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The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis. Basic residues in anion-binding exosite-I (Arg35, Arg36, Arg67, Arg73, Arg75, Arg77A, Lys81, Lys109, Lys110 and Lys149E) were substituted with glutamines and the resultant recombinant mutant thrombins were used to determine kinetic parameters for the cleavage of a peptide (PAR38-60) based on the PAR1 extracellular domain. Compared with wild-type thrombin, replacement of Arg67 and Arg73 had a dramatic effect on the cleavage of PAR38-60 (k(cat)/K(m) = 1.8 x 10(6) and 4.6 x 10(6) vs 9.2 x 10(7) M(-1).s(-1)), whereas the remaining mutations of the anion-binding exosite-I of thrombin had a less pronounced effect, with k(cat)/K(m) values ranging from 3.3 x 10(7) M(-1). s(-1) (R77(a)Q) to 5.8 x 10(7) M(-1).s(-1) (K109Q). The ability of thrombin mutants to activate platelets paralleled that of PAR38-60 cleavage, whereas their ability to clot fibrinogen differed profoundly, as did their susceptibility to hirudin inhibition. Results are interpreted with respect to known interactions of thrombin with thrombomodulin, hirudin, rhodniin and heparin cofactor II. We conclude that the basic residues of anion-binding exosite-I contribute significantly to enhancing the rate of complex formation in two ways; the first (general) ensures electrostatic steering of ligands with complementary electrostatic fields, the second (specific) involves a combination of molecular contacts within the complex that is unique for each ligand.

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We investigated the effect on in vitro platelet function of hirutonin, a modified hirutonin with an RGD-like motif, a pseudo-RGDS peptide and a linear RGDS peptide. Inhibition of expression of surface fibrinogen on ADP-activated platelets with 40 microM of the peptide was as follows: hirutonin 10+/-3%, modified chimeric peptide 26+/-5%, pseudo-RGDS 66+/-11% and linear RGDS 93+/-13%. Both hirutonin and the chimeric peptide significantly inhibited ADP-induced platelet activation as detected by CD62 expression. Unlike the RGDS and pseudo-RGDS controls, neither the chimeric peptide nor the parent hirutonin inhibited ADP-induced platelet aggregation even at 140 microM. The chimeric hirutonin peptide reduced ATP release from ADP-stimulated platelets by 40+/-4%. This inhibition was stronger than that caused by hirutonin (23+/-13%), but less than the RGDS (90+/-2%) and pseudo RGDS-peptides (59+/-11%). Primary platelet haemostasis was slightly but not significantly affected by the peptide at 40 and 80 microM. However, shear-induced platelet adhesion to vWF and especially subsequent aggregate formation was interrupted after the addition of the chimeric peptide. Similar results were obtained with hirutonin. This inhibition was not as marked as with the RGDS- and pseudo-RGDS peptides. Both the parent hirutonin and the chimeric peptide caused prolongation of the clinical coagulation assays aPTT and TT. In conclusion, the chimeric hirutonin peptide with introduction of the RGD motif retained its anticoagulant effect but had little formal disintegrin activity. Instead, it appeared to have novel anti-platelet effects that may be of therapeutic use.

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Glycyrrhizin (GL), an anti-inflammatory compound isolated from Glycyrrhiza glabra, was identified as a new thrombin inhibitor: (a) It prolonged plasma recalcification and thrombin and fibrinogen clotting times, and (b) it inhibited thrombin-induced, but not collagen-, PAF- or convulxin-induced platelet aggregation. On the other hand, GL did not block thrombin's amidolytic activity upon S-2238. Furthermore, the fluorescence emission intensity of dansyl-thrombin was increased upon GL binding. Moreover, GL displaced hirudin as an inhibitor of thrombin-catalyzed hydrolysis of S-2238. Our data provide evidence that GL is a selective inhibitor of thrombin (the first one isolated from plants) that is able to exert its anti-thrombin action by interacting with the enzyme's anion binding exosite 1. A pharmacophoric search identified GL as a sialyl Lewis X (SLe[X]) mimetic compound able to inhibit selectin binding to SLe(X). However, SLe(X) did not affect thrombin clotting activities, which indicates a lack of its interaction with thrombin and distinguishes both molecules. It is suggested that the anti-inflammatory effect of GL may be due to its effective anti-thrombin action.

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A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.

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Hirudin is a potent thrombin inhibitor derived from the leech Hirudo medicinalis salivary gland which has considerable potential for therapeutic use in thrombotic disease. The major risk attendant its use is hemorrhage. This study investigates the hypothesis that the prohemostatic effects of DDAVP infusion can curtail the hemorrhagic effect induced by ongoing hirudin administration. In a randomized and blinded manner, rabbits were exposed to a 15-min intravenous infusion of DDAVP or saline midway through a continuous two-h intravenous infusion of hirudin. Bleeding time was monitored by full thickness ear punctures performed before, during and after hirudin exposure. Hirudin induced a significant hemorrhagic state, manifest as a 7-10-fold prolongation of the primary bleeding time. DDAVP reduced the mean duration of primary bleeding from 10.8 min to 5.9 min (p = 0.001) as well as the number of sites which bled for longer than 6 or 20 min (46% vs 27%, p = 0.002; and 18% vs 5%, p = 0.002, respectively). Although there was no difference in the incidence of spontaneous rebleeding from these sites (44 vs 36%, p = 0.21), rebleeding did not persist as long in animals that received DDAVP (8 vs 16 min, p = 0.005), and fewer sites rebled for longer than 20 min (8 vs 27%, p = 0.027). Results were essentially the same for two different commercial recombinant hirudin preparations. DDAVP appears to attenuate the bleeding caused by continuous hirudin infusion in rabbits and establishes a foundation for clinical assessment in patients.

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Meizothrombin, the stable intermediate product of ecarin-induced prothrombin conversion, was investigated for its ability to bind hirudin in blood. After in vitro pre-incubation of rat plasma with ecarin, the prolongation of the thrombin time caused by hirudin was reduced. The extent of hirudin neutralization was found to be dependent on the duration of incubation with ecarin. In vivo, after bilateral nephrectomy in Wistar rats and following administration of hirudin at a dose of 1 or 5 mg/kg, the blood level of hirudin remained constant after 2 h. After infusion of ecarin following hirudin administration, the hirudin blood level dropped sharply, reaching significantly reduced values, and bleeding stopped. Platelet count and fibrinogen level in plasma remained unchanged in the experiments using ecarin-induced prothrombin conversion intermediate generation. It is concluded that meizothrombin, a naturally occurring prothrombin conversion intermediate, provides an effective agent to neutralize toxic blood levels of hirudin.

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The specific thrombin inhibitor r-hirudin (HBW 023) has been demonstrated to be effective in preventing thrombosis in preclinical models. Up to now, no bleeding complications have been observed using therapeutically effective doses in animals studies. However, in case of inadvertent overdosing the occurrence of undesired impairment of coagulation cannot be excluded. As a potential antidote an activated prothrombin complex concentrate (APC) was tested on its ability to normalize blood coagulation. APC given s bolus injections 5 min and 3.0 mg/kg neutralized the r-hirudin-induced prolongation and 3.0 mg/kg neutralized the r-hirudin-induced prolongation of whole blood coagulation time in rabbits completely within 5 min without any clot formation in the blood vessels or capillaries of the heart, kidneys, or lungs. Furthermore, bleeding time prolongation induced by bolus application of 3.0 and 30.0 mg/kg r-hirudin was significantly inhibited by APC within 5 min. These results suggest that administration of APC may be an effective way to reverse the effects of r-hirudin in the coagulation system in case of inadvertent overdosing of r-hirudin.

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The infusion of a high dose of recombinant desulphatohirudin HVI (CGP 39393) for 40 min at 30 micrograms/kg/min, resulted in a prolongation of bleeding time in the rat when evaluated using transection of the tail. The prolonged bleeding was evident both immediately, and 30 min after cessation of the infusion of hirudin (CGP 39393). Bleeding time returned to normal after 60 min. The effect of several agents, reported to be successful in reducing bleeding tendencies in man, were evaluated in this rat model. The agents were administered immediately following cessation of the CGP 39393 infusion and their ability to normalize the prolonged bleeding-time, observed at 30 min after cessation of the CGP 39393 infusion, determined. Desmopressin (DDAVP), recombinant factor VIII and Vueffe reduced the bleeding time to the control range but did not exert any significant effects on the bleeding time in rats which did not receive CGP 39393. Epsilon-aminocaproic acid (EACA) and recombinant factor VII were ineffective, at the doses used. In conclusion, DDAVP, factor VIII and Vueffe are effective in reversing the effect of direct thrombin inhibition on bleeding in the rat.

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In contrast to thrombin the fibrinogen coagulant effect of the thrombin-like enzyme batroxobin in vitro and in vivo is not inhibited by the specific thrombin inhibitor hirudin. The haemostyptic effect of batroxobin has been studied in rats after bleeding had been induced by corresponding hirudin dosages. Dependent on batroxobin concentration bleeding time was shortened by local application of batroxobin containing solutions. Strong bleeding induced by i.v. injection of 5 mg r-hirudin/kg was stopped almost immediately when a batroxobin concentration of 40 BU/ml was used. Thrombin was less active to stop bleeding after r-hirudin administration than batroxobin.

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Thrombomodulin, a cofactor in the thrombin-catalyzed activation of protein C, blocks the procoagulant activities of thrombin such as fibrinogen clotting, Factor V activation, and platelet activation. The binding site for thrombomodulin within human thrombin has been localized at a region comprising residues Thr147-Ser158 of the B-chain of thrombin. The dodecapeptide sequence, TWTANVGKGQPS, corresponding to these residues inhibits thrombin binding to thrombomodulin with an apparent Ki = 94 microM (Suzuki, K., Nishioka, J., and Hayashi, T. (1990) J. Biol. Chem. 265, 13263-13267). We have found that the inhibitory effect of the dodecapeptide on the thrombin-thrombomodulin interaction is sequence-specific, and that residues Asn151, Lys154, and Gln156 are essential for thrombomodulin binding. The dodecapeptide was also found to directly block thrombin procoagulant activities, fibrinogen clotting (concentration for half-maximum inhibition, 385 microM). Factor V activation (concentration for half-maximum inhibition, 33 microM), and platelet activation (concentration for half-maximum inhibition, 645 microM). This peptide did not block thrombin inhibition by antithrombin III, but blocked thrombin inhibition by hirudin. These findings suggest that the binding site for thrombomodulin in thrombin is shared with the sites for fibrinogen, Factor V, platelets, and hirudin, and that, therefore, the inhibition of thrombin procoagulant activities by thrombomodulin in part results from blocking of the interaction between thrombin and the procoagulant protein substrates by thrombomodulin.

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Recombinant hirudin (r-hirudin) is currently under development as an anticoagulant for use in surgery, therapeutic anticoagulation, disseminated intravascular coagulation and other pathologic states involving the generation of thrombin. Circulating levels of r-hirudin as an antithrombotic agent range from 2 to 20 micrograms/ml (0.1-1.0 mg/kg) as determined in an animal model of stasis thrombosis. In order to establish a relationship between the r-hirudin circulating level and bleeding, we utilized a rabbit ear blood loss model. r-Hirudin did not produce any loss of blood at dosages up to 20 micrograms/ml i.v. (1.0 mg/kg). When the circulating levels were maintained at 20 micrograms/ml for periods of up to 3 h, no increase in blood loss was observed. At 50 and 100 micrograms/ml initial circulating levels (2.5 and 5.0 mg/kg) a dose-dependent increase in the blood loss was observed which was equivalent to that observed with 1.25 and 2.5 mg/kg i.v. heparin. Such levels of r-hirudin are not expected in clinical usage. In contrast to heparin, the anticoagulant actions of r-hirudin were not neutralized by protamine sulfate, platelet factor 4, other polycationic agents and heparinase. In our studies, the blood loss induced by greater than 2.0 mg/kg i.v. dosages of r-hirudin in an animal model was neutralized by the administration of an activated prothrombin complex concentrate at 25 U/kg. In a similar experimental setting, r-factor VIIa was also partially effective. These studies suggest that r-hirudin anticoagulation may not require neutralization, since bleeding effects are not observed at effective antithrombotic dosages in individuals with normal hemostatic status.(ABSTRACT TRUNCATED AT 250 WORDS)

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Hirudin is a specific, potent inhibitor of thrombin that may be a valuable antithrombotic agent. The aim of this study was to investigate the hypothesis that the haemostatic effects of DDAVP counteract the coagulation defect induced by hirudin. The effect of DDAVP was studied in vivo on the anticoagulant action of recombinant hirudin (CGP39393) in vitro. Blood samples were taken at intervals from 10 normal volunteers infused with DDAVP. Factor VIII:C rose from (mean) 0.68 IU/ml before DDAVP to 2.19 and 2.16 IU/ml after 30 and 60 min infusion, respectively. Samples taken during DDAVP infusion showed a dose related decrease in the hirudin (0.5 and 1.0 microM) induced prolongation of the APTT, that occurred at FVIII:C concentrations of up to twice normal. At higher concentrations of hirudin no effect on the APTT occurred. These results demonstrate that DDAVP infusion elevates factor VIII:C levels with an associated significant reduction in the anticoagulant effect of hirudin in vitro.

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Recombinant hirudin (hirudin), which lacks the sulphate group on Tyr-63, has a tenfold-reduced affinity for alpha-thrombin. Incubation of recombinant hirudin with [gamma-32P]ATP and protein tyrosine kinase III from spleen resulted in incorporation of radioactivity into the protein. Phosphatohirudin was purified to homogeneity (overall yield 5%) and shown to contain 1 mol phosphate/mol protein, as a phosphotyrosyl residue at position 63. The kinetics of the inhibition of human alpha-thrombin by phosphatohirudin were determined. It was found that the introduction of the negatively charged phosphate had fully restored the affinity of recombinant hirudin for alpha-thrombin to the level of the wild-type sulphatohirudin. The inhibition constant of phosphatohirudin was 18 fM compared with 20 fM for that of sulphatohirudin. Moreover, the values for the on- and off-rate constants of both forms of hirudin were indistinguishable.

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In the presence of epsilon aminocaproic acid (EACA) thrombin generation in recalcified platelet rich plasma (PRP) was markedly stimulated, as measured by the cleavage of the synthetic substrate S2238. However, thrombin activity remaining after 30 minutes incubation was reduced when compared with control values. The residual activity was shown to be hirudin insensitive and to be associated with a species of higher molecular weight than free thrombin. These results suggested an inhibition of thrombin binding to the antithrombin, alpha 2-macroglobulin (alpha 2M). Preincubation of PRP with EACA reduced the concentration at which EACA elicited its dual effects. Similar results were obtained with the alpha 2M inhibitor, hydrazine. These experiments indicated that alpha 2M may play a more important role in regulating thrombin generation than has been previously recognized.

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A five-step isolation procedure has been developed for the purification of isoforms of hirudin (isohirudins) from whole leeches. The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isohirudins with either N-terminal valine or isoleucine but with identical inhibition characteristics, i.e. specific thrombin inhibiting activities of 680-720 IU/mg and dissociation constants Ki of the thrombin-inhibitor complexes close to 3 X 10(-11) mol/l. The inhibitor with N-terminal isoleucine was designated hirudin PA. This inhibitor contains 66 amino-acid residues and has a molecular mass of 7,087 Da. The complete amino-acid sequence of hirudin PA was established by automated solid-phase Edman degradation of the native and oxidized inhibitor and two of its tryptic fragments. On the basis of the primary structures two types of thrombin inhibitors from the leech can be distinguished, designated hirudin and hirudin PA. The degree of structural homology of both isoinhibitors is approximately 82%; both have a tyrosine-O-sulfate residue near the C-terminus.

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High antithrombin activity was maintained in the salivary gland secretion of starved medicinal leeches (Hirudo medicinalis) despite harvesting of the secretion twice in a month. However, after third or fourth harvesting every month antithrombin activity was no longer detectable. Moreover, in salivary gland secretion high antitrypsin activity was found but no antichymotrypsin activity. On the other hand, intestinal chyme of starved leeches exhibited high antichymotrypsin activity. Low antithrombin and antitrypsin activities were due to contamination by salivary gland secretion. There was a correlation between antichymotrypsin and antithrombin activities of hirudin preparations purified from bdellins and fractionated by isoelectric focusing. Pseudohirudin preparations exhibited neither antithrombin nor antichymotrypsin activities.

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