Summary
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
B2 (TXB2) immunoassay and scanning electron microscopy. Fibrin clots decreased platelet counts,
and released TXB2. Electron microscopy showed platelet aggregates on the clot surface. Argatroban concentration-dependently
inhibited such aggregation with IC50s of 21 nM and 13 nM versus aggregation and TXB2 release respectively. The IC50s of Argatroban against fluid-phase thrombin producing similar aggregation were 12
nM (aggregation) and 33 nM (TXB2). rHV2Lys47 was less active against clot-induced aggregation (IC50 = 1.8 nM) than against fluid-phase thrombin (IC50 = 0.06 nM). Heparin had an IC50 of 0.02 mU/ml against aggregation induced by fluid-phase thrombin, but much greater
concentrations are required to inhibit clot-induced aggregation (IC50 = 48 mU/ml). These data provide a basis for the superiority of direct-acting thrombin
inhibitors over heparin in platelet rich thrombi.
