The anticoagulant effect of recombinant hirudin (rHir) and HirulogTM has been monitored in patients with the activated partial thromboplastin time. Accurate monitoring with this test cannot be achieved if plasmas contain heparin, lupus anticoagulants, low concentrations of fibrinogen or other factors, or elevated fibrinogen-fibrin degradation products (FDP). We have therefore developed a simple, rapid, sensitive clot-based method, the quantitative thrombin time (QTT), to measure levels of rHir and HirulogTM in patient plasma (or whole blood). The QTT is performed by mixing a 1:10 dilution of patient plasma (50 μl) with human fibrinogen (50 μl, 128 mg/dl) at 37° C; the clotting time is initiated by adding human thrombin (50 μl, 5-7.5 U/ml). The concentration of HirulogTM or rHir in plasma can be determined by comparing the QTT in patient plasma with a Standard curve that is generated by adding different concentrations of anticoagulant to pooled normal plasma. Studies with whole blood using the same procedure yield similar results. In the absence of HirulogTM or rHir, the baseline QTT is the same in normal and abnormal plasmas (fibrinogen <150 mg/dl and FDP as high as 1024 μg/ml, elevated FDP alone, lupus anticoagulant, or heparin <0.9 U/ml). When known concentrations of either rHir or HirulogTM are added to abnormal plasmas, the mean observed concentrations as determined by the QTT deviate from the expected values by less than 10% (range 0-19%). The data indicate that the QTT is a simple, rapid, and accurate test for the determination of levels of rHir and HirulogTM in plasma or whole blood.
References
1
Markwardt F,
Hauptman J,
Nowak G,
Kleßen CH,
Walsmann P.
Pharmacological studies on the antithrombotic action of hirudin in experimental animals. Thromb Haemostas 1982; 47: 226-229
4
Doutremepuich C,
Deharo E,
Guyot M,
Lalanne MC,
Walenga J,
Fareed J.
Antithrombotic activity of recombinant hirudin in the rat: a comparative study with heparin. Thromb Res 1989; 54: 435-445
6
Heras M,
Chesebro JH,
Penny WJ,
Bailey KR,
Badimon L,
Fuster V.
Effects of thrombin inhibition on the development of acute platelet-thrombus deposition during angioplasty in pigs. Circulation 1989; 79: 657-665
8
Heras M,
Chesebro JH,
Webster MWI,
Mruk JS,
Grill DE,
Penny WJ,
Bowie EJW,
Badimon L,
Fuster V.
Hirudin, heparin and placebo during deep arterial injury in the pig. Circulation 1990; 82: 1476-1484
9
Kaiser B,
Simon A,
Markwardt F.
Antithrombotic effects of recombinant hirudin in experimental angioplasty and intravascular thrombolysis. Thromb Haemostas 1990; 63: 44-47
10
Freund M,
Cazenave J-P,
Courtney M,
Degryse E,
Roitsch C,
Bernat A,
Delebassée D,
Defreyn G,
Maffrand J-P.
Inhibition by recombinant hirudins of experimental venous thrombosis and disseminated intravascular coagulation induced by tissue factor in rats. Thromb Haemostas 1990; 63: 187-192
11
Haskel EJ,
Prager NA,
Sobel BE,
Abendschein DR.
Relative efficacy of antithrombin compared with antiplatelet agents in accelerating coronary thrombolysis and preventing early reocclusion. Circulation 1991; 83: 1048-1056
12
Sarembock IJ,
Gertz SD,
Gimple LW,
Owen RM,
Powers ER,
Roberts WC.
Effectiveness of recombinant desulphatohirudin in reducing restenosis after balloon angioplasty of atherosclerotic femoral arteries in rabbits. Circulation 1991; 84: 232-243
13
Peters RF,
Lees CM,
Mitchell KA,
Tweed MF,
Talbot MD,
Wallis RB.
The characterization of thrombus development in an improved model of arterio-venous shunt thrombosis in the rat and the effective of recombinant desulphatohirudin (CGP 39393), heparin, and iloprost. Thromb Haemostas 1991; 65: 268-274
16
Cadroy Y,
Maraganore JM,
Hanson SR,
Harker LA.
Selective inhibition by a synthetic hirudin peptide of fibrin-dependent throm-bosis in baboons. Proc Natl Acad Sci USA 1991; 88: 1177-1181
18
Markwardt F.
Pharmacology of hirudin: one hundred years after the first report of the anticoagulant agent in medicinal leeches. Biomed Biochim Acta 1985; 44: 1007-1013
20
Minar E,
Zazgornik J.
Local hirudin application – an aid in preventing occlusion of an arteriovenous fistula in dialysis patients?. Klin Wschr 1985; 63: 190-191
22
Fox I,
Dawson A,
Loynds P,
Eisner J,
Findlen K,
Levin E,
Hanson D,
Mant T,
Wagner J,
Maraganore J.
Anticoagulant activity of HirulogTM, a direct thrombin inhibitor, in humans. Thromb Haemostas 1993; 69: 157
24
Berscheid G,
Grötsch H,
Neubauer H,
Pünter J,
Reindl J,
Seipp P.
Determination of R DNA hirudin and alpha-human thrombin-hirudin complex in plasma samples: enzyme linked immunosorbent assays for hirudin and complex vs chromogenic thrombin substrate assay. Thromb Res 1992; 66: 33-42
25
Bichler J,
Siebeck M,
Fichtl B,
Fritz H.
Pharmacokinetics, effect on clotting tests and assessment of the immunogenic potential of hirudin after a single subcutaneous or intravenous bolus administration in man. Haemostasis 1991; 21: 137-141
26
Bichler J,
Siebeck R,
Maschler R,
Pelzer H,
Fritz H.
Determination of thrombin-hirudin complex in plasma with an enzyme-linked immunosorbent assay. Blood Coagul Fibrinol 1991; 2: 129-133
29
Walenga JM,
Hoppensteadt D,
Koza M,
Wallock M,
Pifarre R,
Fareed J.
Laboratory assays for the evaluation of recombinant hirudin. Haemostasis 1991; 21: 49-63
30
Spannagl M,
Bichler J,
Birg A,
Lill H,
Schramm W.
Development of a chromogenic substrate assay for the determination of hirudin in plasma. Blood Coagul Fibrinol 1991; 2: 121-127
31
Walenga JM,
Hoppensteadt D,
Koza M,
Pifarre R,
Fareed J.
Comparative studies on various assays for the laboratory evaluation of r-Hirudin. Semin Thromb Hemostas 1991; 17: 103-112
32
Riehl-Bellon N,
Carvallo D,
Acker M,
van Dorsselaer A,
Marquet M,
Loison G,
Lemoine Y,
Brown SW,
Courtney M,
Roitsch C.
Purification and characterization of recombinant hirudin produced by Saccharomyces cerevisiae. Biochemistry 1989; 28: 2941-2949
33
Maraganore JM,
Bourdon P,
Jablonski J,
Ramachandran KL,
Fenton JW.
Design and characterization of hirulogs: a new class of divalent peptide inhibitors of thrombin. J Biol Chem 1990; 29: 7095-7101
34
Alving BM,
Barr CB,
Johansen LE,
Tang DB.
Comparison between a one-point dilute phospholipid APTT and the dilute Russell viper venom time for verification of lupus anticoagulants. Thromb Haemostas 1992; 67: 672-678
35
Abdilgaard U,
Lie M,
Ødegãrd OR.
Antithrombin (heparin cofactor) assay with “new” chromogenic substrates (S-2238 and chromozym TH). Thromb Res 1977; 11: 549-552
37
Stone SR,
Dennis S,
Hofsteenge J.
Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex. Biochemistry 1989; 28: 6857-63
40
Krstenansky JL,
Broersma RJ,
Owen TJ,
Payne MH,
Yates MT,
Mao SJT.
Development of MDL 28,050, a small stable antithrombin agent based on a functional domain of the leech protein, hirudin. Thromb Haemostas 1990; 63: 208-214
41
Ginsberg JS,
Hirsh J,
Gent M,
KcKinnon B,
Turpie AGG,
Levine M,
Powers P,
Weitz J,
Findlen K,
Neemeh J,
Buller H,
Adelman B,
Maraganore J,
Fox I.
A Phase II study of hirulog in the prevention of venous thrombosis after major hip and knee surgery. Circulation 1992; 86 (Suppl. 01) 409
43
Lidón R-M,
Adelman B,
Maraganore J,
Théroux P.
Hirulog, a direct thrombin inhibitor, for the management of unstable angina. Circula-tion 1992; 86 (Suppl. 01) 386
44
Sharma GVRK,
Lapsley DE,
Vita JA,
Sharma S,
Coccio E,
Adelman B,
Loscalzo J.
Safety and efficacy of hirulog in patients with unstable angina. Circulation 1992; 86 (Suppl. 01) 386
46
Weitz JI,
Hudoba N,
Massel D,
Maraganore J,
Hirsh J.
Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is not susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-391