Thrombin-induced platelet aggregation and cleavage of [3H]FSBA-labeled aggregin is mediated by platelet calpain. 2. Plasma HMWK inhibits platelet aggregation and cleavage of aggregin in intact platelets, indirectly by thrombin and directly by calpain. 3. Inhibition of thrombin-induced platelet aggregation by HMWK is specific for this proteolytic enzyme. 4. HMWK is the major inhibitor of thrombin-induced activation of platelets in plasma.