Fibrinolytic compromise by simultaneous administration of site-directed inhibitors of thrombin

doi:10.1016/0049-3848(94)90108-2

Thrombosis Research

Volume 74, Issue 3, 1 May 1994, Pages 193-205

https://doi.org/10.1016/0049-3848(94)90108-2 Get rights and content

Abstract

Newly developed synthetic and recombinant thrombin inhibitors possess strong anticoagulant effects. Despite these effects, interactions of these agents with enzymes in the fibrinolytic network result in the modulation of such proteases as t-PA, u-PA and streptokinase. The inhibitory spectrum of several thrombin inhibitors [D-Phe-Pro-Arg-H (GYKI 14166), D-MePhe-Pro-Arg-H (GYKI 14766), Boc-D-Phe-Pro-Arg-H (GYKI 14451), Ac-D-Phe-Pro-boroArg-OH (DuP 714), recombinant hirudin (r-Hir) and unfractionated porcine mucosal heparin complexed with antithrombin III (Heparin/AT-III)] was studied towards various serine proteases such as tissue plasminogen activator (t-PA), plasmin, plasminogen/streptokinase complex, urokinase and kallikrein. Aprotinin was also studied in the same systems as the thrombin inhibitors. All four tripeptide derivatives were found to inhibit t-PA, plasmin and plasminogen/streptokinase complex at micromolar concentrations (IC₅₀: 0.57 mM - 3.3 μM). Boc-D-Phe-Pro-Arg-H and Ac-D-Phe-Pro-boroArg-OH also inhibited urokinase, while Ac-D-Phe-Pro-boroArg-OH inhibited kallikrein as well (IC₅₀: 0.15 mM - 16 μM). In contrast, r-Hir and Heparin/AT-III did not inhibit any of these enzymes at millimolar concentrations (IC₅₀≥ 1 mM). Aprotinin inhibited plasmin, plasminogen/streptokinase complex and kallikrein at micromolar concentrations (IC₅₀: 3.1-0.85 μM). In a rabbit thrombolysis model, where pre-formed clots are lysed by streptokinase, simultaneous administration of D-MePhe-Pro-Arg-H or Ac-D-Phe-ProboroArg-OH, at concentrations ∼ 1 μmol/kg, I.V. resulted in complete inhibition of the fibrinolytic process. Aprotinin at 0.1 μmol/kg, I.V. produced similar inhibition. These results demonstrate that thrombin inhibitors may exert significant antiprotease actions against various fibrinolytic enzymes.

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Cited by (22)

A Mathematical Model of Bivalent Binding Suggests Physical Trapping of Thrombin within Fibrin Fibers
2019, Biophysical Journal
Citation Excerpt :
Because multiple studies have shown allosteric linkage between thrombin exosites and between the exosites and the active site (5,20,41,42), our SSB model could be used to simulate these scenarios and further probe the thrombin binding dynamics. Our model could also be used to study thrombin-specific aptamers (43), thrombin inhibitors sensitive to the enzymes of fibrinolysis enzymes (44,45), and bivalent direct thrombin inhibitors, such as hirudin and bivalirudin (46). To our knowledge, mathematical modeling tools have not yet been applied to these situations.
Thrombin is an enzyme that plays many important roles in the blood clotting process; it activates platelets, cleaves coagulation proteins within feedback loops, and cleaves fibrinogen into fibrin, which polymerizes into fibers to form a stabilizing gel matrix in and around growing clots. Thrombin also binds to the formed fibrin matrix, but this interaction is not well understood. Thrombin-fibrin binding is often described as two independent, single-step binding events, one high-affinity and one low-affinity. However, kinetic schemes describing these single-step binding events do not explain experimentally-observed residency times of fibrin-bound thrombin. In this work, we study a bivalent, sequential-step binding scheme as an alternative to the high-affinity event and, in addition to the low-affinity one. We developed mathematical models for the single- and sequential-step schemes consisting of reaction-diffusion equations to compare to each other and to experimental data. We then used Bayesian inference, in the form of Markov chain Monte Carlo, to learn model parameter distributions from previously published experimental data. For the model to best fit the data, we made an additional assumption that thrombin was irreversibly sequestered; we hypothesized that this could be due to thrombin becoming physically trapped within fibrin fibers as they formed. We further estimated that ∼30% of thrombin in the experiments to which we compare our model output became physically trapped. The notion of physically trapped thrombin may provide new insights into conflicting observations regarding the speed of fibrinolysis. Finally, we show that our new model can be used to further probe scenarios dealing with thrombin allostery.
In vitro and in vivo properties of bicyclic lactam inhibitors: A novel class of low molecular weight peptidomimetic thrombin inhibitors
2000, Thrombosis Research
We have developed potent and selective thrombin inhibitors with a novel non-peptidic structure. A bicyclic lactam was used as the scaffold on which various P1 and P3 motifs were substituted. Herein, we report the in vitro and in vivo properties of four representatives of this novel class of inhibitors. Their Ki values were less than 10 nM, they inhibited equally both free and clot-bound thrombin, and they displayed high level of specificity for thrombin over other serine proteases (trypsin, factor Xa, activated Protein C, and plasmin). They prolonged the clotting time of human plasma to twice the control value in coagulation assays (TT, APTT, and PT) at a concentration below 3 μM. Their anticoagulant activities using rat plasma were similar to, although slightly weaker, than with human plasma. Furthermore, they inhibited thrombin-induced platelet aggregation (human and rat) at concentrations close to their Ki values for thrombin. These molecules demonstrated similar dose response antithrombotic efficacy in rat arterial and venous thrombosis models when given as i.v. bolus followed by infusion. Antithrombotic efficacy of 85% and greater was observed at a dose of 5–7 μM/kg/hour in each model. Bicyclic lactam inhibitor 3, at a dose which caused a complete inhibition of visible thrombus formation in the venous and arterial models of thrombosis, showed a 1.9–2.1 and a 4.0–4.8-fold shift in APTT and TT, respectively. Unfortunately, the bicyclic lactam inhibitors exhibited low oral bioavailability in rats. Therefore, this novel class of bicyclic lactam thrombin inhibitor has the potential to be promising intravenous antithrombotic agents for the treatment of arterial as well as venous thrombosis and warrants further investigation.
Synthetic inhibitors of thrombin and factor Xa: From bench to bedside
1999, Thrombosis Research
The platelet thrombin receptor and postoperative bleeding
1998, Annals of Thoracic Surgery
Background. We hypothesized that small amounts of thrombin desensitize the platelet thrombin receptor during cardiopulmonary bypass (CPB), resulting in postoperative platelet dysfunction and bleeding.
Methods. Seventy-nine patients were entered into a study designed to measure changes in platelet thrombin receptor function during CPB and to correlate them to postoperative bleeding. In addition to measurements of clinical blood loss, platelet function tests of aggregation, activation, and cell–cell adhesion were used. The thrombin receptor agonist peptide (TRAP) was used to activate the platelets. Flow cytometry was used to measure various platelet surface markers and platelet–white cell interactions during CPB.
Results. Compared with preoperative values, both aggregometry and flow cytometry measured a significant reduction of TRAP-induced activation immediately and up to 24 hours after CPB. The response of other activating agents returned to normal by 24 hours. Postoperatively, 8 of 79 patients required excessive blood transfusion (≥10 units of blood products) and had significantly decreased TRAP-induced aggregation response.
Conclusions. Our results show that (1) platelet activation, aggregation, and adhesion to leukocytes induced by TRAP are reduced after CPB, (2) decreased thrombin receptor responsiveness is associated with excessive postoperative blood loss, and (3) because the aggregation and activation responses are different for TRAP and thrombin, there may be a second thrombin receptor on platelets that is protected from damage during CPB. These results imply that prevention of the CPB-induced effects on the thrombin receptor will lessen postoperative morbidity associated with blood transfusion.
Thrombin active site inhibitors
1995, Bioorganic and Medicinal Chemistry
Direct inhibition of protein Ca by site directed thrombin inhibitors: Implications in anticoagulant and thrombolytic therapy
1995, Thrombosis Research
Several synthetic and recombinant antithrombin agents are currently developed for anticoagulation during thrombolytic therapy. Many of these agents are designed to inhibit thrombin at the active site. This active site is similar in enzymes belonging to the family of serine proteases. Since many of the thrombolytic enzymes are serine proteases (the class of enzymes in which thrombin and most of the coagulation enzymes are included), the newly developed antithrombin agents may also inhibit their pharmacologic actions and compromise thrombolytic efficacy. For this reason, it is important to compare the relative antithrombin and antifibrinolytic actions of these agents. A systematic investigation of the antiprotease profile and inhibitory spectrum against fibrinolytic enzymes is also important.
Although several thrombin inhibitors have been compared in their ability to inhibit various enzymes involved in the activation of fibrinolysis (1), no reports on the interactions of these inhibitors with activated protein C are available. Since protein Ca has a dual role, both as an anticoagulant in the coagulation cascade (by inactivating factors Va and VIIIa) and as a profibrinolytic enzyme (by inactivating PAI-1 and PAI-III and thus stimulating the activity of t-PA) (2), it is important that its activity is not compromised by thrombin inhibitors, for optimal coagulation and fibrinolytic state. The current studies are therefore designed to investigate the direct inhibitory effects of synthetic and recombinant antithrombin agents on the amidolytic action of protein Ca. Human activated protein C has been made available by plasma fractionation and thus the inhibitory effects of thrombin inhibitors in a defined biochemical system was possible to investigate.

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PaperFibrinolytic compromise by simultaneous administration of site-directed inhibitors of thrombin

Abstract

Thromb Res

Comparative effects of direct thrombin inhibitory peptides and heparin in global and biochemically defined plasmatic and non-plasmatic systems

Blood

Paper
Fibrinolytic compromise by simultaneous administration of site-directed inhibitors of thrombin