The complete nucleotide sequence of the gene for batroxobin, a thrombin-like snake venom enzyme.
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Amino Acid Sequence↗
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Delayed, reduced or inhibited thrombin production reduces platelet contractile force and results in weaker clot formation.
Clot retraction is a thrombin-dependent, platelet-mediated contraction of the cellular clot mass. In this study, the effects of delayed, deficient and inhibited thrombin generation on the development of platelet contractile force (PCF) and clot elastic modulus (CEM) were measured. When normal citrated whole blood is clotted by the addition of exogenous thrombin (1 U/ml) and calcium (10 mmol/l), PCF and CEM start to develop within the first minute and begin to level off by 1200 s. If identical samples are clotted with batroxobin (0.21 microg/ml) and calcium (10 mmol/l), both PCF and CEM development are delayed approximately 5 min. After 1200 s of clotting, however, values in the batroxobin system approach those seen with exogenous thrombin. If the added calcium concentration is held constant at 10 mmol/l, increasing the exogenous thrombin concentration from 0 to 0.5 U/ml results in increased PCF and CEM values. Above 0.5 U thrombin, the effect plateaus. At exogenous calcium of 10 mmol/l, increasing batroxobin concentrations (0-0.210 microg/ml) caused a 75% increase in PCF and a 55% increase in CEM. The increase in CEM reached a plateau above 0.05 microg batroxobin/ml. The effects of varying calcium concentrations were evaluated at constant batroxobin (0.21 microg/ml) and thrombin (1 U/ml) concentrations. With thrombin, PCF and CEM increased by > 700% as CaCl2 increased from 0 to 5 mmol/l. Above 5 mmol/l, no additional increases occurred. With batroxobin, PCF did not develop at CaCl2 concentrations < or = 2.5 mmol/l. Above 2.5 mmol/l CaCl2, PCF values increased and at 10 mmol/l CaCl2 were equal to those seen with thrombin. CEM in batroxobin-mediated clots peaked at 10 mmol/l CaCl2 but were 40% less than the values found in thrombin-mediated clots. When the thrombin inhibitor P-PACK was added to the batroxobin system, dose-dependent decreases in PCF and CEM were noted. At 120 micromol/l, P-PACK totally suppressed PCF. PCF in blood from a factor VIII-deficient patient varied significantly when clotted with batroxobin versus thrombin. PCF development in factor VIII-deficient blood was normal with thrombin but is delayed and depressed with batroxobin. PCF values in factor VIII-deficient blood did not reach the thrombin value after 1200 s of clotting, and CEM was significantly less. These results confirm that PCF development is thrombin dependent and that delay or reduction of PCF development results in structurally weaker clots.
Adult↗
Suppression of cell-transferred experimental autoimmune encephalomyelitis in defibrinated Lewis rats.
The role of coagulation-fibrinolysis system in experimental autoimmune encephalomyelitis (EAE) was studied by using batroxobin, derived from the venom of the South American pit viper Bothrops atrox moojeni. Batroxobin converts circulating fibrinogen into an insoluble form and causes a profound degree of afibrinogenemia. Batroxobin treatment (30 BU/kg/day) suppressed clinical signs of cell transferred EAE; the mean cumulative clinical score for batroxobin treated rats was 3.97, while saline treated controls scored 6.9 (P < 0.01). Plasma fibrinogen concentration decreased significantly in batroxobin-treated rats. Histologically, the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in batroxobin-treated rats compared to saline-treated control rats, however, deposition of fibrin around the vessels in the spinal cord was markedly suppressed in batroxobin-treated rats. These findings suggest that batroxobin suppresses EAE by preventing fibrin deposition, and provide evidence that CNS-associated deposition of fibrin and ensuing fibrinolysis, together with increased permeability of blood brain barrier (BBB), are related prerequisites for the clinical manifestation of EAE.
Animals↗
Defibrinogenating enzymes.
The venoms from 3 snakes have been shown to induce defibrinogenation: ancrod from the venom of Calloselasma rhodostoma (formerly known as Agkistrodon rhodostoma), batroxobin from the venom of Bothrops atrox moojeni, and crotalase from the venom of Crotalus adamanteus. The purified fractions of ancrod, batroxobin, and crotalase possess coagulant, proteolytic and esterolytic properties, although their primary mechanism of action is a proteolytic effect on circulating fibrinogen. Ancrod cleaves only the A-fibrinopeptides, but not the B-fibrinopeptides, from fibrinogen; this contrasts with thrombin, batroxobin and crotalase, which cleave both fibrinopeptides A and B. Within minutes of administration of ancrod or batroxobin, there is a significant reduction in plasma fibrinogen levels, and these remain exceedingly low with repeated administration (once or twice daily). The rapid fall in plasma fibrinogen levels is accompanied by a slightly delayed but marked rise in the level of fibrinogen-fibrin degradation products. Plasminogen levels are decreased and blood viscosity is reduced, but formed elements in the circulating blood remain unaltered. Ancrod and batroxobin have been investigated in patients with stroke, deep-vein thrombosis, myocardial infarction, peripheral arterial thrombosis, priapism, and sickle-cell crisis; crotalase has not been administered to humans. However, results have been difficult to interpret, and additional well designed trials are needed to better define the optimum role of ancrod and batroxobin in the management of these conditions. Overall, treatment is well tolerated and serious adverse events are infrequent. In the coagulation laboratory, ancrod, batroxobin and crotalase may be used as reagents to perform coagulation studies on specimens of blood that contain heparin. These venom fractions can be substituted for thrombin in performing the thrombin time and in removing fibrinogen from plasma for accurate determination of fibrinogen-fibrin degradation products.
Ancrod↗
[Relationship between the content of fibrinogen in middle ear effusion and the effect of treatment on secretory otitis media].
OBJECTIVE: To study the relationship between the fibrinogen content in middle ear effusion (MEE) and the protracted inflammation of secretory otitis media (SOM) and to explore the possible mechanism of batroxobin in treatment of SOM. METHODS: The fibrinogen content of middle ear effusion from 156 patients with SOM was investigated with concretion technique at different stages. After two times punctuation of tympanum, the recurrence patients were randomly divided into two groups: batroxobin and dexamethasone group, and 0.5 ml (2 BU/ml) batroxobin or dexamethasone (2 mg/ml) was injected into middle ear cavity. The therapeutic effects were investigated. RESULTS: The concentration of fibrinogen in the recurrence group of SOM patients was higher than that in the cured group, and even higher in the second recurrence group than in the first recurrence group (P < 0.01). There was significantly different (P < 0.001) in the therapeutic efficacy between the batroxobin group (91.6%) and the dexamethasone group (62. 5%); the difference of the fibrinogen content in MEE and air conduction of pure tone audiometry at frequencies (0.5, 1.0, 2.0 kHz) between the two groups after treatment were also significant (P < 0.01). CONCLUSIONS: Fibrinogen may play a significant role in the occurrence and development of secretory otitis media. Batroxobin had better therapeutic effect on SOM than dexamethasone. The mechanism of batroxobin in the treatment of SOM may be that the batroxobin can relief the depressant effect of fibrinogen on surface active agents of the Eustachian tube and prevent the fibrinogen from turning into insoluble fibrin polymer by means of fibrinolysis.
Adolescent↗
Effects of venom defibrase on brain edema after intracerebral hemorrhage in rats.
We evaluated the effects of defibrase DF-521 batroxobin on reducing brain edema formation and the expression of ICAM-1, complement C3d and C9 in the perihematomal area after intracerebral hemorrhage (ICH) in rats. A rat ICH model, involving infusion of autologous blood into the right basal ganglia, were used in this study. The animals were sacrificed at 24 and 72 hours after ICH to determine the water content of the brain tissue with wet/dry weight measurement. While the expression of ICAM-1 and complement C3d was detected using immuno-histochemistry, and C9 was detected semi-quantitatively with Western blot analysis in the perihematomal area. Perihematomal brain edema was reduced after intraperitoneally injection of DF-521 batroxobin 24 and 72 hours after intracerebral hemorrhage. Immunohistochemistry showed that there were less ICAM-I positive cells were found around the hematoma after intraperitoneally injection of DF-521 batroxobin 24 and 72 hours after ICH. Immuno-histochemistry also showed that C3d deposition reduced significantly, and the Western blot analysis also showed the content of C9 protein declined around the hematoma in DF-521 batroxobin treatment group at 72 hours after ICH. Defibrase DF-521 batroxobin down-regulate ICAM-1 and complement C3d and C9 expression in the perihematomal area, and attenuate brain edema formation in ICH rats.
Animals↗
New model for in vivo studies of pharmacological interventions with endogenous fibrinolysis: effects of thrombin inhibitors.
A new rat model for investigation of the interactions of thrombin inhibitors with endogenous fibrinolysis in vivo is described. The method utilizes the thrombin-like snake enzyme batroxobin, which mainly cleaves the fibrinopeptide A from fibrinogen and activates factor XIII only to a slight degree. Compared to thrombin-formed fibrin, batroxobin-formed fibrin is more readily lysed by plasmin, since it only cross-links fibrin to a minor extent. Radiolabeled fibrinogen (125I) was given intravenously to monitor the effects of batroxobin on fibrinogen and the effects of plasmin on the fibrin formed. Batroxobin was given intravenously in a dose that converted most fibrinogen to fibrin. Five to 10 minutes after batroxobin administration, 125I-activity in the blood decreased, indicating the disappearance of fibrinogen from the circulating blood. At the same time, the 125I activity as measured with a gamma counter increased over the lungs. The fibrin formed in the microvasculature of the lungs started an endogenous fibrinolysis. This could be seen as reappearance of the 125I activity in the blood from fibrin degradation products accompanied by a decrease in the 125I activity over the lungs. When the rats were given tranexamic acid, the endogenous fibrinolysis was markedly decreased, measured as 125I activity in the blood, over the lungs and in lung parenchyma samples. The thrombin inhibitor DuP 714, which apparently inhibits not only thrombin but also plasmin and tissue plasminogen activator, completely prevented the fibrinolytic phase. The selective thrombin inhibitor argatroban, on the other hand, led to shortened lysis time. It is concluded that the present method provides a convenient and accurate means of studying pharmacological interventions with endogenous fibrinolysis. The differential effects of selective and nonselective thrombin inhibitors on endogenous fibrinolysis at comparable levels of thrombin inhibition are clearly demonstrated.
Animals↗
Fragmentation of actin by thrombin-like snake venom proteases.
The effect of thrombin-like snake venom proteases (Ancrod of Agkistrodon rhodostoma and Batroxobins of Bothrops moojeni and Bothrops marajoensis) on skeletal muscle actin was studied and compared to the thrombic cleavage of this protein. Only EDTA-pretreated G- and F-actin were split by thrombin and Ancrod, while Batroxobins hydrolyzed native G-actin, too. The time course of digestion was followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A split product of 37 500 daltons appeared first which was cleaved further resulting in three lower molecular weight fragments. The sodium dodecyl sulfate gel pattern of thrombic fragmentation was well distinguishable from those caused by Ancrod and Batroxobins. The first split products of Batroxobin digestion--a smaller peptide and the 37 500 dalton fragment--were isolated and by estimating their N-, and C-terminal end groups and amino acid compositions the peptide bond hydrolyzed first was located in the primary structure of actin. It was established that while thrombin split off two actino-peptides (at Arg(28)-Ala(29) and Arg(39)-His(40) from the N-terminal end of the molecule only Arg(39)-His(40) was cleaved by Batroxobins.
Actins↗
Thrombin-like snake venom proteinases.
Proteinases affecting one or several physiological thrombin substrates are current components of Crotalidae and Viperidae venoms. Enzymes causing in vitro coagulation of fibrinogen without affecting other thrombin-susceptible blood constituents as well as enzymes affecting platelets, F. V. VIII and XIII with only minor action on fibrinogen have been isolated. Fibrinogen affecting proteinases may catalyze the release of either fibrinopeptide (Fp) A (e.g. ancrod, batroxobin) or Fp B (Agk, contortrix proteinase) or of both Fp A and B (B. gabonica proteinase). Some of these enzymes are inhibited by AT III-heparin complex (e.g. Agk. contortrix proteinase) some are not inhibited by either AT III-heparin or hirudin (e. g. batroxobin). The application of Fp A releasing venom proteinases into animals causes transformation of fibrinogen into fibrin I monomer which is rapidly degraded by fibrinolysis and thereby leads to a state of afibrinogenaemia. The administered enzyme is gradually bound to serum proteinase-inhibitors and inactivated. A species dependent interaction between venom enzyme, fibrinogen and serum proteinase inhibitors creates specific differences in dose response relationship. Thus, batroxobin isolated from B. moojeni (HOGE) proved to be a superior defibrinogenating agent in man, as compared to the closely related enzyme isolated from B. atrox (L.). LD50 of B. atrox venom in previously batroxobin defibrinogenated mice is not significantly different as compared to normal animals, indicating an only minor role of batroxobin in Bothrops venom poisoning.
Aging↗
Fibrin deposition in the central nervous system correlates with the degree of Theiler's murine encephalomyelitis virus-induced demyelinating disease.
We examined the role of coagulation-fibrinolysis system in Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). The degree of fibrin deposition around the vessels in the spinal cord was significantly higher in susceptible SJL/J mice on 30 days post intracerebral injection (i.c.) than resistant C57BL/6 mice on 30 days post i.c. or mock infected SJL/J mice. Treatment with batroxobin (30 BU/kg/day), which is a thrombin-like defibrinogenating enzyme, causing a profound degree of afibrinogenemia, suppressed clinical signs of TMEV-IDD. Plasma fibrinogen concentration was significantly decreased in batroxobin-treated mice. Histologically, though the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in batroxobin-treated mice compared to saline-treated control mice, fibrin deposition was markedly suppressed in batroxobin-treated mice. These findings suggest that batroxobin suppresses TMEV-IDD through its defibrination effect, and provide evidence that CNS-associated deposition of fibrin and ensuing fibrinolysis, together with increased permeability of the blood-brain barrier (BBB), are prerequisite events for clinical manifestations of TMEV-IDD.
Animals↗
The contribution of residues 192 and 193 to the specificity of snake venom serine proteinases.
Snake venom serine proteinases, which belong to the subfamily of trypsin-like serine proteinases, exhibit a high degree of sequence identity (60-66%). Their stringent macromolecular substrate specificity contrasts with that of the less specific enzyme trypsin. One of them, the plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA), which shares 63% sequence identity with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom, specifically activates plasminogen to plasmin like tissue-type plasminogen activator (t-PA), even though it exhibits only 23% sequence identity with t-PA. This study shows that TSV-PA, t-PA, and batroxobin are quite different in their specificity toward small chromogenic substrates, TSV-PA being less selective than t-PA, and batroxobin not being efficient at all. The specificity of TSV-PA, with respect to t-PA and batroxobin, was investigated further by site-directed mutagenesis in the 189-195 segment, which forms the basement of the S(1) pocket of TSV-PA and presents a His at position 192 and a unique Phe at position 193. This study demonstrates that Phe(193) plays a more significant role than His(192) in determining substrate specificity and inhibition resistance. Interestingly, the TSV-PA variant F193G possesses a 8-9-fold increased activity for plasminogen and becomes sensitive to bovine pancreatic trypsin inhibitor.
Amino Acid Sequence↗
A novel method to assess platelet inhibition by eptifibatide with thrombelastograph.
We examined a novel method to detect platelet inhibition with thrombelastography (TEG). We hypothesized that this method would be suitable for monitoring the antiplatelet effects of eptifibatide (Integrilin). Whole blood from healthy volunteers was anticoagulated with 3.2% citrate or unfractionated heparin (7 IU/mL). For the platelet aggregation test, both citrate and heparinized samples were spiked with increasing concentrations of eptifibatide (0, 0.2, 0.4, 0.8, 1.6, and 4 microg/mL). Conventional kaolin TEG was performed with citrated samples, and batroxobin-modified TEG was performed with heparinized samples, which were spiked with eptifibatide at concentrations of 0, 0.4, 0.8, 1.6, 4, 8, and 24 microg/mL. Adenosine 5'-diphosphate-induced platelet aggregation was reduced to 6.4% +/- 2.9% (citrate) and 10.3% +/- 4.8% (heparin) with eptifibatide at the concentration of 4 mug/mL. The kaolin TEG showed a decrease in maximum amplitude (MA) only at the eptifibatide concentration of 24 mug/mL and no change in alpha angle, whereas with the batroxobin-based TEG, the difference in MA and alpha angle was observed at concentrations >/=0.8 microg/mL. Additionally, the time to achieve maximum MA was much shorter for batroxobin TEG than for kaolin TEG. We conclude that the batroxobin-modified TEG is a sensitive method that detects platelet inhibition induced by eptifibatide.
Adenosine Diphosphate↗
Defibrinogating therapy for peripheral circulatory disturbance in patients with vibration syndrome.
This study examines the effects of batroxobin (a defibrinogating drug) on the peripheral circulatory disturbance of 118 patients with vibration syndrome. In 53 cases, batroxobin was intravenously administered once a day for three successive days in the order of 20, 10, and 5 batroxobin units (BU), and 5 or 10 BU were supplemented every day or every second day, respectively, for four weeks. In these cases, the blood fibrinogen and viscosity decreased. A significant decrement in the vascular resistance of the fingers also occurred after each administration. The other 65 cases were subjected to an intravenous administration of 20 BU initially and an additional 10 BU every 2 d for four weeks. For all of the 118 cases the results of skin temperature measurement, finger plethysmography, and nail compression in conjunction with cold provocation showed statistically significant improvement after the therapy. The transcutaneous partial pressure of oxygen and finger blood flow measured in 22 and 27 cases, respectively, were also significantly improved. A sensation of warmth in the whole body appeared in 70.3% of the 118 cases after the therapy. The results suggest that defibrinogation by batroxobin is useful for improving the peripheral circulatory disturbance of the vibration syndrome.
Batroxobin↗
Comparison of the platelet aggregation induced by three thrombin-like enzymes of snake venoms and thrombin.
Platelet aggregation induced by three thrombin-like enzymes of snake venoms was compared with that by thrombin. Acutin was isolated from Agkistrodon acutus venom and thrombocytin and batroxobin were from Bothrops atrox venom. The fibrinogen-clotting activities were 700, 170 and 7 U/mg for batroxobin, acutin and thrombocytin, respectively. They induced aggregation and ATP release of washed rabbit platelets. The aggregating activity of thrombin was 10(2), 10(4) and 10(5) times more potent than those of thrombocytin, acutin and batroxobin, respectively. Platelet-activating potency of the thrombin-like enzymes was correlated with their effectiveness on the retractility and elasticity of the clots. Platelet aggregation induced by thrombin or thrombocytin could be inhibited by heparin with antithrombin III while that by acutin or batroxobin could not. Indomethacin showed weak inhibition on the aggregation while the ADP-scavenging system, creatine phosphate/creatine phosphokinase, inhibited the aggregation induced by the three thrombin-like enzymes but not that by thrombin. Platelet aggregation induced by the thrombin-like enzymes could not be inhibited by PAF antagonists-BN 52021, kadsurenone or L-652,731. In the presence of EGTA, only thrombin could induce ATP release from platelets. Thrombin-like enzymes and low concentration of thrombin did not form thromboxane B2. Nitroprusside and prostaglandin E1 completely inhibited the aggregation, mepacrine and imipramine showed marked inhibition while verapamil had only weak inhibition. It is concluded that the aggregation induced by the thrombin-like enzymes is different from that of thrombin and mainly due to ADP released from platelets.
Adenosine Triphosphate↗
[Thrombolytic agents].
The clinical efficacy of thrombolytic agents, urokinase, lysyl-plasminogen and batroxobin, was evaluated on the patients with deep venous thrombosis. In the group treated with combination of batroxobin, lysyl-plasminogen and urokinase, significantly better efficacy was obtained over the urokinase group in terms of judgement on venography and clinical improvement. Plasma fibrinogen, plasminogen and alpha 2-plasmin inhibitor were markedly reduced by batroxobin infusion. Fibrinolytic activity measured by plasminogen-free fibrin plate method was detected in 89% of blood samples taken after 120,000 I.U. urokinase infusion following lysylplasminogen administration. These results suggested that even moderate dosage of urokinase could induce fibrinolytic activity under conditions of low alpha 2-plasmin inhibitor and fibrinogen levels caused by batroxobin and additional lysylplasminogen.
Batroxobin↗
Fibrinogen Magdeburg I: a novel variant of human fibrinogen with an amino acid exchange in the fibrinopeptide A (Aalpha 9, Leu-->Pro).
INTRODUCTION: The exchange of Aalpha 16, Arg for Cys or His is the most common molecular defect in dysfibrinogenemia directly affecting the thrombin cleavage site involved in fibrinopeptide A (FPA) release. Other amino acid exchanges within the fibrinopeptide A have been only rarely reported. MATERIALS AND METHODS: In clinically asymptomatic dysfibrinogenemic patients with low functional plasma fibrinogen (Fg) levels and prolonged thrombin time but normal or slightly prolonged batroxobin (reptilase) time, mutation analysis was carried out by direct sequencing of the coding regions of the three fibrinogen genes. Isolated fibrinogen was functionally characterized for thrombin- or batroxobin-induced fibrinopeptide release and fibrin formation. Fibrinogen and fibrinopeptides were structurally studied by electrophoretic techniques or high-performance liquid chromatography. RESULTS AND CONCLUSIONS: Molecular analysis revealed heterozygosity for a novel missense mutation T1182C in the FGA gene causing the amino acid exchange Aalpha 9, Leu-->Pro. Fibrin generation induced by thrombin was moderately impaired, whereas batroxobin-induced fibrin formation was almost normal. Release of the abnormal fibrinopeptide A by thrombin was delayed but fibrin monomer aggregation was almost normal. Cleavage of Aalpha chains by batroxobin was only slightly delayed. Fibrinopeptides A of the patient fibrinogen did not show any gross abnormality in chromatographic behaviour. This new molecular variant designated fibrinogen Magdeburg I supports the view that amino acid residue Leu-9 in the Aalpha chain as part of a small hydrophobic cluster is involved in the interaction with an apolar binding site of thrombin, thus adding to our understanding of the thrombin-fibrinogen interaction crucial in coagulation.
Child, Preschool↗
Adjunctive Therapy with an Antithrombotic Drug Can Prevent Reocclusion and Induce Residual Thrombus Reduction After Percutaneous Transcatheter Angioplasty of the Thrombotic Lesions.
Acute reocclusion after successful angioplasty is a severe complication. The preventive effects of heparin, the synthetic antithrombin, argtroban, and the defibrinogenating agent batroxobin on reocclusion after balloon angioplasty of thrombotic occlusions were evaluated in canine iliac arteries. After the 2-hour-old thrombus was evaluated by angiography and angioscopy, percutaneous transcatheter angioplasty (PTA) was performed on the thrombotic stenosis. We used one of the three agents, heparin (100 U/kg), the antithrombin argatroban (0.3 mg/kg), or the defibrinogenating agent batroxobin (0.3 U/kg). Then angioscopy and angiography were performed before, just after, and 2 hours after PTA. After PTA, angiography revealed a marked reduction in percent stenosis in all groups (from 88 +/- 8% to 24 +/- 4% in the heparin group, from 79 +/- 7% to 26 + 11% in the argatroban group and from 89 +/- 12% to 32 +/- 7% in the batroxobin group). At 2 hours after PTA, angiography demonstrated a greater reduction in percent stenosis with argatroban (from 26 +/- 11% to 9 +/- 3%) and batroxobin (from 32 +/- 7% to 10 +/- 8%), and maintenance of percent stenosis reduced by PTA with heparin (from 24 +/- 5% to 28 +/- 9%) when compared with the significant reversal of percent obstruction in the control side. Angioscopic visualization also demonstrated a similar trend. These results show that these antithrombotic drugs have a preventive effect on reocclusion after balloon angioplasty for thrombotic obstruction.
Journal Article↗
Binding of alpha-thrombin to fibrin depends on the quality of the fibrin network.
Binding of human alpha-thrombin to fibrin was studied in a purified system at pH 7.35, I 0.08 and 37 degrees C. Binding experiments with active thrombin resulted in fibrin clots of variable quality, depending on the thrombin concentration: opaque gels composed of 'coarse' network were produced at low thrombin concentrations, while increasing concentrations of thrombin led to more translucent 'fine' gels. Scatchard analysis showed a non-linear dependence of thrombin binding to fibrin, suggesting the existence in fibrin(ogen) of multiple classes of binding sites for thrombin. Binding of catalytic-site-inhibited thrombin was investigated in clots of defined quality produced with three different concentrations of a thrombin-like enzyme, batroxobin (EC 3.4.21.29). Straight lines of different slopes were established by Scatchard analysis of binding data at each fixed batroxobin concentration. These results favour a model according to which binding affinity for thrombin depends on the thickness of fibrin bundles. Labelled active-site-inactivated thrombin incorporated in batroxobin-induced clots was only sparingly released during incubation for 24 h in the presence of a 200-fold excess of unlabelled thrombin, indicating that thrombin binding to fibrin is not reversible and that Scatchard analysis is not appropriate for quantification of binding parameters. Irreversible binding of thrombin appears to reflect trapping of thrombin molecules within fibrin fibres. The amount of trapped thrombin depends on the quality of the fibrin fibres, which in turn is determined by the concentration of the clotting enzyme.
Amino Acid Chloromethyl Ketones↗