Platelet Aggregation Induced In Vitro by Rabbit Plasma Clot-associated Thrombin, and Its Inhibition by Thrombin Inhibitors

Abstract

SummaryThe activation of rabbit platelets by rabbit plasma clots, and the inhibition of clot-associated thrombin by heparin:antithrombin III, recombinant hirudin (rHV2Lys47) and argatroban, a low molecular weight thrombin inhibitor, was studied.Plasma clots caused the aggregation of platelets suspended in a plasma-free medium as assessed by single platelet counting, and by scanning electron microscopy (platelet aggregates present on the clot surface). Platelet aggregation, induced by clot-associated thrombin, was inhibited by argatroban with an IC50 of 14 ± 3 nM compared to an IC50 of 12 ± 2 nM when human thrombin in solution titrated to give the same decrease in the platelet count as plasma clots was used. rHV2Lys47 also inhibited aggregation induced by clot-associated thrombin with an IC50 of 1.6 ± 0.4 nM compared to 1.6 ± 0.5 nM with thrombin in solution. Heparin was less active against clot-associated thrombin (IC50 = 69 ± 9 mU/ml) than against thrombin in solution (IC50 = 15 ± 5 mU/ml).This study shows that plasma clot-bound thrombin activates platelets and that direct-acting thrombin inhibitors such as argatroban and rHV2Lys47 are more effective than heparin:antithrombin III in inhibiting this phenomenon.

The Heparin Binding Properties of Heparin Cofactor II Suggest an Antithrombin-like Activation Mechanism

“…Although HCII deficiency has not been established as a significant risk factor for thrombosis (7), recent studies suggest that HCII may play a role in preventing arterial thrombosis (7)(8)(9)(10) and may be uniquely capable of inhibiting clot-bound thrombin (11). Clot-bound thrombin is resistant to heparin anticoagulant therapy because of the obligate co-occupation of AT and thrombin on the same heparin chain (12,13).…”

Role of clot‐associated (‐derived) thrombin in cell proliferation induced by fibrin clots in vitro

“…The proliferation of CCL39 cells induced by fibrin clots was inhibited by both antithrombin III alone and by heparin in the presence of 20 n M antithrombin III. This latter result is in complete contrast to previous studies on the pharmacology of clot‐associated thrombin, where its amidolytic and platelet activating activity inter alia are resistant to heparin/antithrombin III (Mirshahi et al ., 1989; Weitz et al ., 1990; Berry et al ., 1994; Arocas et al ., 1996; Lunven et al ., 1996; Gandossi et al ., 1998) and was at first sight surprising to us. However, in this study, the incubation period of the thrombin with the cells (48 h) was considerably longer than the incubation periods used to study the amidolytic and platelet activating activity of clot associated thrombin (3–20 min, op.…”

“…In addition, fibrin clot‐bound thrombin retroactivates the coagulation cascade (Kumar et al ., 1994) and enhances platelet pro‐coagulant activity (Kumar et al ., 1995). Fibrin and plasma clot‐bound thrombin can also induce platelet aggregation, which is inhibited by direct thrombin inhibitors such as argatroban, recombinant hirudin and Bothrojaracin, but is resistant to heparin:antithrombin III (Arocas et al ., 1996; Lunven et al ., 1996; Gandossi et al ., 1998).…”

Thrombin Inhibitors and Anti-Factor Xa Agents in the Treatment of Arterial Occlusion

“…Indeed, argatroban may be more effective than hirudin at inhibiting clot-bound thrombin, due, at least in part, to its smaller size [46]. A relatively potent inhibitor of platelet activation and aggregation [47,48], Argatroban, has a short half-life and is metabolized principally by the liver. It does not appear to accumulate in renal failure, although dose adjustment is mandated for patients with altered hepatic function.…”