JP2009539757A - ADAMTS13-containing composition having thrombolytic activity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising ADAMTS13 having thrombolytic activity, and a method for treating or preventing disorders associated with the formation and / or presence of one or more thrombus, and one or It relates to a method of disrupting one or more thrombus in a patient in need of more than one thrombus disruption. Furthermore, the present invention provides for treating or preventing disorders associated with the formation and / or presence of one or more thrombus and in patients in need of one or more disruption of thrombus. It relates to the use of a pharmaceutically effective amount of ADAMTS13 for the preparation of a pharmaceutical composition for disrupting one or more thrombus.

Description

(Field of Invention)
The present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of ADAMTS13 having thrombolytic activity, a method of treating or preventing disorders associated with the formation and / or presence of one or more thrombus, and 1 It relates to a method for disrupting one or more thrombus in a patient in need of disruption of one or more than one thrombus. Furthermore, the present invention provides for treating or preventing disorders associated with the formation and / or presence of one or more thrombus and in patients in need of one or more disruption of thrombus. It relates to the use of a pharmaceutically effective amount of ADAMTS13 for the preparation of a pharmaceutical composition for disrupting one or more thrombus.

(Background of the Invention)
Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized by thrombotic microangiopathy, thrombocytopenia and microvascular thrombosis that can cause varying degrees of tissue ischemia and infarctions. Clinically, TTP patients are diagnosed by symptoms such as thrombocytopenia, dividing red blood cells (red blood cell fragments) and increased lactate dehydrogenase levels (Moake JL. Thrombotic microangipathies. N Engl J Med. 2002; 347: 589-). . 600; Moake JL von Willebrand factor, ADAMTS-13, and thrombotic thrombocytopenic purpura Semin Hematol 2004; 41:.... 4-14; Sadler JE, Moake JL, Miyata T, George JN Recent advances in thrombotic thrombocytopenic purpura Hematolog (Am Soc Hematol Educ Program) 2004:... 407-423; Sadler JE New concepts in von Willebrand disease Annu Rev Med 2005; 56:. 173-191). In 1982, Moake et al. Found abnormally large von Willebrand factor (UL-vWF) multimers in the plasma of patients with chronic relapsed TTP (Moake JL, Rudy CK, Troll JH, Weinstein MJ, . Colannino NM, Azocar J, Seder RH, Hong SL, Deykin D. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura N Engl J Med 1982; 307:. 1432-1435). The link between UL-vWF and TTP was supported by an independent finding by Furlan et al. And Tsai and Lian that most patients suffering from TTP are deficient in plasma metalloproteases that cleave vWF. (Furlan M, Robles R, Solenthaler M, Wassmer M, Sandoz P, Laemmle B. Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura Blood 1997; 89:.. 3097-3103; Tsai HM, Sussman, II, Ginsburg D, Rankhof H, Sixma JJ, Nagel RL Proteolytic cleavage of recombinant type 2A von Willebrand factor mutants R834W and R834Q:... Inhibition by doxycycline and by monoclonal antibody VP-1 Blood 1997; 89:. 1954-1962; Tsai HM, Lian EC Antibodies to von Willebrand factor-cleaving protease in act thrombolytic turbopura purpura. N Engl J Med. 1998; 339: 1585-1594). Belongs to ADAMTS family, ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats) and protease called is a glycosylated protein primarily 190kDa produced by the liver (Levy GG, Nichols WC, Lian EC, Foroud T, McClintic JN, McGee BM, Yang AY, Siemeniak DR, Stark KR, Gruppo R, Sarode R, Shrine SB, Chandrasek. , Ginsburg D, Tsai HM. .. Tations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura Nature 2001; 413: 488-494; Fujikawa K, Suzuki H, McMullen B, Chung D. Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family. Blood. 2001; 98: 1662-1666; Zheng X, Chung D, Takayama TK, Majerus EM, Sadler JE. , Fujikawa K. Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura J Biol Chem 2001; 276:.. 41059-41063; Soejima K, Mimura N, Hirashima M, Maeda H, Hamamoto T , Nakagaki T, Nozaki C. A novel human metalloprotease synthesized in the liver and secreted into the blood: positively, the world. factor-cleaving protease? J Biochem (Tokyo). 2001; 130: 475-480; Gerritsen HE, Robles R, Lammle B, Furlan M .; Partial amino acid sequence of purified von Willebrand factor-cleaving protease. Blood. 2001; 98: 1654-1661). Mutations in the ADAMTS13 gene have been shown to cause TTP (Levy GG, Nichols WC, Lian EC, Foround T, McClintic JN, McGee BM, Yang AY, Siemeniak DR, StarkR R, Grupro R, GruproS, GruproS). , Chandrasekaran V, Stabler SP, Sabio H, Bouhassira EE, Upshaw JD, Jr., Ginsburg D, Tsai HM Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenia purpura Nature 2001; 413:... 488-494) . Idiopathic TTP, often caused by autoantibodies that inhibit ADAMTS-13 activity, is a more common disorder that occurs in adults and older children and recurs at regular intervals in 11% to 36% of patients. get (Tsai HM, lian EC Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura N Engl J Med 1998; 339:... 1585-1594; Furlan M, Lammle B. Deficiency of von Willebrand factor-cleaving protease in family and acquired thrombotic . Hrombocytopenic purpura Baillieres Clin Haematol 1998; 11:. 509-514). Non-neutralizing autoantibodies can also inhibit ADAMTS activity by inducing clearance from the circulation (Scheiflinger F, Knobl P, Tattner B, Primeauer B, Mohr G, Dockal M, Dorner F, Rieger M. Nonneutralz. and IgG antigens to von Willebrand factor-cleaving protease (ADAMTS-13) in a patient with thrombotic purpura. Blood: 2003-102: 432; Plasma ADAMTS13 activity in healthy adults ranges from 50% to 178% (Moake JL. Thrombotic tropotopic purpura and the hemolytic urine syndrome. 14: 2). In most patients with familial TTP or acquired TTP, plasma ADAMTS13 activity is absent or less than 5% of normal. Mortality is greater than 90% without treatment, but plasma therapy has reduced mortality to approximately 20% (Moake JL. Thrombolytic turbopura and the hematologic uremic syndrome. 1433).

  VWF synthesized in megakaryocytes and endothelial cells is stored as ultra-large vWF (UL-vWF) in platelet α-granules and Weibel-Parade bodies, respectively. (5. Moake JL, Rudy CK, Troll JH, Weinstein MJ, Colannino NM, Azocar J, Seder RH, Hong SL, Deykin D. Unusually large plasma factor VIII:. Von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura N Engl J 1982; 307: 1432-1435; Wagner DD, Olmsted JB, Marder VJ.Immunolocalization of von Willebrand protein in Weibel-Pladebo. J: Cell Biol. 1982; 95: 355-360; Wagner DD, Bonfanti R. von Willebrand factor and the endotherum. DD. Von Willebrand factor released from Weibel-Parade bodies bounds more avidly to extracellular matrix third tangled constitutive 69.31. 4;.. Tsai HM, Nagel RL, Hatcher VB, Sussman, II Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases Biochem Biophys Res Commun. 1989; 158: 980-985; Tsai HM, Nagel RL, Hatcher VB, Sussman, II. Multimeric composition of endothelial cell-derived von Willebrand factor. Blood. 1989; 73: 2074-2076). Once secreted from endothelial cells, these UL-vWF multimers are cleaved by ADAMTS13 in the circulation into a series of smaller multimers at specific cleavage sites within the vWF molecule (Tsai HM, Nagel RL, Hatcher). .. VB, Sussman, II Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases Biochem Biophys Res Commun 1989; 158:. 980-985; Dent JA, Galbusera M, ruggeri .. ZM Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit J Clin invest 1991; 88:. 774-782; Furlan M, Robles R, Affolter D, Meyer D, Baillod P, Lammle B. Triplet structure of von Willebrand factor reflectors proteolytic degradation of high molecular weight multimers. Proc Natl Acad Sci US A. 1993; : 7503-7507). Proteases cleave at Tyr842-Met843 binding in the central A2 domain of the mature vWF subunit and require zinc or calcium for activity (Dent JA, berkowitz SD, Walle J, Kasper CK, Ruggeri ZM. Identification of a. cleavage site directing the immunological detection of molecular abbreviations in type IIA von Willebrand factor.Proc Natl Acad Sci. vWF is present in the form of “thread-like balls” and filaments as confirmed by an electron microscope (Slayter H, Loscalzo J, Buckenstedt P, Handin RI. Native conformation of human von Willis and aisland roskin prosthesis). -Light light scattering. J Biol Chem. 1985; 260: 8559-8563). In addition, atomic force microscopy has confirmed that vWF exists in a spherical three-dimensional structure under static conditions and in an unfolded filamentous state after exposure to shear stress (Siedrecchi CA, Lestini BJ, Kottke-Marchant KK, Eppell SJ, Wilson DL, Marchant RE. Shear-dependent changes in the three-dimensional structure of humans. This can also occur in vivo when one end of the vWF filament is anchored to a surface.

  UL-vWF multimers present in the Weibel-Parade body bind to platelets more tightly (via GPIbα) than plasma vWF when released by activated endothelial cells (27. Arya M, Anvari B, Romo GM). , Cruz MA, Dong JF, McIntire LV, Moake JL, Lopez JA Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex; studies using optical tweezers Blood 2002; 99:... 3971- 397). It has been shown in vitro that platelets align as beads to released UL-vWF on the endothelial surface (Dong JF, Moake JL, Nolasco L, Bernard A, Arceneaux W, Shrimpon CN, Schade AJ, McInire LV, Fujikawa K, Lopez JA.ADAMTS-13 rapidly cleaved newly secreted ultrawide von Willebrand factor multi-on-the-end. These secreted UL-vWF multimers are immobilized on the cell surface as long filamentous structures. These multimers are then cleaved by ADAMTS13 as ADAMTS13 is secreted from stimulated endothelial cells (Dong JF, Moake JL, Bernard A, Fujikawa K, Ball C, Nolasco L, Lopez JA, Cruz MA. ADAMTS-13 metalloprotease interacts with the endothelial cell-derived ultra-large von Willebrand factor. J Biol Chem. 2003; 278: 29633-29639).

  The thrombus in TTP patients is almost free of fibrin and consists mainly of vWF and platelets, suggesting vWF-mediated platelet aggregation as a cause of thrombosis (30. Asada Y, Sumiyashi A, Hayashi T, Suzuki Z, J (Immunohistochemistry of vasculal ratio in thrombotropic thrombotopic purpura, with special reference to factor VIII related to VIII 38). Patients with recurrent TTP have ultra large multimers in their plasma. The UL-vWF multimer accumulates over time. This is because the persistence of the inhibitor (anti-ADAMTS13Ab) decreases ADAMTS13 activity. UL-vWF multimers are overly active and unfold as a result of shear stress causing platelet aggregation, resulting in intravascular thrombosis (Non-Patent Document 1; Non-Patent Document 2).

It is believed that the presence of excessively reactive UL-vWF multimers in plasma due to ADAMTS13 deficiency may be associated with an increased risk of arterial thrombosis associated with coronary heart disease.
Tsai HM. Von Willebrand factor, ADAMTS13, and thrombotrophic purpura. J Mol Med. 2002; 80: 639-647 Tsai HM. Defectiency of ADAMTS-13 in thrombotic and thrombotopic purpura. J Thromb Haemost. 2003; 1: 2038-2040; discussion 2040-2035

  Therefore, there is a strong need to provide new compositions that can prevent and / or treat thrombi caused by certain disorders in patients.

(Summary of the Invention)
One object of the present invention is to provide a pharmaceutical composition having thrombolytic activity. The pharmaceutical composition comprises a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof, and optionally one or more pharmaceutically acceptable carriers and / or dilutions. Contains agents. The composition may also contain one or more additional active ingredients. Furthermore, the present invention provides a method of treating or preventing disorders associated with the formation and / or presence of one or more thrombus and in patients in need of one or more thrombus disruption. It relates to a method of disrupting one or more thrombus. Examples of disorders associated with the formation and / or presence of one or more thrombus are hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, arterial thrombosis, acute myocardial infarction (AMI) Stroke, sepsis, and disseminated intravascular coagulation (DIC). Accordingly, one or one in a patient in need of treatment or prevention of a disorder associated with the formation and / or presence of one or more than one thrombus, and one or more than one thrombus disruption. In the preparation of a pharmaceutical composition for disrupting more than one thrombus, a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof can be used. The pharmaceutically effective amount of the ADAMTS13 or biologically active derivative thereof can range, for example, from 0.1 mg to 20 mg per kg body weight.

(Detailed description of the invention)
One aspect of the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof having thrombolytic activity.

  The term “thrombolytic activity” as used herein means the destruction of one or more thrombus. Suitable methods for determining thrombolytic activity are well known in the art. For example, suitable methods are determination of whole blood clot lysis using tPA or streptokinase, or determination of thrombolysis in vivo.

  The term “disintegration” as used herein refers to partial or complete disintegration, dissolution, dissolution, destruction, and / or lysis of a thrombus. ).

  The term “thrombosis” as used herein includes blood clots (particularly platelet-containing blood clots), microthromboses, and / or emboli. The thrombus may or may not adhere to arterial or venous blood vessels and may partially or completely block blood flow in the arterial or venous blood vessels.

  The term “biologically active derivative” as used herein means any polypeptide having substantially the same biological function as ADAMTS13. The polypeptide sequence of a biologically active derivative can include one or more amino acid deletions, additions and / or substitutions, each of which is absent, present and / or substituted, respectively. It does not have any substantial negative effect on the biological activity of the polypeptide. The biological activity of the polypeptide includes, for example, decreased or delayed platelet adhesion to the endothelium, decreased or delayed platelet aggregation, decreased or delayed formation of platelet strings, decreased or delayed thrombus formation, It can be measured by reduced or delayed thrombus growth, reduced or delayed vascular occlusion, proteolytic cleavage of vWF, and / or thrombus collapse.

  The terms “ADAMTS13” and “biologically active derivative” also encompass polypeptides obtained by recombinant DNA technology, respectively. Recombinant ADAMTS13 (“rADAMTS13”) (eg, recombinant human ADAMTS13 (“r-hu-ADAMTS13”)) can be produced by any method known in the art. One particular example is disclosed in WO 02/42441, which is hereby incorporated by reference with respect to methods for producing recombinant ADAMTS13. This may include (i) production of recombinant DNA by genetic manipulation (eg, by reverse transcription of RNA and / or amplification of DNA), (ii) prokaryotic cells by transfection (ie, by electroporation or microinjection) or Introduction of recombinant DNA into eukaryotic cells, (iii) culture of the transformed cells (eg in a continuous or batch mode), (iv) ADAMTS13 (eg constitutive or upon induction) Expression and (v) isolation of the ADAMTS13 (eg, by collecting the transformed cells or from the transformed cells) is substantially (vi) (eg, by anion exchange chromatography or affinity chromatography). To obtain purified recombinant ADAMTS13, For conducting in order, it may include any method known in the art. The term “biologically active derivative” also refers to chimeric molecules (eg, to improve biological / pharmacological properties, such as the half-life of ADAMTS13 in the circulatory system of mammals, particularly humans). , ADAMTS13 in combination with Ig (or biologically active derivatives thereof). Ig may also have binding sites for optionally mutated Fc receptors.

  The rADAMTS13 can be produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically effective ADAMTS13 molecule. Examples of eukaryotic cells are mammalian cells (eg, CHO, COS, HEK293, BHK, SK-Hep, and HepG2). There are no particular limitations on the reagents or conditions used to produce or isolate ADAMTS13 according to the present invention, and any system known in the art or commercially available can be utilized. In one embodiment of the invention, rADAMTS13 is obtained by a method as described in the state of the art.

  A wide range of vectors can be used for the preparation of rADAMTS13 and can be selected from eukaryotic and prokaryotic expression vectors. Examples of vectors for prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc. Here, promoters used in prokaryotic expression include lac, trc, trp, recA, araBAD, etc. Is mentioned. Examples of vectors for eukaryotic expression include: (i) vectors such as pAO, pPIC, pYES, pMET that use a promoter such as AOX1, GAP, GAL1, AUG1, etc. for expression in yeast; ii) vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc. using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh etc. for expression in insect cells; and (iii) mammals For expression in animal cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, etc. using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV and β-actin, And viral systems (eg vaccinia virus, adeno Ban virus, herpes virus, vectors derived from such a retrovirus).

  Optionally, the pharmaceutical compositions of the present invention also contain one or more pharmaceutically acceptable carriers and / or diluents.

  The pharmaceutical composition of the present invention also inhibits one or more additional active ingredients (eg antithrombin factor, factor that stimulates ADAMTS13 production / secretion by the treated patient / individual, ADAMTS13 degradation, Therefore, factors that increase their half-life, factors that increase ADAMTS13 activity (eg, by binding to ADAMTS13 and thereby inducing activated conformational changes), or inhibiting ADAMTS13 clearance from the circulation, thereby Factors that increase its plasma concentration). Examples of antithrombin factors include antiplatelet substances, t-PA, aspirin and heparin.

  The invention further relates to a method of treating or preventing a disorder associated with the formation and / or presence of one or more thrombus, the method comprising the step of administering to the patient a composition according to the invention. . The disorder may be due to a genetic deficiency, inflammatory disease, stroke or septic condition. Examples of disorders associated with the formation and / or presence of one or more thrombus are hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, arterial thrombosis, acute myocardial infarction (AMI) Stroke, sepsis, disseminated intravascular coagulation syndrome (DIC), and venous thrombosis such as deep vein thrombosis or pulmonary embolism.

  Another aspect of the invention relates to a method of disrupting one or more thrombus in a patient in need of one or more than one thrombus disruption, the method according to the invention Administering a composition.

  The route of administration of the composition of the present invention does not exhibit a particular limitation and can be, for example, subcutaneous or intravenous. The term “patient” as used herein includes mammals (especially humans).

  Furthermore, the present invention provides a pharmaceutically effective amount of ADAMTS13 or a biological thereof in the preparation of a pharmaceutical composition for treating or preventing a disorder associated with the formation and / or presence of one or more than one thrombus. To the use of active derivatives.

  Another aspect of the present invention is a pharmacy for the preparation of a pharmaceutical composition for disrupting one or more thrombus in a patient in need of one or more than one thrombus disruption. Use of an effective amount of ADAMTS13 or a biologically active derivative thereof.

  The pharmaceutically effective amount of the ADAMTS13 or biologically active derivative thereof can range, for example, from 0.1 mg to 20 mg per kg body weight.

  The invention is further illustrated in the following examples. The following examples are not any limitation on the present invention.

Example 1: Endothelial activation resulting in thrombus formation in microvenules of Adamts13-/-mice
In Adamts13 − / − mice, more adhesion / translocation in venules (200-250 μm) activated with the calcium ionophore A23187 (Weibel-Parade body Parade) at low shear rate (approximately 100 S ⁻¹ ) Observed in comparison with WT. We investigated whether activation of microvenule endothelium by A23187 could result in platelet aggregation resulting in thrombus formation. A23187 does not peel the endothelium (Andre P, Denis CV, Walle J, Saffaripour S, Hynes RO, Ruggeri ZM, Wagner DD. Platelets adhere to von Willebrand factor presented by the endothelium in stimulated veins, and It is rearranged, Blood.2000; 96: 3322-3328). The investigated shear rate (200-250 s ⁻¹ ) and all venule diameters were similar in Adamts13 − / − and WT mice (Table 1). In the microvenules of Adamts13 − / − mice, platelet aggregation resulting in thrombus formation was observed 45 seconds to 1 minute after local perfusion of A23187 (FIG. 1). This thrombus was unstable and was swept into the bloodstream, leading to frequent emboli, resulting in downstream occlusion. However, this occlusion lasted for only 3-4 seconds and the venules then reopened. Thrombus formation was no longer observed at the site of stimulation 2 minutes after A23187 perfusion. In identically treated WT mice, a string of platelets and very small platelet aggregation could be observed attached to the endothelium for 1 second, but this did not result in thrombus formation. Adamts13 − / − mice or arterioles running parallel to venules in WT did not show any platelet aggregation or thrombus formation. No platelet strings were observed in arterioles. These observations indicate that ADAMTS13 inhibits platelet aggregation and therefore prevents thrombus formation in microvenules.

Example 2: Antibody to ADAMTS13 induces platelet string formation in WT venules
Previous studies have shown that most patients with acquired forms of TTP have autoimmune inhibitors against ADAMTS13 (Tsai HM, Lian EC. Von Willebrand factor cleavage in acute thrombotic idiopathic thrombocytopenic purpura. Antibody to Protease N Engl J Med.1998; 339: 1585-1594; Furlan M, Robles R, Galbusera M, Remuzzi G, Kyrle PA, Brenner B, Krause M, Scherrer I, Aumann M Lammle B. von Willebrand factor cleaving protease in thrombotic idiopathic thrombocytopenic purpura and hemolytic-uremic syndrome N Engl J Med. 1998; 339: 1578-1584). Polyclonal anti-human ADAMTS13 Ab (dissolved in PBS) was injected into WT 2 hours before surgery. After local perfusion of A23187 in both control mice (PBS infused) and anti-ADAMTS13 Ab infused, many platelets adhered / translocated onto the endothelium, and platelet adhesion between 45 seconds and 1 minute. A peak was reached, which gradually decreased with time. However, more platelet adhesion was observed 4 minutes after A23187 application with Ab-injected WT when compared to controls (FIG. 2A). This phenomenon observed was similar to Adamts13 − / −. A string of platelets varying from 20-40 μm adhered to one end of the endothelium and waves in the bloodstream were observed. Platelet strings were either not observed in mice injected with PBS or control Ig, or remained very short (less than 2 seconds). In WT mice (CASA / Pk) on a genetic background marked by elevated vWF levels treated with inhibitory antibodies, platelet strings are still longer (varies between 30-60 μm), which are predominantly It was shown that it was formed by vWF polymer. (In some mice, these platelet strings were anchored to the endothelium for up to 10 seconds (FIG. 2B) and then flushed.)
(Example 3: ADAMTS13 inhibitor causes thrombus formation in microvenules in WT mice)
In WT mice injected with anti-human ADAMTS13-Ab 2 hours prior to surgical preparation, 4 of 6 mice had microthrombi formed on the vessel wall 45 seconds to 1 minute after local perfusion of A23187 (Figure 3). The appearance of this microthrombus was similar to that observed in Adamts13 − / − mice (FIG. 1). Inhibitory antibodies were then injected into WT mice on a C57BL / 6 background. Microthrombi were formed in 3 out of 5 mice. In control WT mice, fine platelet aggregation could be observed adhering to the endothelium, but no thrombus formation occurred (n = 3).

Example 4: Histamine induces platelet strings in the venules of Adamts13-/-mice, while recombinant ADAMTS13 inhibits its formation
Histamine produced during inflammation is a secretion promoter for the Weibel-Parade body and stimulates the endothelium. Endogenous platelets were labeled by injecting Rhodamine 6G prior to surgery. 1 mM (200 μl) of histamine was administered i.v. in Adamts 13 − / − mice (n = 5) and WT mice (n = 5) 15 min prior to surgical preparation. p. Injected and visualized venules at a shear rate of about 100- ^S . In WT, platelet strings were not observed or present very shortly (less than 5 seconds, FIG. 4A), whereas in Adamts13 − / − mice, platelet strings that fluctuate between 20-100 μm are approximately 1 minute. It was observed anchored to the endothelium (FIG. 4B). In some mice, some strings appeared to coalesce and formed aggregates that were later released into the bloodstream (FIG. 4C). Injection of recombinant human ADAMTS13 (r-hu ADAMTS13) into Adamts13 − / − mice (n = 4, 3 venules per mouse) inhibited platelet strings in all 12 venules examined (FIG. 4D) Therefore, the activity of ADAMTS13 at low shear force was shown.

(Example 5: Platelet binding to the subendothelium is increased in Adamts13 − / − mice)
Iron (III) chloride damage leads to endothelial detachment and exfoliates the subendothelium (Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD. Both von Willebrand factor and fipurinogen Sustained platelet thrombus formation in arterioles of mice lacking J. J Clin Invest. 2000; 106: 385-392). After injury with arterial shear, platelet subendothelial interactions are initiated by GPIb-vWF interactions and then propagated by other receptors (Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes). RO, Wagner DO, persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen, J Clin Invest. 2000; 106: 385-392). In both WT and Adamts13 − / − mice, platelet-vessel wall interactions began rapidly after application of iron (III) chloride to arterioles. After injury, the number of animals with more than 100 platelets deposited in 2-3 minutes was higher in Adamts13 − / − mice (FIG. 5A). In Adamts13 − / −, 7 out of 12 mice showed more than 100 platelets deposited on the vessel wall compared to 3 out of 10 in WT mice. These results were statistically significant (P <0.05, FIG. 5A).

Example 6: Accelerated thrombus formation in damaged arterioles of Adamts13-/-mice
VWF present in plasma is either constitutively synthesized by endothelial cells or secreted in the form of an unusually sized multimer from platelet α-granules and endothelial Weibell-Palar bodies upon activation. It is. UL-vWF is the most sticky and active form of vWF and can lead to platelet aggregation, which results in thrombosis if not processed by ADAMTS13. In the iron chloride thrombus formation model in Adamts13 − / − mice, the thrombus grew faster. Because thrombus larger than 30 μm was observed at 6.64 ± 0.93 min compared to 10.78 ± 0.80 min in WT mice, and these results were statistically significant (P <0.005, FIG. 5B). This thrombus grew to an occluded size in 10.56 ± 0.72 minutes in Adamts13 − / − mice, while in WT, the blood vessels remained open (P <0.0005, FIG. 5C and 5D). For WT, the average vascular occlusion time was 16.69 ± 1.25 after injury (P <0.0005). All blood vessels were occluded at the site of injury. Arteriole shear rates and diameters studied were similar for Adamts13 − / − mice and WT mice (Table 2). Of note, in the arterioles of Adamts13 − / − mice, the mean time for thrombus (> 30 μm) formation and mean occlusion time were less for any individual WT mouse (FIGS. 5B and 5C). . Thrombus (> 30 μm) was formed in 4 (40%) of 10 Adamts13 − / − mice, whereas 2 out of 11 (18.2%) in WT mice showed embolization. It was observed that Thus, Adamts13 − / − thrombus appeared to be slightly more unstable than WT. The embolization observed in Adamts13 − / − mice did not lead to downstream occlusion.

(Example 7: ADAMTS13-deficiency increases thrombus growth in an αIIbβ3 integrin-dependent manner)
In vitro flow chamber study, blocking antibody to αIIbβ3 (JON / A) (Bergmeier W, Schulte V, Blockoff G, Bier U, Zirgibl H, Nieswandt B. Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody. Cytometry 2002; 48: 80-86) was performed with whole blood in the presence or absence. To quantify the size of the thrombus, the surface area covered by fluorescently labeled platelets was determined. Adamts13 − / − blood forms a thrombus significantly larger than WT when perfused on collagen for 2 minutes at a shear rate of 1500 s-1 (44.66 ± 3.63% vs 20.22 ± 3.88%). P <0.0005) again demonstrated the key role of ADAMTS13 in limiting thrombus growth. In the presence of blocking antibodies against αIIbβ3, only single platelets adhered to the collagen surface and thrombus formation was completely inhibited in both WT and Adamts13 − / − mouse blood (3.01 ± 0.97% Vs. 2.82 ± 0.39%, P> 0.05). Furthermore, β3 integrin-deficient mice of ADAMTS13 inhibitory antibodies (Hodivala-Dilke KM, McHugh KP, Tsakiris DA, Rayburn H, Crowley D, Ullman-Cullre M, Ross FP, Coller BS, Temu BS, Integr BBS, TeIr Bet. -Deficient mice were tested to see if injection into (which is a model of Grantsman platelet asthenia gravis showing placental defects and reduced survival) can induce thrombus formation after chloride infusion injury. In the injured arterioles of 3 − / − mice (3 mice evaluated), no thrombus could be detected regardless of the presence of anti-ADAMTS13 (not shown). Together, these results show that at the arterial shear rate described above, extra-large vWF increases thrombus growth in an αIIbβ3-dependent manner.

Example 8: Injection of recombinant human ADAMTS13 in Adamts13 − / − mice or WT mice inhibits thrombus growth by destabilizing platelet aggregation or thrombus
Cleavage of the vWF subunit into proteolytic fragments by recombinant human ADAMTS13 (r-hu ADAMTS13) has been shown in vitro (Plaimauer B, Zimmermann K, Volkel D, Antonio G, Kerschbamer R, Jenab P, Furian M, Ger Cloning, expression, and functional characterization of Lammie B, Schwartz HP, Scheiflinger F. von Willebrand factor-cleavable protease (ADAMTS13), Blood. 2002; 100: 3626-3632). r-hu ADAMTS13 has been shown to correct vWF cleavage defects in hereditary TTP plasma (Antoine G, Zimmerman K, Primeauer B, Growrowitzer M, Studt JD, Laemmie B, Scheiflinger F. ADAMTS13 gene defect in two siblings with sexual purpura, Br J Haematol 2003; 120: 821-824). ADAMTS13 negatively regulates thrombus growth because accelerated thrombus growth in Adamts13 − / − mice was observed, and therefore r-hu ADAMTS13 infusion in Adamts13 − / − mice can delay thrombus formation Hypothesized. The concentration of circulating human protein was approximately 8.8 U / ml 17 minutes after injection of r-hu ADAMTS13 into Adamts13 − / − mice and 1.1 U / ml 53 minutes after injection. These times correspond approximately to the start of iron chloride damage and the end of the experiment. In 5 of 13 Adamts13 − / − mice injected with r-hu ADAMTS13, the damaged arterioles did not occlude until 40 minutes when the experiment was terminated (FIG. 7A). The effect of injected r-hu ADAMTS13 was greater than that of endogenous ADAMTS13 in WT mice. This is because in this injury model, all WT vessels occluded in less than 24 minutes (FIG. 5C). Mean occlusion time was significantly prolonged compared to control mice injected with buffer (P <0.0005). In all mice whose arterioles did not occlude, thrombus formed but was unstable and collapsed (FIG. 7C). This phenomenon of thrombus formation and collapse was present during the entire period of observation. Infusion of pre-injury r-hu ADAMTS13 in WT mice (C57BI / 6J) caused a significant delay in occlusion time, and half of the arterioles did not occlude by 40 minutes, whereas WT injected with vehicle All arterioles of the mice were occluded by 15 minutes (FIG. 7B, P <0.008). Thus, ADAMTS13 appears to have significant antithrombogenic potential even in WT mice with normal levels of endogenous ADAMTS13 protein.

Example 9: Injection of recombinant mouse ADAMTS13 inhibits thrombus growth in WT mice
r-mouse ADAMTS13 (2.6 / kg mouse) was injected into WT mice 5 minutes prior to iron chloride injury (iv). The model described above was used with slight modifications as described below. FIG. 8 shows that 4 out of 9 WT mice injected with r-ADAMTS13 did not occlude damaged arterioles in 40 minutes observation time (mean occlusion time = 27.04 ± 3.84 minutes). On the other hand, it shows that all WT mice injected with buffer were occluded (mean occlusion time = 15.15 ± 1.18 min, P = 0.006). Western analysis of plasma samples taken at the end of the experiment shows that shorter occlusion times (similar to WT) in several mice injected with r-ADAMTS13 were due to increased clearance of this recombinant protein. It was. Thus, murine ADAMTS13 appears to have the same antithrombotic ability as human ADAMTS13 in WT mice with normal levels of endogenous ADAMTS protein.

  These results indicate that ADAMTS13 has antithrombogenic and thrombolytic activity.

(Experimental procedure)
(animal)
The mice used in the experiment were Adamts13 +/− mice on the C57BL / 6J / 129Sv background (Mototo DG, Chauhan AK, Zhu G, Homerester J, Lamb CB, Desch KC, Zhang W, Tsai HM, WagneD, WagneD, WagneD. Shigatoxin induces thrombotic thrombocytopenic purpura in genetically susceptible Adamts13-deficient mice (J Clin Invest 2005; 115: 2752-2761). Mice with a pure C57BL / 6J background were purchased from Jackson Laboratory, Bar Harbor, ME, and β3 integrin − / − mice on the Balb / C background were a gift from Richard Hynes (MIT). The mice used for in vivo staining microscopy were both male and female young mice (about 4 weeks old) weighing 14-18 grams. Infused platelets were isolated from 4-6 month old mice of the same genotype. Animals were housed and housed at the CBR Institute for Biomedical Research, and all experimental procedures were approved by the Animal Care and Use Committee.

(material)
Calcium ionophore A23187 and iron chloride are available from Sigma Chemicals, St. From Louis, MO.

(Blood sampling and platelet preparation)
Blood was collected from the posterior orbital venous plexus by puncture and collected in 1.5 ml polypropylene tubes containing 300 μl heparin (30 U / ml). Platelet rich plasma (PRP) was obtained by centrifugation at 1200 rpm for 5 minutes. Plasma and buffy coat containing significant RBCs were gently transferred to a new polypropylene tube and centrifuged again at 1200 rpm for 5 minutes. PRP was transferred to a new tube containing 2 μl of PGI ₂ (2 μg / ml) and incubated at 37 ° C. for 5 minutes. After centrifugation at 2800 rpm, the pellet was washed with 1 ml modified Tyrode's-HEPES buffer (137 mM NaCl, 0.3 mM Na ₂ HPO ₄ , 2 mM KCl, 12 mM NaHCO ₃ , 5 mM m HEPES, 5 mM with ₂ μl PGI _2. Glucose, 0.35% BSA) and incubated at 37 ° C. for 5 minutes. The suspended pellet was centrifuged at 2800 rpm for 5 minutes. To remove PGI ₂ , the washing step was repeated twice and platelets were fluorescently labeled with calcein AM (Molecular Probes, Eugene, OR) 0.25 mg / mL for 10 minutes at room temperature.

(Production and purification of polyclonal anti-ADAMTS13 IgG)
Polyclonal rabbit anti-human ADAMTS13 IgG was produced by Baxter Bioscience, Vienna Austria. This antibody was obtained by immunization of New Zealand white rabbit with r-hu ADAMTS13 tagged with 6 His residues at the purified C-terminus. Two rabbits were immunized by injection of 20 μg r-hu ADAMTS13 (6-His) in 200 μl complete Freund's adjuvant. These animals were boosted at weeks 2, 4 and 6 by injecting 20 μg r-hu ADAMTS13 (6-His) in 200 μl complete Freund's adjuvant. After 8 weeks, the rabbits were sacrificed and exsanguinated. IgG antibodies were purified by Protein G affinity chromatography (HiTrap Protein G HP column; Amersham Bioscience, Piscataway, NJ, USA) and formulated in PBS>
(Thrombosis in microvenules)
In vivo staining microscopy is described in Frenette PS, Johnson RC, Hynes RO, Wagner DD. Role of platelets on simulated endothelium in vivo: interactions mediated by endothelial P-selection. Proc Natl Acad Sci USA, 1995; 92: 7450-7454. Briefly, mice are anesthetized with 2.5% tribromoethanol (0.15 ml / 10 g), and an incision is made through the abdominal wall, the mesentery is stripped, and a 25-30 μm diameter mesentery The venules were examined. The exposed mesentery was kept moist by periodic perfusion with PBS (no Ca ²⁺ or Mg ²⁺ ) warmed to 37 ° C. The mesentery was transluminescent with a 12V, 100W, DC stabilized power supply. The shear rate is Frentte PS, Moyna C, Hartwell DW, Low JB, Hynes RO, Wagner DD. Platelet-endothelial interaction in inflamed mesenteric venules. Blood. 1998; 91: 1318-1324. Calculated using an optical Doppler velocimeter as described in. The venules were visualized using a Zeiss (Germany) Axiovert 135 inverted microscope (objective 10 × and 32 ×) connected to a SVHS video recorder (AG-6730; Panasonic, Tokyo, Japan). One venule was selected per mouse and taken for 3 minutes for baseline before A23187 perfusion (30 μl of 10 Mmol / L) and monitored for 10 minutes.

(Platelet adhesion in large venules)
In vivo staining microscopy was performed as described above, except that 200-300 μm diameter mesenteric venules were examined. Fluorescent platelets (1.25 × 10 ⁹ platelets / kg) were infused through the tail vein. One venule per animal was imaged for baseline for 3 minutes prior to A23187 perfusion (30 μl of 10 μmol / L solution), and after imaging returned to baseline, platelet adhesion and rolling Continued until. Purified rabbit polyclonal anti-human ADAMTS13 antibody (5 mg / kg mouse) was dissolved in PBS. Control rabbit IgG (Sigma, St. Louis, MO) was in PBS. 200 μl of 1 mM histamine (Sigma) i. p. Injected and stimulated endothelium. 100 μl (0.2 mg / ml) Rhodamine 6G i. v. Infused and labeled endogenous platelets and leukocytes prior to surgery and imaging.

(Thrombosis in arterioles)
Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest. 2000; 106: 385-392 was used with slight modifications (Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD. Von Willebrand factor and fibrinogen. Sustained platelet thrombus formation in arterioles of mice lacking J. J Clin Invest. 2000; 106: 385-392). Briefly, mice were anesthetized with 2.5% tribromomethanol (0.15 ml / 10 g) and fluorescent platelets (1.25 × 10 ⁹ platelets / kg) were injected through the posterior orbital venous plexus of the eye. An incision was made through the abdominal wall to expose the mesentery and the approximately 100 μm arteriole was examined. The exposed mesentery was kept moist by periodic perfusion with PBS (no Ca ²⁺ or Mg ²⁺ ) warmed to 37 ° C. The mesentery was transluminated as described above and the shear rate was calculated. The arteriole is a 100-W HBQ fluorescent lamp source (Optical Hgip, with a narrow band fluorescein isothiocyanate filter set (Chroma Technology, Brattleboro, VT) and a silicon-enhanced tube camera C2400 (Hamamatsu, Tokyo, Japan). , NY) and visualized using the same microscope described above. Whatman paper saturated with iron chloride (10%) was applied topically, which induced vascular damage and endothelium exposure. The paper was removed after 5 minutes and the blood vessels were monitored for 40 minutes after injury or until occlusion. One arteriole was selected per mouse.

(Quantitative analysis of arteriole thrombosis)
Analysis of the recorded tape was performed with the genotype hidden. The parameters applied to explain the characteristics of thrombus formation are: (1) a single, determined as the number of fluorescent platelets deposited on the 250 μm vessel wall (observed on a video monitor) during 1 minute. One platelet-vessel wall interaction. (2) Time required for formation of a thrombus larger than 30 μm. (3) Thrombus stability by determining the number of thrombi with a diameter greater than 30 μm that form emboli away from the field of view prior to vascular occlusion. (4) Blood vessel occlusion time, ie, the time required to stop blood flow for 10 seconds, and (5) Site of blood vessel occlusion, ie, the site of injury or downstream.

(Recombinant human ADAMTS13 injection)
Recombinant human ADAMTS13 has been described by Primerau B, Zimmermann K, Volkel D, Antonio G, Kerschbaumer R, Jenab P, Furlan M, Gerritsen H, Lammle B, Schwarz HP, and Scheffling. Cloning, expression, and functional characterization of von Willebrand factor-cleavable protease (ADAMTS13). Blood. 2002; 100: 3626-3632. Recombinant human ADAMTS13 protein was dissolved in 150 mmol NaCl / 20 mmol histidine / 2% sucrose / 0.05% Crillet 4HP (Tween 80), pH 7.4 (Baxter Bioscience, Vienna, Austria). Recombinant human ADAMTS13 is prepared by i. v. Injection (3460 U / kg mouse). The levels of human ADAMTS13 antigens are as follows: Rieger (Rieger M, Kremer Hovinga JA, Konetschny C, Herzog A, Koller L, Weber A, Remuzzi G, Dockal M, Primerer B and ScheF. The relationship between ADAMTS13 activity and ADAMTS13 antigen levels in patients of) determined by using a slight modification of the ELISA method described by Thrombosis and Hemostasis 2006; 95 (2): 212-20) and r -Hu ADAMTS13 activity is determined by Gerritsen (Gerritsen HE, Truesk PL, Schwarz HP, Laemmi B, and Furlan M. von Willebrand factor (vWF) -cleavable protease, assay based on reduced collagen binding affinity of degraded vWF: a tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). 1999; 82: 1386-1389). 1 U corresponds to the level of ADAMTS13 activity in pooled normal human plasma.

(Recombinant mouse ADAMTS13 injection)
Recombinant mouse ADAMTS13 is from Bruno K, Volkel D, Primeauer B, Antoine G, Table S, Motto DG, Lemmerhirt HL, Dorner F, Zimmermann K, and Scheiflinger F. Cloning, expression and functional characterization of full-length mouse ADAMTS13. J Thromb Haemost 2005; 3 (5): 1064-1073. Recombinant mouse ADAMTS13 protein was dissolved in 150 mmol NaCl / 20 mmol histidine / 2% sucrose / 0.05% Crillet 4HP (Tween 80), pH 7.4 (Baxter Bioscience, Vienna, Austria).

(Flow chamber research)
Flow chamber studies were performed using Bergmeier (Bergmeier W, Burger PC, Piffath CL, Hoffmeister KM, Hartwig JH, Nieswandt B, and Wagner DD. In vitro-aged or damaged blood platelet function recovery. Blood 2003; 102: 4229-4235). Briefly, platelets were isolated from heparinized whole blood, washed in modified Tyrode-HEPES buffer and labeled with 2.5 μg / mL calcein. Platelet-poor whole blood was reconstituted with labeled platelets prior to perfusion in a parallel-plate flow chamber system coated with 100 μg / mL collagen Horm (NYCOMED, Munich, Germany) for 1 hour at room temperature. Where indicated, samples were pretreated with 30 μg / ml JON / A (emfret Analyticals, Wuerzburg, Germany) for 10 minutes prior to perfusion. Platelet adhesion was visualized with an Axiovert 135 inverted microscope (Zeiss). The percent of the surface area covered by fluorescent platelets was analyzed by NIH Image 1.61 software with the genotype hidden solids.

(Statistical analysis)
Results are reported as mean ± SEM. Statistical significance of differences between means was assessed by Student's t test.

(Discussion of experimental results)
Experimental results have defined an important role for ADAMTS13 in preventing thrombus formation in activated microvenules and preventing excessive thrombus formation in damaged arterioles of mice. Using in vivo staining microscopy, activating venules (25-30 μm) in vivo has been shown in ADAMTS13 − / − mice to result in platelet aggregation leading to microthrombus formation. Microthrombi do not form in the venules of the same treated WT mice. When released from the activated endothelium, these microthrombi can migrate downstream and cause occlusion in any of the small capillaries that they cannot pass, thus leading to organ ischemia. Patients with TTP often have microthrombi that form in the microvasculature of organs such as the brain, heart, pancreas, spleen, kidney and mesentery. Various factors, including drugs, antibodies and immune complexes, such as viruses, bacterial shiga toxins, ticlopidine and clopitodogel may possibly trigger vascular activation that induces Weibel-Parade body release. In arterioles also treated with A23187 (having higher shear stress) no thrombus was observed. This is likely because the Weibel-Parade body was not released in these blood vessels, or vWF was washed too quickly from the endothelial surface, promoting platelet adhesion.

  Autoantibodies that neutralize human ADAMTS13 are a major cause of the acquired type of TTP. Injection of anti-ADAMTS13 antibody in WT mice resulted in sustained adhesion of platelets to secreted vWF and platelet string formation on stimulated endothelium (FIG. 2A) similar to that observed in Adamts13 − / − mice. . Platelets of platelets that vary in length from about 20-70 μm can be observed anchored to the endothelium (FIG. 2B). Platelet string and aggregation were frequently observed in Adamts13 − / − mice when challenged with secreted promoters of Weibel-Parade bodies such as histamine, inflammatory cytokine TNF-α and activated platelets. Infusion of r-hu ADAMTS13 protein in Adamts13 − / − mice challenged with histamine inhibits platelet string formation. These strings are cut at the upstream end.

  Activation of microvenules (25-30 μm) with A23187 results in platelet aggregation leading to thrombus formation in Adamts13 WT mice injected with anti-ADAMTS13-Ab (FIG. 3). However, these thrombus occluded rapidly, similar to those in Adamts13 − / − mice. Microthrombus formation can also be induced in WT mice on a C57BL / 6 background injected with inhibitory antibodies. Thus, mice injected with anti-ADAMTS13 Ab are a good model for acquired TTP in many aspects. Furthermore, the role of ADAMTS13 in preventing platelet aggregation in the circulation is shown.

  VWF, GPIbα and αIIbβ3 through its receptor contribute to platelet function in initiating platelet aggregation and in the progression of thrombus formation. The experimental observation that platelet-endothelial interactions are sustained and that endothelial activation produces microthrombi in Adamts13 − / − mice has led to the hypothesis that ADAMTS13 deficiency can accelerate thrombus formation in damaged arterioles. Indeed, the absence of ADAMTS13 promoted all aspects of thrombus growth. Unexpectedly, even more platelets were deposited on the naked vessel wall 2-3 minutes after injury in Adamts13 − / − mice when compared to WT (FIG. 5A). Since early platelet deposition in arterioles is vWF-dependent, it can be demonstrated that plasma ADAMTS13 reduces vWF uptake into the basement membrane when it is exposed to blood, or that it is already in the extracellular matrix. It means that any of the existing vWF is digested. Rapid thrombus growth and occlusion in Adamts13 − / − mice indicates that ADAMTS13 can cleave vWF matilmer incorporated into the thrombus.

  It was suggested that cleavage of VWF domain A2 by ADAMTS13 is facilitated by binding of VWF to GPIbα. Therefore, VWF-GPIb interaction within the thrombus can negatively regulate thrombus formation. Thrombus formation under venous and arterial flow conditions is also dependent on the major integrin αIIbβ3. The current example in arterial shear rate shows that ADAMTS13 regulates a thrombus that grows only when platelets in the thrombus express active β3 integrin. Under these in vitro and in vivo experimental conditions, ADAMTS13-deficiency did not promote major thrombus growth when major platelet integrins were absent or inhibited (FIG. 6).

  In order to inhibit the rapid thrombus growth observed in Adamts13 − / − mice, r-huADAMTS13 was injected into Adamts13 − / − mice and WT mice prior to injury. The anti-thrombogenic effect of r-huADAMTS13 varied between animals, although highly statistically significant (FIGS. 7A and 7B). One mouse did not respond to r-huADAMTS13 treatment. In these mice, r-huADAMTS13 was inactivated by thrombin proteolysis and plasmin may have been generated at the site of vascular injury. IL-6 and large amounts of vWF released after inflammation or injury can also reduce ADAMTS13 activity. In blood vessels that did not occlude, the phenomenon of thrombolysis and modification is observed (FIG. 7C). These findings indicate that ADAMTS13 has both anti-thrombogenic and thrombolytic activity. Possible mechanisms include thrombus in the thrombus, as ADAMTS13 cleaves UL-vWF multimers into smaller fragments that are less sticky or to cleave attached platelets into strings. Direct cleavage of the cross-linking vWF molecule. In vivo, ADAMTS13 is active in both low venous shear stress conditions and high arterial stress conditions. It cleaves platelet strings and regulates platelet interactions with “activated” vessel walls within venules, prevents thrombus in activated microvenules, and modulates thrombus response in damaged arterioles To do.

Congenital TTP patients are currently being treated by serum exchange, while acquired TTP patients are receiving plasma sputum where autoantibodies are removed from the plasma. ADA
In addition to TTP, the anti-thrombogenic and thrombolytic effects of MTS13 protein can be achieved, for example, by recombinant ADAMTS13 in genetic defects, inflammatory diseases, septic conditions, or venous thrombosis such as deep vein thrombosis or pulmonary embolism. FIG. 5 shows that it can be used to treat patients suffering from thrombotic disorders caused by them. The thrombolytic activity of ADAMTS13 indicates that this protein can be used in clogged arteries or in combination with other treatments to break down the thrombus after a minor stroke.

  In summary, the above experimental data indicate that the metalloprotease ADAMTS13 negatively regulates thrombosis, indicating that this molecule has antithrombogenic activity. Therefore, the recombinant human ADAMTS13 protein with thrombolytic activity can be used as a therapeutic agent to treat thrombogenic disorders.

FIG. Thrombus formation in microvenules. After making an incision through the abdominal wall and exposing the mesentery of a living mouse, mesenteric venules with a diameter of about 25-30 μm were visualized. One minute after local administration of A23187, thrombus formation was observed in Adamts13 − / − mice (n = 5). Arrows indicate microthrombi. Microthrombus formation was not observed in similarly treated WT mice (n = 5). Thus, stimulation of the Weibel-Parade body can result in spontaneous thrombus formation in Adamts13 − / − mice without vascular injury. FIG. ADAMTS13 inhibitors increase platelet adhesion and filament formation on the vessel wall. Fluorescently labeled platelets representing approximately 2.5% of total platelets were observed in mesenteric venules (200 μm to 250 μm) in live mice before (baseline) and after perfusion of A23187 did. A: Platelets began to adhere to the endothelium 30 to 45 seconds after perfusion. In WT (anti-human ADAMTS13 Ab injected) mice (n = 4), more platelets adhered to the vessel wall after 4 minutes compared to WT (PBS injected) controls (n = 4). The arrow indicates a filamentous material of platelets of 20 μm or more that has one end attached to the endothelium and undulates blood flow. The time when it is inserted in the lower right corner indicates the time after perfusion of A23187. The bar shown in the center of the panel is (about) 50 μm. B: In 5 of 7 WT mice injected with an inhibitor against ADAMTS13, platelet filaments from μm to about 2535 μm long remained on the vessel wall by 10 seconds. The time when it is inserted in the lower right corner indicates the time after perfusion of A23187. The bar shown in the center of the panel is (about) 525m. FIG. Thrombus formation in microvenules of WT mice injected with ADAMTS13 inhibitor. Mesenteric venules having a diameter of about 25 to 30 μm were observed. One minute after local perfusion of A23187, thrombus formation was observed in 4 of 6 Adamts13 WT mice injected with ADAMTS13 inhibitor. Microthrombus formation was similar to that confirmed in Adamts13 − / − mice (FIG. 1). Arrows indicate microthrombi. No microthrombus was formed in WT mice (n = 5) injected with PBS. FIG. Recombinant ADAMTS13 inhibits platelet filaments in Adamts13 − / − mice. Rhodamine 6G was used to label endogenous platelets and leukocytes. 1 mM histamine (200 μl) was administered i.v. 15 min before surgery. p. Administered and visualized three mesenteric venules of approximately 200-300 μm in diameter per mouse. A: Platelet filaments were not confirmed in Adamts13 WT mice (n = 5). B: Platelet filaments (indicated by arrows) were confirmed in Adamts13 − / − mice (n = 5). C: As indicated by the arrows, platelet filaments can form platelet aggregates in Adamts13 − / − mice. D: Injection of recombinant human ADAMTS13 protein inhibits platelet filaments in Adamts13 − / − mice (n = 4). FIG. Quantitative analysis of platelet adhesion and thrombus formation in small arteries of WT and Adamts13 − / − mice. A: The number of fluorescent platelets deposited per minute was counted at 1 minute intervals for the total number of platelets 2-3 minutes after injury). For statistics and average, more than 100 platelets were considered 100. The absence of ADAMTS13 in plasma clearly affects the initial platelet interaction with the subendothelium. Compared to WT, Adamts13 − / − had more platelets deposited on the vessel wall (P <0.05). B: Thrombus (> 30 μm) appears in ADAMTS13 − / − mice (mean = 6.64 ± 0.93) earlier after injury compared to WT (mean = 10.78 ± 0.80) Is statistically significant (P <0.005). This indicates that cleavage of UL-vWF multimers by ADAMTS13 delays thrombus formation. C: The occlusion time when the blood flow completely stopped for 10 seconds was determined. Both WT and Adamts13 − / − mice were occluded at the site of injury; however, the occlusion time was greater in Adamts13 − / − mice compared to WT mice (mean = 16.69 ± 1.25 min). Short (average = 10.56 ± 0.72). This difference was statistically very significant (P <0.0005). D: After ferric chloride injury, fluorescently labeled platelets representing approximately 2.5% of total platelets were observed in mesenteric small arteries of live mice. Blood flow was from left to right. The time when it is inserted in the lower right corner indicates the time after injury. In WT, single adherent platelets are confirmed in the small arteries 4 minutes after injury, but in the small arteries of Adamts13 − / − mice, thrombus (approximately 30 μm) can be confirmed at the same time point. In Adamts13 − / − mice, the vessels were occluded in 10 minutes by thrombus at the site of injury, but the small arteries of WT mice remained open at that time. The photographs are representative of 10 mice of each genotype. FIG. Integrin αIIbβ3 inhibition blocks ADAMTS13 − / − platelet thrombus formation in collagen under small artery shear rate conditions. Adamts13 + / + and Adamts13 − / − whole blood was perfused over the collagen surface for 2 minutes at a shear rate of 1500 s-1. A: A representative image is shown. Upper panel—untreated whole blood, lower panel—pretreated with blocking antibody against αIIbβ3 (JON / A). B: Quantification of the surface area covered by platelets after 2 minutes of perfusion. Four frames from different areas of the flow chamber were analyzed for each blood sample. Data show mean percentage of surface area covered by fluorescent platelets ± SEM (n = 3-4). FIG. Infusion of recombinant human ADAMTS13 inhibits thrombus growth. Recombinant human ADAMTS13 was injected (iv) into Adamts13 − / − mice 15 minutes prior to ferric chloride injury. The occlusion time (blood flow completely stopped for 10 seconds) was determined. A: Five of the 13 Adamts13 − / − mice injected with recombinant human ADAMTS13 did not occlude with small arteries during the observation time up to 40 minutes (mean occlusion time = 23.80 ± 3.71 minutes) ), But all 10 Adamts13 − / − mice injected with recombinant buffer alone were occluded (mean occlusion time = 11.17 ± 0.87); (P = 0.005). B: WT (C57BI / 6J) mice injected with either r-hu ADAMTS13 (mean occlusion time = 27.99 ± 4.72 min) or buffer only (mean occlusion time = 13.12 ± 0.55 min) Occlusion time in damaged small arteries. C: Representative fluorescent images of damaged arterioles of Adamts13 − / − mice treated with r-huADAMTS13 are shown. The arrow indicates a thrombus that collapses. FIG. Injection of recombinant mouse ADAMTS13 inhibits thrombus growth in WT mice. Recombinant mouse ADAMTS13 (2.6 mg / kg mouse) was injected (iv) into WT mice 5 minutes prior to ferric chloride injury. Of the 9 WT mice injected with recombinant mouse ADAMTS13, 4 did not occlude damaged arterioles with an observation time of 40 minutes (mean occlusion time = 27.04 ± 3.84 minutes) but buffered All WT mice injected with fluid were occluded (mean occlusion time = 15.15 ± 1.18 min, P = 0.006). FIG. Table 1: Hemodynamic parameters before and after application of A23187 in arterioles (Figure 1); Table 2: Hemodynamic parameters before and after application of ferric chloride in small arteries (Figure 5).

Claims (13)

A pharmaceutical composition having thrombolytic activity comprising a pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof. The pharmaceutical composition according to claim 1, wherein the ADAMTS13 is recombinant human ADAMTS13 or a biologically active derivative thereof. The pharmaceutical composition according to claim 1, wherein the biologically active derivative is a chimeric molecule comprising ADAMTS13 or a biologically active derivative thereof and Ig or a biologically active derivative thereof. The pharmaceutical composition according to claim 1, further comprising one or more additional active ingredients. Said further active ingredient is selected from the group consisting of an antithrombin factor; a factor that stimulates ADAMTS13 production / secretion; a factor that inhibits ADAMTS13 degradation; a factor that increases ADAMTS13 activity; and a factor that inhibits ADAMTS13 clearance from the circulation. The pharmaceutical composition according to claim 4. 6. The pharmaceutical composition according to claim 5, wherein the antithrombin factor is selected from the group consisting of an antiplatelet substance, t-PA, aspirin and heparin. A method of treating or preventing a disorder associated with the formation and / or presence of one or more thrombus comprising administering to a patient the composition of claim 1. The disorders include hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, arterial thrombosis, acute myocardial infarction (AMI), stroke, sepsis, disseminated intravascular coagulation syndrome (DIC), and deep vein thrombosis 8. The method of claim 7, wherein the method is selected from the group consisting of venous thrombosis such as symptom or pulmonary embolism. A method for disrupting one or more thrombus in a patient in need of one or more than one thrombus disruption comprising the step of administering to the patient the composition of claim 1. The method of inclusion. A pharmaceutically effective amount of ADAMTS13 or a biologically active thereof for the preparation of a pharmaceutical composition for treating or preventing a disorder associated with the formation and / or presence of one or more thrombus Use of derivatives. The disorders include hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, arterial thrombosis, acute myocardial infarction (AMI), stroke, sepsis, disseminated intravascular coagulation syndrome (DIC), and deep vein thrombosis 11. Use according to claim 10, selected from the group consisting of venous thrombosis such as symptom or pulmonary embolism. A pharmaceutically effective amount of ADAMTS13 or an organism thereof for the preparation of a pharmaceutical composition for disrupting one or more thrombus in a patient in need of one or more than one thrombus disruption Use of biologically active derivatives. Use according to claim 10 or 12, wherein the pharmaceutically effective amount of ADAMTS13 or a biologically active derivative thereof is in the range of 0.1 mg to 20 mg per kg body weight.

Images