SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991
Laboratory Use of Hirudin
The inhibition of thrombin by hirudin is facilitated by binding to the protein recognition sites and to the apolar binding site of the enzyme molecule. The catalytic center of thrombin remains unaffected. Through this binding, hirudin inhibits the enzymatic and nonenzymatic actions1-3 of the multifunctional proteinase throm bin (Tables 1,2). The inhibition of thrombin by hirudin is very selective (Table 3). Thrombin-related coagulation fac tors, sensitive to antithrombin III/heparin, are not af fected by hirudin. Its action is restricted to α-thrombin, to the thrombin precursor meizothrombin,4 to the prote olytic degradation product gamma-thrombin,5 and to thrombin-α2-macroglobulin complexes.6 Hirudin may therefore be used as a versatile tool selectively to prevent or to interrupt thrombin action in diagnostic and prepar ative procedures. An extensive review of the multiple nonclinical applications of hirudin has been presented by Walsmann.7 Since hirudin forms an almost stoichiometric equimolar complex with thrombin, it may be used for the quantitative determination of thrombin or prothrombin as well as for the screening of disturbed prothrombin activation in plasma. It may also serve as a rapid test to verify the therapeutic range of oral anticoagulation. Hirudin has been used in numerous studies on the kinetics of fibrinopeptide release catalyzed by thrombin or thrombinlike enzymes,8 on the structure of fibrin,9,10 and on the interaction between fibrinogen fragments and fibrin mono- and oligomers.11 Hirudin has been applied to quench thrombin on activation of Factor XIII in experi ments on fibrin cross-linkage and interactions between fibrinogen, fibrin, and fibronectin.12-14
From Pentapharm Ltd., Basle, Switzerland. Reprint requests: Dr. Stocker, Pentapharm Ltd., CH-4002 Basle, Switzerland.
A further research application of hirudin is its use in studies on thrombin binding to membrane receptors of platelets, endothelial cells, leukocytes, fibroblasts, and malignant cells. Hirudin prevents binding of thrombin to neuroblastoma cells, thereby inhibiting the synthesis of cyclic guanosine monophosphate.15 Hirudin can be added in excess to blood, plasma, or cells containing test mixtures to prevent thrombin action immediately after its generation. It may be added to quench thrombin on extensive or limited action, and it may be utilized to neutralize and likewise to titrate a precisely defined amount of thrombin. Hirudin can also be used in an immobilized, matrix-bound form to capture and remove undesired thrombin in reaction mixtures or purification processes. In plasma coagulation factor testing with synthetic chromogenic substrates, addition of hirudin to the reac tion mixture can improve the specificity of the assay by excluding substrate hydrolysis catalyzed by thrombin, meizothrombin, and α2-macroglobulin thrombin.16 Three particular applications for hirudin merit spe cial attention: 1. The use of hirudin as an inhibitor of meizothrombin 2. The use of hirudin for the discrimination between thrombin and other plasma proteinases 3. Hirudin as a blood anticoagulant for testing cell and corpuscle functions.
MEIZOTHROMBIN INHIBITION When rabbit brain extract (tissue factor) is added to heparinized blood or plasma (100 U heparin/ml), a clot is formed in a few minutes. This gel formation cannot be attributed to α-thrombin, since this would be inhibited rapidly by heparin antithrombin III (AT III)-complexes. Addition of iodoacetamide (0.5 mM/liter) to block the alternative, thiol-dependent pathway of fibrinogen gela tion did not prevent coagulation in heparinized brain
Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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KURT STOCKER, Ph.D.hon.
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991 TABLE 3. Hirudin Specificity
TABLE 1. Protein Substrates for Thrombin Fibrinogen
Albumin
Prothrombin
Secretin
Enzyme
Inhibition by Hirudin
Inhibition by AT III/Heparin
Clotting Factors V, VII, VIII, IX, X, XIII
Chymotrypsinogen
Alpha-thrombin
Yes
Yes
Cholecystokinin
Gamma-thrombin
Slight
Yes
Protamine
Meizothrombin
Yes
Slight
Trypsinogen
Alpha2M-thrombin
Yes
No
Catalase
Ancrod
No
No
Protein C Antithrombin III Complement components C3, C5, C9 Platelet membrane glycoproteins Thrombospondin Thrombosthenin M Thrombosthenin A Procollagen Collagen types I, III, V Fibronectin Laminin
Casein
Batroxobin
No
No
Myoglobin
Staphylothrombin
No
Slight
Lysozyme
Factor XIIa
No
Yes
Ribonuclease
Factor XIa
No
Yes
Gonadotropin (chorionic)
Factor Xa
No
Yes
Histone
Factor IXa
No
Yes
Somatotropin
Activated protein C
No
No (slight)
Apolipoprotein
Cell surface proteins
Transmidase (spleen)
Myosin Troponin C
Transamidase (membrane bound)
Calmodulin Adapted from Muszbek and Laki.1
extract containing blood. Clot formation was, however, quenched by the addition of hirudin to the text mixture. This phenomenon can be explained by the presence of meizothrombin, an activation product formed by cleav age of the Arg323-Ile324 bond of the prothrombin molecule leading to the exposure of the enzymatic TABLE 2. Cell and Tissue Functions Modulated by Thrombin Proliferation Fibroblasts Splenocytes Secretion Platelets Endothelial cells Prostaglandin synthesis Platelets Endothelial cells Fibroblasts Neuronal cells Chemotaxis Monocytes Neutrophils Contractility Smooth muscle Neurotransmitter synthesis Neuroblastoma cells Permeability increase Endothelium
thrombin center. Meizothrombin was first detected and characterized as a product of action of snake venom on prothrombin. Its formation is catalyzed independently from Factor V, phospholipids, and calcium by the prothrombin activator Ecarin, a metalloproteinase from the Echis carinatus snake venom. However, as investi gated by Rosing et al 17,18 and Krishnaswamy et al, 19 meizothrombin is not only formed by the snake venom but also under physiologic conditions, by the action of prothrombinase, generated by interaction of Factor Xa, Factor Va, phospholipids, and calcium. In vitro coagu lation of heparinized blood on addition of brain extract suggests that intravascular activation of prothrombin may lead to the formation of meizothrombin, which therefore may represent a marker for this pathologic event. Meizothrombin is unstable and undergoes rapid autocatalytic conversion to α-thrombin. Like thrombin, meizothrombin clots fibrinogen, exerts other thrombin actions, and is inhibited by hirudin and by low molecular weight synthetic thrombin inhibitors. Like α-thrombin, meizothrombin is very slowly inhibited by AT III. Although heparin potentiates α-thrombin inhibition, it does not enhance meizothrombin inhibition by AT III. For test procedures that do not require prolonged reaction times, the weak inhibition of meizothrombin by heparin may be neglected compared with the strong and fast inhibition by hirudin (Fig. 1). The different sensitivity to AT III-heparin complexes can be utilized to discriminate α-thrombin from meizothrombin for quantitative test purposes. 4,17,18 Meizothrombin may be present in blood samples of heparin-treated patients and in plasma prepared from blood that has been heparinized in vitro. In such samples, fibrinogen-fibrin parameters, thrombin-activated cofac tors, and platelet functions may be affected by meizo-
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Cloacin
Actin
LABORATORY USE OF HIRUDIN—STOCKER
115
thrombin during plasma processing and storage. Addi tion of hirudin should therefore improve the quality of assays for fibrinogen, soluble fibrin, fibrinopeptides, and fibrinogen degradation products. During separation and purification steps in the preparation of clotting factors from heparinized blood, meizothrombin may be formed, may act on thrombinsusceptible bonds, and may "disappear" unrecognized after its autocatalytic conversion to α-thrombin. Inhibi tion of meizothrombin by hirudin could therefore im prove the yield and quality of the isolated thrombinsensitive products. Hirudin can be used as a capturing agent for coating microtiter plates in immunologic meizothrombin assay procedures. Since meizothrombin is an early product of prothrombin activation, a hirudin tolerance test could be a highly sensitive indicator of disturbed prothrombin activation. This test would be more sensitive than a corresponding heparin tolerance test that reacts only on the late prothrombin activation product, α-thrombin.
FUNCTIONAL MEIZOTHROMBIN ASSAY IN HUMAN BLOOD
reversible synthetic thrombin inhibitor Nα-(2-naphthylsulfonylglycyl)-D,L-amidinophenylalanine piperidide (NAPAP, Pefabloc TH), which, however, in the final enzyme reaction mixture had to be diluted to an inactive level. For meizothrombin determination, 9 volumes of blood were collected in tubes provided with 1 volume of a solution consisting of NAPAP (20 nmol/ml) and heparin (100 U/ml). Separation of plasma from NAPAP- and heparincontaining blood samples was not possible without loss of meizothrombin. This loss of meizothrombin activity could not yet be elucidated; adsorption of meizothrombin or meizothrombin-NAPAP complexes to erythrocytes might be an explanation. To overcome this drawback, an adequate volume of whole blood was diluted with aprotinin-containing buffer (to exclude plasma kallikrein) and incubated with a chromogenic thrombin substrate for a defined time period at 37°C. The reaction was then stopped by the addition of trichloroacetic acid (TCA), which at the same time caused complete protein precipitation. The precipitate was removed by centrifugation and the released pNA measured at 405 nm in the supernatant.
Procedure To prevent autocatalytic transformation of the heparin-insensitive meizothrombin to the heparin-sensitive α-thrombin during collection and processing of blood samples, the enzymatic activity of meizothrombin has to be quenched. Trials to interrupt autocatalytic meizo thrombin transformation by pH reduction in blood sam ples and subsequent potency measurements at optimum pH for chromogenic substrate hydrolysis have failed. Meizothrombin, experimentally induced by brain extract in a heparinized blood sample, could be stabilized by the
For test A, 0.05 ml patient blood or blood experi mentally activated with brain extract, anticoagulated by means of heparin (10 U/ml) and NAPAP (2 nmol/ml), was mixed with 1.65 ml aprotinin (1 U/ml) containing Tris-imidazole buffer, pH 8.4, ionic strength 0.3, and 0.2 ml Tos-Gly-Pro-Arg-pNA (Chromozym TH) 4 µmol/ml; the mixture was incubated for exactly 30 minutes at 37°C. For test B, a similar reaction mixture was prepared, but 0.01 ml hirudin (2000 ATU/ml) was
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FIG. 1. Activation of prothrombin to meizothrombin and α-thrombin: ac tion and inhibition.
SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991
added prior to the chromogenic substrate. After incubation, 0.1 ml TCA (50%) was added to stop the reaction and to precipitate protein and lipid material. The precipitate was separated by 5 minutes centrifugation at 4000 x g. Absorbance (A405) of the clear supernatant was measured and (DA) per minute calculated. The difference in p-nitroaniline release expressed in DA405/per minute between test A and test B represents the amount of meizothrombin activity present in the test mixture. Amidolytic activity was calculated according to the following equation:
where V1 is the test volume, V2 is the sample volume and A is the millimolar absorbance coefficient of pNA at 405 nm (10.3), 1 mU is the amount of meizothrombin that hydrolyzed 1 µmol of substrate per minute under standard conditions. Meizothrombin activity experimentally generated in heparined human blood is presented in Table 4.
DISCRIMINATION BETWEEN THROMBIN AND OTHER PLASMA PROTEINASES The selective inhibitor effect of hirudin may be used in chromogenic substrate assays to inhibit thrombin in the complex proteinase mixtures that result from a thromboplastin- or partial thromboplastin-induced activation of the clotting system in plasma. Hirudin cannot only improve the possibly insufficient specificity of chromogenic substrates, but may also serve for kinetic studies of plasma zymogen activation. It may, in addition, be used for the identification and characterization of thrombin- or meizothrombin-mediated feedback reactions within the intrinsic clotting system. Thus, zymogen activation in plasma, on addition of a partial thromboplastin reagent (cephalin/ellagic acid),
TABLE 4. Meizothrombin Experimentally Generated in Heparinized Human Blood Brain Saline extract (% Rabbit Brain Powder)
Meizothrombin (mU/ml) After 1 Min 37°C
5.0
55
2.5 1.0
93 19 4
0.5 0