Modulation of serine proteases-mediated platelet activation by novel direct thrombin inhibitors

ABSTRACT

Novel direct thrombin inhibitors [DTIs], such as bivalirudin, are replacing heparin in several clinical scenarios. In particular, DTIs are indicated for the treatment and thromboprophylaxis of patients with heparin-induced thrombocytopenia [HIT]. In interventional cardiology, DTIs have several advantages over heparin, and offer a clinical benefit equivalent to that of a combination of heparin and antiplatelet agents. We hypothesize that this benefit results from the ability of DTIs to inhibit platelet activation by activated serine proteases. This study represents the development of a modified [14]C-serotonin release assay [SRA] to investigate the relative inhibitory effects of three DTIs [argatroban, bivalirudin and hirudin] on thrombin- and factor Xa-mediated [14]C-serotonin release [SR] in plasma-free systems. Washed platelets were isolated from blood of healthy volunteers. The [14]C-SRA test was similar to that used to detect heparin-PF4 antibody-mediated platelet activation, except that it was used to evaluate the ability of DTIs to modulate protease-induced SR responses. The inhibitory effects of DTIs were determined at protease concentrations that induced >/= 50% SR. Serine proteases induced SR from platelets in a concentration-dependent manner. Human thrombin was found to be more potent than bovine thrombin [2-3 times for 50-80% SR]. Bovine factor Xa [>/= 0.2 nKat/ml] produced a comparable [50- 80%] SR response. All three DTIs effectively blocked serine protease-mediated platelet activation in a concentrationdependent manner. The optimum inhibitory concentrations of bivalirudin on SR was tilde100 nM for human thrombin and bovine factor Xa and almost double for bovine thrombin; well below plasma concentrations necessary for effective anticoagulation for percutaneous coronary interventions. Wide variations in the inhibitory effects of each DTI on thrombin- and factor Xa-mediated platelet activation were noted, which was partly dependent on the donor platelets and stability of the proteases/inhibitors. It is concluded that DTIs can directly inhibit serine proteases- mediated platelet activation responses