fibrinopeptide-a and hirugen

Clinical trials evaluating direct thrombin inhibitors in unstable coronary artery disease (CAD) have been disappointing. The hypothesis tested in the present study was that these agents may inhibit the anticoagulant effect of thrombin to a further extent than the procoagulant effect of thrombin.. We studied both reversible and irreversible thrombin inhibitors and compared the effects of each inhibitor on activated protein C (APC) generation vs. the effect on fibrinopeptide A (FPA) generation. A mixture of protein C, thrombin inhibitor, fibrinogen, fibrin polymerisation blocker and thrombin was incubated with thrombomodulin (TM)-expressing human saphenous vein endothelial cells (HSVECs). The inhibitors investigated were melagatran, inogatran, hirudin, hirugen, D-Phe-D-Pro-D-arginyl chloromethyl ketone (PPACK), and antithrombin (AT) alone or in combination with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH).. All agents, except hirugen, inhibited APC and FPA generation in a dose-dependent manner. FPA inhibition/APC inhibition ratios, based on IC50 for inogatran, melagatran, hirudin, PPACK, AT, AT-UFH and AT-LMWH were 1.73, 0.85, 0.55, 2.1, 0.5, 0.65 and 3.1 respectively.. All agents, except hirugen, inhibited APC and FPA generation approximately to a similar extent. Thus, it can be inferred that the poor efficacy of thrombin inhibitors in recent clinical trials in patients with unstable CAD is unlikely to be a consequence of their effects on the protein C system.

Topics: Amino Acid Chloromethyl Ketones; Anticoagulants; Azetidines; Benzylamines; Clinical Trials as Topic; Coagulants; Coronary Artery Disease; Fibrinopeptide A; Glycine; Hirudins; Humans; Peptide Fragments; Piperidines; Protein C; Thrombin

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate d-Phe-Pro-Arg-pNA. The K(d) for the glycocalicin-thrombin interaction was 1 microm at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 microm at 100 mm NaCl (Gamma(salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K(d) values for their interactions with GPIb alpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K(d) values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate d-Phe-Pro-Arg-pNA.

Topics: Fibrinopeptide A; Heparin, Low-Molecular-Weight; Hirudins; Humans; Peptide Fragments; Platelet Glycoprotein GPIb-IX Complex; Recombinant Proteins; Sodium Chloride; Thrombin

The Ser195Ala mutant of human alpha-thrombin was complexed with fibrinopeptide A(7-22) (FPA) in an effort to describe the (P1'-P6') post-cleavage binding subsites of the fibrinogen-recognition exosite and define more clearly the nature of the Michaelis complex and the scissile peptide bond bound at the catalytic site. The thrombin mutant, however, has residual catalytic activity and proteolysis occurred at the Arg16-Gly17 bond. Thus, the structure of the thrombin complex determined was that of FPA(7-16) bound at the active site, which is very similar to the ternary FPA(7-16)cmk-human thrombin-hirugen complex (r.m.s.d. approximately 0.4 A; Stubbs et al. , 1992). It is further shown by subsidiary experiments that the cleavage is the result of residual catalytic activity of the altered catalytic machinery.

Topics: Alanine; Amino Acid Substitution; Binding Sites; Crystallography, X-Ray; Fibrinopeptide A; Hirudins; Humans; Models, Molecular; Peptide Fragments; Point Mutation; Protein Conformation; Serine; Software; Thrombin

To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.

Topics: Amino Acid Sequence; Animals; beta-Thromboglobulin; Fibrinolytic Agents; Fibrinopeptide A; Hirudins; Male; Molecular Sequence Data; Papio; Peptide Fragments; Peptides; Platelet Factor 4; Recombinant Proteins; Thrombin

Hirudin and hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(54-65)] abrogate the enzyme activities of alpha-thrombin by binding the enzyme simultaneously at its catalytic centre and fibrin(ogen)-recognition exosite. In contrast, hirugen [hirudin-(54-65)] binds alpha-thrombin solely at the fibrin(ogen)-recognition exosite, and competitively inhibits fibrinopeptide A release. To investigate the extent to which the fibrin(ogen)-recognition exosite is involved when alpha-thrombin catalyses the amplification reactions of coagulation, we compared the abilities of hirudin, hirulog-1 and hirugen to inhibit simultaneously Factor X, Factor V and prothrombin activation. Whereas 0.1 microM-hirudin and 0.1 microM-hirulog-1 (i.e. less than 10% of the concentration of prothrombin in plasma) inhibited Factor X, Factor V and prothrombin activation, 10 microM was the minimum concentration of hirugen to achieve a similar anticoagulant action. Concentrations of hirudin and hirulog-1 equimolar to and 5 times greater than those of alpha-thrombin respectively abrogated Factor V activation by exogenous alpha-thrombin. In contrast, a 500-fold molar excess of hirugen could not. The inability of hirugen to inhibit the activation of the three clotting factors effectively suggests that the fibrin(ogen)-recognition exosite does not play a mandatory role when thrombin activates Factor V.

Topics: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Blood Coagulation; Factor V; Factor X; Fibrinopeptide A; Hirudins; Humans; Molecular Sequence Data; Peptide Fragments; Prothrombin; Rabbits; Recombinant Proteins; Thrombin

Propagation of venous thrombi or rethrombosis after coronary thrombolytic therapy can occur despite heparin administration. To explore potential mechanisms, we set out to determine whether clot-bound thrombin is relatively protected from inhibition by heparin-antithrombin III but susceptible to inactivation by antithrombin III-independent inhibitors. Using plasma fibrinopeptide A (FPA) levels as an index of thrombin activity, we compared the ability of thrombin inhibitors to block FPA release mediated by fluid-phase thrombin with their activity against the clot-bound enzyme. Incubation of thrombin with citrated plasma results in concentration-dependent FPA generation, which reaches a plateau within minutes. In contrast, there is progressive FPA generation when fibrin clots are incubated with citrated plasma. Heparin, hirudin, hirudin dodecapeptide (hirugen), and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK) produce concentration-dependent inhibition of FPA release mediated by fluid-phase thrombin. However, heparin is much less effective at inhibiting thrombin bound to fibrin because a 20-fold higher concentration is necessary to block 70% of the activity of the clot-bound enzyme than is required for equivalent inhibition of fluid-phase thrombin (2.0 and 0.1 U/ml, respectively). In contrast, hirugen and PPACK are equally effective inhibitors of fluid- and solid-phase thrombin, while hirudin is only 50% as effective against the clot-bound enzyme. None of the inhibitors displace bound 125I-labeled thrombin from the clot. These studies indicate that (a) clot-bound thrombin is relatively protected from inhibition by heparin, possibly because the heparin binding site on thrombin is inaccessible when the enzyme is bound to fibrin, and (b) clot-bound thrombin is susceptible to inactivation by antithrombin III-independent inhibitors because the sites of their interaction are not masked by thrombin binding to fibrin. For these reasons, antithrombin III-independent inhibitors may be more effective than heparin in certain clinical settings.

Topics: Amino Acid Chloromethyl Ketones; Antithrombin III; Blood Coagulation; Fibrin; Fibrinopeptide A; Heparin; Hirudins; Humans; In Vitro Techniques; Kinetics; Peptide Fragments; Protease Inhibitors; Protein Binding; Solubility; Thrombin