeutically effective amount of the peptide of Claim 1 is administered to said mammal.

71. A method of blocking tissue uptake and clearance of heparin or other glycosaminoglycans in a mammal to increase heparin half-life in circulation, wherein a therapeutically effective amount of the peptide of Claim 2 is administered to said mammal.

72. A method of blocking tissue uptake and clearance of heparin or other glycosaminoglycans in a mammal to increase heparin half-life in circulation, wherein a therapeutically effective amount of the peptide of Claim 3 is administered to said mammal.

73. A method of blocking tissue uptake and clearance of heparin or other glycosaminoglycans in a mammal to increase heparin half life in circulation, wherein a therapeutically effective amount of the peptide of Claim 7 is administered to said mammal.

74. A method of blocking tissue uptake and clearance of heparin or other glycosaminoglycans in a mammal to increase heparin half-life in circulation, wherein a therapeutically effective amount of the peptide of Claim 8 is administered to said mammal.

75. A method of blocking tissue uptake and clearance of heparin or other glycosaminoglycans in a mammal to increase heparin half-life in circulation, wherein a therapeutically effective amount of the peptide of Claim 9 is administered to said mammal.