Inhibition by Argatroban, a specific thrombin inhibitor, of platelet activation by fibrin clot-associated thrombin.

Clot-associated thrombin retains amidolytic activity, and is resistant to inhibition by heparin, but not to low molecular weight thrombin inhibitors. We show that clot-associated thrombin induces platelet aggregation, is resistant to heparin :antithrombin III, less so to recombinant hirudin (rHV2Lys47) but not to argatroban, an active-site directed thrombin inhibitor. Fibrin clots prepared with human fibrinogen and thrombin were used to aggregate rabbit washed platelets assessed by single platelet counting, thromboxane B 2 (TXB 2 ) immunoassay and scanning electron microscopy. Fibrin clots decreased platelet counts, and released TXB 2 . Electron microscopy showed platelet aggregates on the clot surface. Argatroban concentration-dependently inhibited such aggregation with IC 50 s of 21 nM and 13 nM versus aggregation and TXB 2 release respectively. The IC 50 s of Argatroban against fluid-phase thrombin producing similar aggregation were 12 nM (aggregation) and 33 nM (TXB 2 ). rHV2Lys47 was less active against clot-induced aggregation (IC 50 = 1.8 nM) than against fluid-phase thrombin (IC 50 = 0.06 nM). Heparin had an IC 50 of 0.02 mU/ml against aggregation induced by fluid-phase thrombin, but much greater concentrations are required to inhibit clot-induced aggregation (IC 50 = 48 mU/ml). These data provide a basis for the superiority of direct-acting thrombin inhibitors over heparin in platelet rich thrombi.