CA2137342A1

CA2137342A1 - Test for quantitative thrombin time

Info

Publication number: CA2137342A1
Application number: CA 2137342
Authority: CA
Inventors: Thomas Reid; Barbara Alving
Original assignee: Individual
Current assignee: United States Department of the Army
Priority date: 1992-06-05
Filing date: 1993-06-03
Publication date: 1993-12-23
Also published as: AU4405293A; WO1993025220A1; JPH08501682A; AU4768193A; EP0643727A1; EP0643727A4; WO1993025578A1

Abstract

2137342 9325578 PCTABScor01 A quantitative method for determining the plasma levels of thrombin-specific inhibitors is based on the quantitative thrombin time using plasma dilutions, excess fibrinogen and thrombin. The plasma dilutions and excess fibrinogen act in concert to eliminate the effects that coagulopathies have on standard coagulation tests.
The method is relatively simple and provides superior results to standard conventional tests. The method is suitable for performance in clinical hematology laboratories on a routine basis using commercially available instrumentation.

Description

21373~2 W093/25~78 PCT/~lS93/0~31 Title: TEST FOR Q~ANTI~ TIVE THROMBIN TIME

Fiel~ of the inye~tion:
This invention relates t~ laboratory testing useful in monitoring of anticoagulant therapy. More specifically, this invention relates to assays for the anticoagulants that are known to inhibit the enzyme thrombin.
BACKGRO~ND OF THE INVENTION
Presently, thrombin-specific inhibitors are in clinical tria~s as anticoagulant drugs for the treatment of arterial (after coronary artery angioplasty) and deep venous thrombosis.
The specificity of these drugs is reflected in the dissociation constants ranging from 2 picomoles/L - 2 nanomoles~L. The thrombin specific inhibitors have demonstrated efficacy in animal models for both treatment and prevention of arterial and venous thrombosis. Recombinant hirudin, derived from the medicinal leech Hirudo medicinalis, is the thrombin-specific inhibitor most studied. Effective doses and their range for therapeutic plasma levels in humans have been determined. The low and high ends of the range are used for venous and arterial thrombosis, respectively.
Several U.S. patents have been issued on anti-thrombin agents for use as medicinals or bound to implantable materials.
U~S. Patent 4,944,943 to Eschenfelder, et al. teaches use of hirudin and t-PA for treatment of thrombosis. U.S. Patent 4,952,562 to Klein, et al, discloses anti-thrombotic peptides and pseudopeptides. U.S. Patent 5,019,393 to Ito, et al.
teaches use of implantable materials having a thrombogenesis inhibitor immobilized thereon. U.S. patent 5,053,453 to Ku discloses hirudin or hirudin derivatives covalently linked to support materials to avoid formation of thrombi. U.S. Patent . 5,640,814 to Klein, et al. discloses an anti-thrombotic peptide for administration as a medicinal. U.S. ~atent 5,087,613 to . Courtney, et al. discloses hirudin variants for use as inhibi-tors of thrombin act~vity. U.S. Patent 5,095,092 discloses a process for isolation and purification of hirudin. None of the cited patents disclose a quantitative thrombin-time test as ,~

W093/~78 PCT/US93/0531~

2~3~ 3 4~ 2 taught herein.
, Several methods are presently uæed to assess whether the I patient taking the new thrombin7specifîc inhibitors is ¦ therapeutically anticoagulated. ~t is possible to measure J 5 plasma thrombin-specific inhib~tors levels by high performance liquid chromatography (HPLC). Though accurate, HPLC is generally available only in large referral hospitals and commercial labs and requires a high level of staff expertise.
Even when available in large referral hospitals, the technique is time consuming. Hence, there is delay in obtaining results.
This delay is unacceptable when a clinical practitioner is attempting to closely monitor and control the progress of the patient.
Another approach is to monitor the prolongation of the 1~ activated partial thromboplastin time (APTT)~ The APTT is a standard screening test of the coagulation system. This test is commonly used to follow the degree of anticoagulation in patients receiving the anticoagulant heparin. However, in the absence of specific thrombin inhibitors a variety of coagulo-pathies ~e.g. isolated or multiple factor deficiency, abnormal fibrinogen, decreased fibrinogen concentration or antiphos-pholipid antibodies] tend to prolong the APT~ results. Some patients with coagulopathies associated with the nephrotic syndrome or the lupus anticoagulant also develop thrombosis requiring treatment with anticoagulants. In these cases, the APTT is already prolonged and cannot be used to monitor anticoagulation with thrombin specific inhibitors.
Another clotting assay, the standard thrombin time, is routinely used in a qualitative fashion in clinical laborato-ries to determine the presence of heparin and abnormal or low levels of fibrinogen. The standard thrombin time is not effective and has not been used to quantitate the concentra-tions of thrombin-specific inhibitors in the blood because of the inherent sensitivity of the standard thrombin time, as presently developed, to factors other than these inhibitors (Walenga JM et al. Seminars in Thrombosis Hemostasis 17:103, 1991). In addition, plasmas obtained from patients with liver - 21373~2 W0~3J~78 PCT/US~3/0531~

disease, dysfibrinogenemia, h~pofibrinogene~ia, or who have received fibrinolytic therapy are likely to produce a prolonged standard thrombin time.
Besides HPLC and the APTT, further methods including amidolytic assays for thrombin and immunologic assays for the hirudin-thrombin complex h~ve been suggested. However, the techniques are not easily applied to the clinical laboratory.
Because of the potential therapeutic ef~icacy of the thrombin-specific inhibitors, a new and improved laboratory test for determining plasma levels that can be performed in clinical laboratories with presently available instrumentation is needed. Furthermore, such a new process capable of providing-results that are easily interpreted by clinical practitioners would be of great utility.
~ummarY Of The Invention A novel clinically useful test for monitoring patients on thrombin-specific inhibitor (TSI) therapy, a quantitative thrombin time tQTT) for the management of anticoagulation with thrombin-specific inhibitors, based on the specificity of the TSI for thrombin is disclosed and claimed herein.
The object of the present invention is to provide a method to measure thrombin time that will not be affected by presence of abnormal plasmas which cause a prolongation of standard tests (APTT and standard thrombin time, vide supra), but will be sensiti~e to the presence of thrombin-specific inhibitors.
Another object of the invention is to provide a method from j which unknown plasma concentrations of thrombin-specific I inhibitors can be determined by generating a standard curve for the therapeutic range of the inhibitor using the QTT. A
related object is to provide a method which can be readily performed on laboratory instruments commonly used for measuring the standard thrombin time~
8rief Desori~tion O f The Drawin~s , Fig. l shows the effect of concentration of purified : 35 fibrinogen on the quantitative thrombin time in plasma and abnormal plasmas.
Fig. 2 shows the effect of heparin concentration on the 2 ~3~ 3 42 4 QTT.
Fig. 3 is the standard curve for recombinant hirudin using the quantitative thrombin time.
Fig. 4 shows the effect of recombinant hirudin concentra-tion on the APTT.
Detailed De~Gri~tio~ of The Invention ~`~
This invention provides to a noyël method for the quantitative determination of plasma-~ levels of thrombin-¦ specific inhibitors by specifically measuring thrombin time in ¦ 10 the presence of exogenous fibrinogen and thrombin. Normal ¦ plasmas and plasmas with a coagulopathy have the same baseline for thrombin-specific coagulation time when tested by the QTT.
! By eliminating the interfering effects of factors in the plasmas fram patients with with coagulopathy, the prolongation of the QTT will depend only on the presence thrombin-specific j anticoagulant present in the sample and will, therefore, provide a method for evaluation of the anticoagulant indepen-dent of other~plasma abnormalities that cou}d affect the thrombin time. To eliminate the effect of abnormalities such as antiphospholipid antibodies, and factor deficiencies, the - phospho~ipid based APTT was abandoned in favor of the thrombin-based assay which is independent of phospholipid and coagula-tion factors other than fibrinogen. Only fibrin degradation products, heparin, and abnormal fibrinogens or a decreased fibrinogen potentially present in plasma could interfere with the assay. The effect of these abnormalities can be removed by diluting plasma l:l0 in buffer and adding excess fibrinogen.
- While the test developed has been shown to be effective when using other thrombin-specific inhibitors, the inhibitor exemplified in the testing disclosed herein was recombinant hirudin. Quantification was tested by adding recombinant hirudin to normal plasma at different concentrations within the known therapeutic range. The QTT was then tested with these plasmas and a linear relationship was shown between the log ~5 - (QTT) and concentration of recombinant hirudin. This allowed the determination of concentrations of recom~inant hirudin in - plasma. The specificity of the QTT was assessed by adding a W093/2~78 2 1 3 7 3 g 2 PCT/US93~0~31~

known concentration of recombinant hirudin to patient plasma samples demonstrating a coagulopathy (prolonged APTT and/or standard thrombin time). The expected and observed values of recombinant hirudin levels were then compared and shown to be compatible with use of the QTT in the clinical monitoring of patients on recombinant hirudin~ It was shown, by comparative evaluation, that thé APTT does not provide the specificity provided by the inventive method.
The technigue was performed using two different commer-cially available instruments and e~uivalent results were obtained.
Metho~s ~n~ materials:
Materials:
Plasmas were those submitted to the coagulation laboratory for evaluation of abnormal screening coagulation studies and from healthy ~olunteers who gave informed consent under a protocol approved by th`e Human Use Committee of the Walter Reed Army Institute of Research. ~uman alpha-thrombin (specific activity 2,000-4,000 NIH units/mg protein), recombinant hirudin (specific activity 8,500 units/mg protein) and reptilase (ATROXIN) were purchased from Sigma Chemical Company (St Louis, MO). Human fibrinogen was supplied by KabiVitrum (Stockholm, Sweden). APTT reagent was purchased from Organon Teknika Corporation (Durh~m, NC). Sodium heparin (l000 U/mL) was purchased from Elkins-Sinn, Incorporated (Cherry Hill, NJ).
Blood sample ~re~aration:
Blood samples were collected into plastic tubes containing balanced citrate as the coagulant in a ratio of one-part anticoagulant to nine parts blood. Each sample was centrifuged a 4C and l0,000 x g for 20 minutes to obtain platelet-poor plasma which was then stored at -70C until ready for use.
. Standard thrombin time: ~
The thrombin time was measuring the time to clot formation after adding l00 ~L of a 5 U/mL solution of alpha thrombin to l00 ~L of plasma and l00 ~L VS buffer (vide infra). Either a dataclot-2 fibrometer (Helena Laboratories, Beaumont, TX) or an ST4 coagulation instrument (Diagnostica Stagoj Parsippany ~, ~313 ` PCT/~S93/0531~

NJ) was used.
ouantitative thrombin time (T~l:
¦ Human alpha-thrombin was diluted to a concentration of 2.5-20 U/mL in veronal-saline-calcium (VSC) buffer (2.8mM
sodium diethylbarbiturate, lOOmM sodium chl;~ride, 25mM calcium chloride, pH 7.35) that also contained 1~ human serum albumin.
I Fibrinogen concentrations (0.1-18~M) were made in VS buffer (as ! vsc buffer except the buffer contained no calcium, and sodim ~ chloride concentration was 144mM). Plasma samples were diluted ¦ 10 1:10 in VS buffer. The plasma dilutions, alpha-thrombin and , fibrinogen solutions were incubated separately at 37C for six j minutes before using. Equal volumes of plasma samples and fibrinogen were mixed and incubated at 37C for one minute.
200 ~L of the plasma-fibrinogen mixture were removed and placed in an assay well. The thrombin time was measured as the time ¦ to clot formation after adding 100 ~L of the alpha-thrombin solution. Instruments used for the standard thrombin time were also used for the QTT.
~j APT~ a~av:
The AETT assay was done using a CP-8 photooptic coagula-tion profiler tBIODATA Corporation, Willow Grove, PA). 100 ~L
~; - of plasma was incubated with 100 ~L APTT reagent for six minutes. The APTT was measured as the time of clot formation I after adding 100 ~L 25 mM CaC12.

3 25 Reptilase_time:
~¦ The reptilase time was done using a dataclot-2 fibrometer.
- 100 ~L of reconstituted reptilase was added to 200 ~L of plasma and the clotting time was recorded.
Preparation of plasma samples for standard curve determination:
Recombinant hirudin (lyophilized preparation) was diluted ;in normal pooled plasma to a concentration of 50 ~g/mL.
Further dilutions in normal plasma were made to obtain concentrations within the therapeutic range (0.1-3~g/mL).
- Standard_curve:
The log of the QTTs were calculated and a standard curve was generated after linear regression by plotting the log (QTT) vs concentration of recombinant hirudin (~/mL). From the W093/25~78 2 1 3 7 3 ~ 2 PCT/US93/0531~

standard curve, an unknown plasma le~ l of.rec.~inant hirudin can be determined.
Pre~ar~ti~n of Dlasma sam~les for determinina effect of hemarin concentration on the OTT:
Heparin (initial concentration lOO0 u/mL) was diluted in normal pooled plasma to a concentration of lO U/mL. Further dilutions in plasma were made to obtain heparin concentrations of O.l - 8 U/mL. -` ~: The OTT in normal ~lasma samDles:
The results of the QTT from tw.enty normal individuals (ten male, ten femaIe) are shown in Table l; all had a normal standard thrombin time and APTT.

T~ble l.
8ummary stati~tic~ ba~e~ on pla~m~ f~om t~enty norm~l ~ndi~:~du~l~, ten m~le ~ ten fem~le. All pl~ma 8~mple~ ha~
~o ~ : APTT nd st~n~r~ thro~bin time, u~ing st~n~r~
pro¢o~ures~ e~t~st).
~ , . ., : l : ~e~n`~Qu~t~tat~v-: 13.9 20~ ~ hro~b~ T~, seeo~
~ ~ l ~ ~ COD~ 0.14 ~,, l o~ ~ ¦ 0.66 ~r~g ~(~ a~ 2~ -), 12.6 - 15.2 ; ~ ~oco~- , , : _r.~.s~ f~
~ : ~ = tion; c root mean :~ ~ square:difference : ~ : :
~ : Erfect~of fibrinoaen concentration on the OTT:
:~- ~ 30 : The effect of concentration of purified fibrinogen on the quantitative thrombin time in plssma and abnormal plasmas was . determined. Different fibrinogen concentrations were used in the guantitative thrombin time to determine at which concentration the effects of the abnormi'~ plasmas were eliminated.
,. ~, .
~: ~ ~
: Procedure:
. . ~
. Plasmas were diluted l:l0 in buffer; l00 ~L of the diluted . plasma was added to l00 ~L purified ~uman fibrinogen at .
40~ different concentrations (0.l - 18 ~M) and incubated for 30 3. :.
:,'-' W093/2~57X ~ 3 4~ PCT/VS93/0531~

seconds; 100 ~.L of 5U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured. The addition of excess fibrinogen in the assay eliminated the effects of abnormal fibrinogens (Fig 1) and;~ow fibrinogen (Fig S 1, insert). Plasmas with an abnormal f~inogen (dysfibrino-genemia: standard thrombin time = 45.2 a~a 18.5 sec ~normal 11-14], reptilase time = 22.6 and 15.5 ~ ec [normal 8-11]), low fibrinogen (hypofibrinogenemia: fibrinogen = 38 mg/dL ~normal 200-400]) and plasma with increased fibrin degradation products (FDP = 1024 ~g/mL lnormal <10 ~g/.~L]) and low fibrinogen (fibrinogen = 112 mg/dL) were evaluated~ The optimal fibrino-gen concentration was determined to be 9-18 ~.M. This concen-tration was more than sufficient to overcome the effects of abnormal plasma samples. ~e~ct of dilutina plasma (in buffer) on the OTT:
Dilutin.g the plasma samples 1:10 in buffer mitigated the ~ effects of fibrin degradation products and therapeutic heparin ;~ levels (Table 2). The presence of antiphospholipid antibodies did not interfere with the assay.
Procedure:
Plasmas were diluted 1:10 in buffer; 100 ~L of the diluted plasma was added to 100 ~L. 9 ~N purified human fibrinogen and incubated for 30 seconds; 100 ~.L of 5 U/mL human alpha-thrombin was added to start the reaction; the clotting time was measured.
, ~ .
.

,1 Y
1 ~ .

W093/2~78 213 7 3 q 2 PCT/US93/0~31~

T~bl~ 2.
Coagulopathies that might otherwise interfere with standard clotting tests, do not interfere with the quantitative thrombin time.-. __ , . ......... _ . . .
Qu~ntitative 8t~n~ar~
. Thrombin Thrombi~
CoaouloPathv Timeb, seo TimeC, sec APT~d ~ec I _ _ _ Abnormal fibrinogen14.2 59.1* 29.4 (dysfibrinogenemia) e i ~f 14.8 45.2* 35.7*
I .
.. 9 14.4 18.5* 43.3*
_ ,Ih 13.6 16.0* 37.5*
_ Increased fîbrin 14.2 23.5* 43.0*
.degradation products' .. i . 14.2 22.4* 74.8*. . .
Multiple Fac~ 13.8 13.8 57.6*
Deficiencies' ~eL-ri~ e~e = DY 14.4 > 120* 64.2*

Low fibrinogenm 14.2 2S.8* > 120*
Antiphospholipid 14.2 12.3 50.1*
Antibody .. 14.4 13.5 44.4*
. _ 13.2 12.2 57.9*
.. 13.2 13.7 65.3*
' ~ 13.6 12.8 90.0*
~ __ _ Low factor XII" 14.2 14.0 45.2*

'* Abnormal value. b No~ lal: 12.6-15.2-s !conds; c nor~ ~ 14 ¦ 30 seconds; d normal: 22-34 seconds. ' reptilase time = 29.4 sec (normal 8-11); f reptilase time = 22.6; ~ reptilase time =
! IS 5; h reptilase time = 15.2; i fibrin degradation products =
128 ~g~mL (normal < 10), fibrinogen = 225 mg/dL (normal 200-400); ~ fibrin degradation products = 1024 ~g/mL, fibrinogen =
112 mg/dL; k factor II - 37~, factor X = 16% (normal 50-150);
0.5 U heparin/mL; m fibrinogen = 38 mg/dL; n factor XII < 10%
. (-ormal 50-150).
. Effect of heparin concentration on the OTT:
. . Normal pIasma supplemented with heparin at various concentrations ~-ere tested in the QTT . Procedure: Plasma was W093/25578 ~313 ~ PCT/~S93/0531~

supplemented with porcine heparin at different concentrations (0.1-10 U/mL); these plasmas were diluted 1:10 in buffer; 100 ~L of t~e diluted plasma was added to 100 ~L 9 ~M purified human fibrinogen and incubated for 30 seconds; 100 ~L of 5 U/mL
S human alpha-thrombin was added to start the reaction; the ~clotting time was measured. The QT~'became sensitive to heparin at 0.8 U/mL (Fig 2). The the~ eutic range for heparin is O.3-0.5 U~mL. ~ ~
; Standard curve for recombinant hi ~ din usina the OTT:
The quantitative thrombin time was determined for different known concentrations of recombinant hirudin in plasma. A standard curve was developed by plotting the LOG
(quantitative thrombin time) vs concentration of recombinant hirudin. Procedure: Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-1.75 ~g/mL); these plasmas were diluted 1:10 in buffer; 100 ~L of the diluted plasma wa~ added to 100 ~L 9 ~M purified human fibrinogen and incubated for 30 seconds; 100 ~L of human alpha-thrombin (5 U/mL) was added to start the reaction; the clotting time was measured.
Several thrombin concentrations were tested in developing assays for recombinant hirudin. Low thrombin concentrations were too sensitive to high therapeutic plasma levels of recombinant hirudin whereas high thrombin concentrations were insensitive to low therapeutic levels. There was no thrombin concentration that was effective over the entire therapeutic ; range of recombinant hirudin. Therefore, a thrombin concentra-tion (5 U/mL) sensitive at the low therapeutic range t0.1-1.75 ~g/mL) of recombinant hirudin was selected ~Fig 3). The hirudin-free plasma data point was not included in the standard curve. To assay hirudin concentrations greater tAan 1.75 ~g/mL, a 1:1 dilution in pooled plasma was required before the 1:10 dilution in the assay. The optimal conditions for the QTT
were: 100 ~L 1:10 plasma dilution, 100 ~L 9-18 ~M fibrinogen and 100 ~L alpha-thrombin (5 U/mL).
.-" ' ' ' ' . .
. ' , W093/25~78 PCT/US93/0531~

Validitv of the OTT in determinina ~lasma concentrations of recom~inant hirudin:
The validity of the QTT in determining plasma recombinant hirudin levels was assessed by adding known concentrations of j 5 recombinant hirudin to abnormal and normal pooled plasma. The 3 QTT was measured and the recombinant hirudin concentration determined from the standard curve described above. Table 3 demonstrates that a number of coagulopathies that may prolong the standard thrombin time do not interfere with the measure-ment of recombinant hirudin levels as determined by the QTT.
Procedure:
Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-1.75 ~g/mL): these plasmas were diluted 1:10 in buffer; 100 ~L of the diluted plasma was added to 100 ~L 9 ~M purified human fibrinogen and incubated for 30 seconds; lOO ~L of SU/mL human alpha-thrombin was added to~start the reaction: the clotting time was measured.
Standard curve for recombinant hirudin usina the APTT:
The effect of recombinant hirudin concentrations on the Z APTT ~was measured. Normal plasma was supplemented with recombinant hirudin at various concentrations and the APTT was measured.
Procedure:
Plasma was supplemented with purified recombinant hirudin at different concentrations (0.1-4 ~g/mL); 100 ~L of the APTT
reagent was added to 100 ~L of the plasma supplemented with recombinant hirudin and incubated for 6 minutes; 100 ~L of 25 mM CaCl2 was added to ætart the reaction; the clotting time was measured. Fig 4 demonstrates that the APTT is not useful at the low end of the therapeutic ranges for recombinant hirudin.
Procedure:
-~ Plasma was supplemented with purified recombinant hirudin ~ ~, .
~t d~fferent concentrations (0.1-4 ~g/mL); 100 ~L of the APTT
~'A ' ~ ~ ' reagent was added to 100 ~ of the plasma supplemented with recom~inant hirudin and incubated for 6 minutès; ~00 ~L of 25 : mM CaC12 was added to start the reaction; the ciot~ng time was measured.

, ~

W093/25S78 2~3~ 3 42 PCT/US93/0531~
,:

T~blo 3.
M-~sur-~conaentr~t~ on of QTT, r~combi~t Coaoulo~at~Y secon~ h~ru~in. ua~m~ % Error-. _ Antithrombin IIIb 22.6 1. ~i 11 deficiency .
~o= ~ceo~ Xll' 23.8 " 1.21 3.1 .~
Abnormal 21.7 ; ~1.04 17.0 : 10 fibrinogen : dysfibrinogenemiad ~ 27.8 1.49 19.5 : nf 23.8 1.21 3.2 ~ _ .
Elevated fibrin degradation 24.0 1.22 2.2 products9 nh 22.4 1.1 12.3 ni 25.5 1.34 7.0 Low fibrinogeni 24.4 1.25 0 20 ~ Heparin therapyk 26.1 1.38 10.4 Multiple factor 24.6 1.27 1.6 :~: :def iciencies I : ~
Antiphospholipid 24.4 1.25 0 Antibody 1~ _ _ . ..
~ .. 22.4 1.11 12.3 ~ .. 22.5 1.1 11.7 : ~
. . - . . ... . . . . . _ .
ecombinant hlrudln was added to a 1 plasma samples at a final concentration of 1. 25 ~g/mL.
' \exDected-measured\ x 100 = \1. 25 ua/mL - measured\ x 100 expected . 1. 25 ~g/mL
b Antithrombin III = 54% (normal 88-140~; c factor XII < 10~
(normal 50-150); d standard thrombin time =18.5 sec (normal 11-14), reptilase time = 15.5 sec (normal 8-11); e standard thrombin time s 45.2 sec, reptilase time ~ 22.6 sec; f standard ~; 35 thrombin time e 18.1 sec, reptilase time = 13.1 sec; ~ fibrin degradation products ~ 1024 ~g/mL (normal ~10), fibrinogen =
183 mg/dL (normal 200-400): h fibrin degradation products = 256 g/mL, fibrinogen ~ 360 mg/dL; i fibrin degradation products ~ = 1024 ~g/mL, fibrinogen = 112 mg/dL; j fibrinogen = 38 mg/dL;
`~ ~ 40 k 0.5 U heparin/mL;.l factor II = 37%, factor X = 16% (normal i 50-150).

.
Table 4 conclusively shows that the presence of a lupus anticoagulant, abnormal fibrinogens, heparin and fibrin W0~3/25578 2 1 3 7 3 4 2 PCT/US93/0531;

degradation products interfere with the APTT assay for the recombinant hirudin.
Table ~.
Coagulopathies that interfere with the APTT, interfere with the determination of plasma Ievels of recombinant hirudin as performed with the APTT.-., ..
11~8u~ ~
concentr~tion of APTT, r-corbi~nt Coaoulopathy secon~s ~iru~in, ug~mL % ~rror~
Low fibrinogen' >300 I
AbnormaI fibrinogen (dysfibrino- 105.8 1.93 54 lS 1 genemia) d ~ne ~ 132.0 3.66 169 Increased fibrin ~
degradation 182.1 6.97 458 productsf .
.
~2~0~ ~ 9 - 108.8 2.13 70 ¦ ~Neparin~therap~ 300 _ _ Low ~factor XIIi 103.4 1.77 42 ¦ Antiphospholipid 117.7 ~ 2.72 118 I Antibody ~ n 115 . 6 ~ ~ 2.58 106 ~ . l ~ ~ ~ ~ .................. 162.4 5.67 354 ? : ~ _~_ 1~ ~
- RecomDlnant~nlrualn was aaaea to all plasma samples at a flnal concentration of 1.25 ~g/mL. - indicates value could not be determined.
b \expected-measured\ x 100 - \1.2S ug/mL - measured\ x loO
expected 1.25 ~g/mL
c fibrinogen - 38 mg/dL (normal 200-400): d standard thrombin time s 45.2 sec tnormal 11-14), reptilase time = 22.6 sec (norma} 8-11); ' standard thrombin time = 18.5 sec, reptilase time = 15.S sec; f fibrin degradation products = 1024 ~g/mL
(normal ~ 10), fibrinogen = 112 mg/d~; 9 fibrin degradation products = 2S6 ~g/mL, fibrinogen = 360 mg/dL; h 0. 5 U hepa-rin/mL; i factor XII < 10% (normal S0-lS0).
Comparison of results from the OTT and APTT usina abnormal 40 - lasmas suD~lemented with recombinant hirudin.
~ The~ mean errors for determining the concentration of ; ~ ~ recombinant hirudin in abnormal plasma using the QTT were less than 10%. In contraæt, using the same plasmas, the measured concentration of recombinant hirudin using the standard APTT

!: :

2 ~3~ 3-42 14 showed-no agreement with the expected value (Table 5).
T~ble S.
~u~ry of Data for vali~ation of t~- QTT in d~ff~r-nt ¢linical conditions. ~;

. OTT ;~ ;PTT
¦ r.m.s.' diff-rence ~ 0.12 2.82 r-corbin~t hirudin I0 concentratiOn, ~g~m~.
I l r.~.~. error, % 9.8 225 ~_ ~root mean square The thrombin-specific inhibitor hirudin and its analogues have demonstrated efficacy in the prevention of thrombosis in preclinical studies. The anticoagulant effects have been monitored by the APTT because the standard thrombin time is too sensitive to the agents to be of clinical utility. The APTT
has two major problems that limit it's usefulness in monitoring 20~ recombinant hirudin: - l) patients with a prolonged baseline APTT cannot be followed (this also holds true for patients -~ receiving heparin therapy); and 2) there is a nonlinear dose-response at low therapeutic levels of recombinant hirudin with regard to the APTT (Fig 4). The clinical consequence of using the APTT to monitor therapy in the low therapeutic range is excessive anticoagulation for the specific clinical condition.
~ We have developed a quantitative throm`bin time that eliminates -; the inherent difficulties associated with the APTT and the standard thrombin time. The problems which the invention is -~ 30 designed to solve are the sensitivity of standard tests to therapeutic heparin levels, low levels of fibrinogen, abnormal fibrinogens and fibrin degradation products, lupus anticoagu-lant (antiphospholipid antibodies), `and low plasma factor levels.
Some of the novel aspects of the invention include 1) the addition of fibrinogen in excess to compensate for abnormal or low fibrinogen and 2) the plasma samples are diluted l.lO in buffer which effectively prevents interference of fibrin degradation products, abnormal fibrinogen and heparin. There ,:
, W093/25578 PCT/~S93tO~31~

are several distinct advantages of the invention. The,method is simple and can be done in any lab that performs thrombin times routinely (most hospitals). The procedure is specifical-ly adapted for use on instruments presently available to S clinical laboratories. ~The newer automated equipment and methods used in determinatiDn of thrombin time may be uæed for practice of the invention. The method is also quick and requires no additional expertise above that already available in the clinical laboratory. A small amount of patient material is needed t2S-50 ~L of plasma for 4 tests); where standard tests require lOO ~L for a single test. The method allows quantitation of plasma levels of rec~mbinant hirudin and other thrombin-specific inhibitors for use with other thrombin-s,pecific inhibitors (e.g. synthetic or semisynthetic analogues of recombinant hirudin). The mean error is less than 10%. The standard curve is linear in the therapeutic range, where standard tests are either not linear in the low therapeutic range (ARTT) or are too sensitive to recombinant hirudin (standard thrombin time). The method is more sensitive to thrombin-specific inhibitors in comparison to standard tests, as demonstrated by the response of the clotting times to recombinant hirudin concentrations. Finally, the test is inexpensive and fast in comparison to HPLC.
While normal human thrombin was used in the procedure as exemplified, mutant or normal thrombin from other sources may be used in the QTT so long as the al'ternate thrombin provides , a reliable dosage curve when tested against the specific thrombin-specific inhibitor for which concentration is being determined. Similarly, the fibrinogen can be from any source so long as it provides a reliable dosage curve when tested against the TSI.
The materials for the QTT may be sold as kits. The ' following components could be provided in the kit:
, . ' l) One or more vial(s) of lyophilized plasma containing a known amount of thrombin-specific inhibitor (TSI) which may ,.
, , contain,protamine sulfate should be provided. The contents of ' , the vial(s) may be reconstituted with water so that the final .
.

w093/25~78 ~3~ 3 4~ PCT/US93/0531~

concentration of the TSI is such that serial dilutions allow for TSI within the therapeutic range. The addition of the protamine sulfate may be used to effectively remove heparin from the reaction. If protamine sulfate`is used, a preferred concentration in the final diluted ~ containing solution would be about 120 ~g/mL. However~ some instances the TSI
may be available to laboratories as a stock solution(s).
2) One or ~ore vials of lyophilized alpha-thrombin which may be formulated with a stabilizing agent such as albumin and may contain buffer should be provided. The thrombin could be reconstituted with water to provide the desired concentration.
- 3) one or more vial~s) of lyophilized fibrinogen to be recon-stituted to a final concentration of about 4.75 ~M (160 mg/dL).
The plasma may contain a neutralizing agnet such as protamine sulfate. The kit can, if desired, hold a separate vial of protamine sulfate to be used with samples when the patient is on heparin therapy.

4) Vials of buffer for diluting samples may be included.
Using the kit items, the following procedure may be followed.
In a preferred embodiment the standard curve may be developed to identify effective levels of TSI by the following method:
a. Preparing serial dilutions of a thrombin-specific inhibitor in normal plasma in known concentrations to provide samples (though a kit containing varying amounts of TSI within the appropriate range will avoid this procedure).
b. Diluting the samples in buffer to provide l:lO dilutions.
c. Nixing the dilutions with a 9-18 ~M solution of purified human fibrinogen to provide samples.
d. Adding a 5 U/mL solution of purified human alpha-thrombin to the samples of step c to provide solutions.
~ e. Measuring the clotting times of the solutions in step d.
! f. Plotting a standard curve from the results obtained from step e.
The level of TSI concentration in patient plasma is tested in the following manner to determine the TSI level in the patient:
l. Diluting in buffer a plasma sample from patients receiving a thrombin-specific inhibitor to provide a l:lO plasma to !

W093~2~78 2 1 3 7 3 4 2 PCT/US93/0531~

buffer concentration.
2. Mixing the patient samples with a solution of fibrinogen.
3. Adding a thrombin solution to the patient samples prepared in step 2.
4. Measuring the clotting times of the solutions of step 3.

5. Determining the co~centration of the inhibitor in the patient samples using the standard curve previously developed.
The invention may also be practiced using whole blood rather than patient plasma.
Vials containing varying amounts of the TSI required for generating the standard curve would be made by diluting the TSI
to provide varying dilutions for use in determining the curve.
The normal plasma is reconstituted by dilution with buffered water (or the buffer may be added into the diluent) to provide varying concentrations of TSI. A second set of containers containing only plasma is prepared for purposes of comparison.
A third container is prepared containing l mL of patient plasma with unknown concentration of TSI. To each sample is added 475 ~L of reconstituted fibrinogen solution and 50 ~L reconstituted thrombin. The clotting time is then measured. Steps a to f may be used as a research tool to evaluate activity of TSI's.
However, as more is known about the effect on QTT of various concentrations of a particular TSI, the running of a,standard to obtain the curve may be unnecessary. In such instances, the ~ 25 QTT alone without the comparison tsting will be sufficient to : allow practitioners to evaluate concentration of TSI in the blood.
As an alternative, the kit may contain only fibrinogen and thrombin, whilst one or more stock solutions containing appropriate concentrations of TSI in plasma is maintained for use in the test. (It would also be possible to have one stock . concentration at highest level to be teæted, whic~ would be diluted to provide lower concentrations for use to provide the appropriate curve.) The extent to which stock solutions are used or multiple vials of TSI containing differing amounts of -TSI are provided will depend on the number of tests done in any particular health care center and the quality of staff ' - .

W093~25578 2~3~ ~ 42 PCT/US93/05315 ^ 18 available to provide testing services. All of the components, ¦ plasma, thrombin, and fibrinogen, should be used fresh or be ¦ frozen to avoid loss of potency. All of the agents used, including plasma with TSI, norm~a~ plasma, thrombin, and ¦ 5 fibrinogen may be provided as ly~o~ ilized materials in vials ; ~for reconstitution. once reconstituted, the active components j are easily inactivated.
Among the uses for which the invention iæ suitable include quantitative clinical monitoring of patients anticoagulated with the thrombin-specific agents and laboratory research in animal models of thrombosis.

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Claims

WHAT IS CLAIMED:

1. A method for quantitating plasma levels of specific inhibitors of the enzyme thrombin, comprising the steps of:
a. Preparing serial dilutions of a thrombin-specific inhibitor in known concentrations to provide samples, b. Mixing the samples prepared in step a with purified human fibrinogen, c. Adding purified human alpha-thrombin to the samples prepared in step b, d. Measuring the clotting times of the solutions in step c, e. Plotting a standard curve from the effect of TSI based on results obtained from step d, f. Diluting a plasma sample from patients receiving a thrombin-specific inhibitor, g. Mixing the patient samples with a solution of fibrinogen of step f, h. Adding a thrombin solution to the patient samples prepared in step g, i. Measuring the clotting times of the solutions of step h, j. Determining the concentration of the inhibitor in the patient samples using the standard curve.

2. The method according to Claim 1, wherein step f comprises retaining sensitivity (graph linearity) at low therapeutic levels of the inhibitor.

3. The method according to Claim 1, wherein wherein, addition-ally, the effect of therapeutic heparin levels in the plasma is avoided by addition of a neutralizing agent.

4. A method of claim 3 wherein the neutralizing agent is protamine sulfate.

5. A method of evaluating appropriate dosage of a thrombin-specific inhibitor comprising the steps of:
a. Preparing serial dilutions of a thrombin-specific inhibitor in known concentrations to provide samples, b. Mixing the samples prepared in step a with purified human fibrinogen, c. Adding purified human alpha-thrombin to the samples prepared in step b, d. Measuring the clotting times of the solutions in step c and e. Plotting a standard curve from the effect of TSI based on results obtained from step d.

6. A kit comprising:
1. at least one vial of lyophilized fibrinogen, 2. at least one vial containing thrombin, and 3. instructions for evaluating QTT.

7. A kit of claim 6 which, additionally, contains at least one vial of plasma.

8. A kit of claim 6 containing, additionally, at least one vial of a thrombin-specific inhibitor.

9. A method of measuring quantitative thrombin time compris-ing the steps of:
(1) preparing a composition comprising plasma in buffer to obtain a concentration of about 1:10 plasma to buffer;
(2) preparing a fibrinogen mixture comprising fibrinogen concentration of about 0.1 to 18 µM fibrinogen in buffer;
(3) preparing a composition containing a concentration of about 2.5 to 20 U/mL alpha-thrombin in buffer;
(4) preparing a mixture comprising equal amounts of the compositions obtained in steps (l) and (2);
(5) placing in an assay well an aliquot of the mixture prepared in step (4) and an aliquot of the mixture prepared in step (3);
and (6) determining the amount of time required for clot formation.

10. A method of claim 9 wherien the mixture prepared in step (2) is at a concentration of 9 to 18 µM fibrinogen in buffer.

11. A composition of matter comprising plasma, purified human fibrinogen, and alpha-thrombin in a buffer.

12. A composition of claim 11 wherein the alpha-thrombin is human alpha-thrombin.

13. A composition of claim 11 wherein the fibrinogen is present in a concentration of 0.1 to 18 µM.

14. A composition of claim 13 wherein the fibrinogen is present in a concentration of between 3 µM and 18 µM.

15. A method of claim 9 wherein, in step (5), the ratio of amount of reactants is 2 parts of the mixture obtained in step (4) to 1 part of the mixture obtained in step (3).