Computer-assisted data analysis of binding isotherms (LIGAND) has shown that human platelets have binding sites for alpha-thrombin of high (Kd 0.3 nM), moderate (Kd 10 nM), and low affinities (Kd 3 microM). Application of similar techniques has shown that TLCK-thrombin does not, whereas PPACK-thrombin does, bind to the high-affinity binding site accessible to alpha-thrombin, but that both bind to the moderate and low-affinity sites. Treatment of platelets with Serratia marcescens protease destroys the high-affinity site but does not affect moderate-affinity binding. In accordance with this model, both modified thrombins compete with alpha-thrombin for platelet activation at the moderate-affinity site, but only PPACK-thrombin competes at the high-affinity site. These results establish that platelet activation by either low or moderate concentrations of thrombin are receptor-mediated events and explain the paradox of the differential effects of TLCK-thrombin on the binding and activation of platelets by alpha-thrombin.