Activation of Platelets by a - Thrombin Is a Receptor - mediated Event D - PHENYLALANYL - L - PROLYL - L - ARGININE

Competition binding studies have been carried out to evaluate the antagonism of TLCK-thrombin (N“-tosylL-lysine chloromethyl ketone-treated thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated thrombin) with athrombin using computer-assisted analysis of the binding isotherms (LIGAND). &-Thrombin bound to high, moderate, and low affinity sites as previously described (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). PPACK-thrombin bound to all three sites accessible to a-thrombin (ICl, 7 nM; R1, 20 sites/platelet; Kz, 3 nM; Rz, 1800 sites/platelet; K3, 510 nM; R3, 84,000 sites/platelet) as well as to a separate fourth site ( I C x , 0.4 nM; R,, 20 sites/platelet) for PPACK-thrombin that was not accessible to a-thrombin. In contrast, TLCK-thrombin did not bind to the high affinity site for a-thrombin but bound to the moderate and low affinity sites for a-thrombin with similar affinity (IC2, 2 nM; Rz, 890 sites/platelet; K3, 900 nM; R3, 100,000 sites/platelet) and to another site (K,, 0.03 nM; R,, 10 sites/platelet) which was not accessible to a-thrombin. As predicted from these binding studies, TLCKthrombin did not compete with a-thrombin for platelet activation at concentrations as high as 1000 nM (500fold excess). In contrast a 300-fold excess of PPACKthrombin (670 nM) totally inhibited platelet activation by 2 nM thrombin. These results demonstrate 1) that the high affinity binding site for thrombin on human platelets is a classical receptor, occupancy of which is necessary for platelet activation by low concentrations of thrombin; 2) that TLCK-thrombin does not occupy this high affinity site and hence cannot inhibit platelet activation by a-thrombin; and 3) that PPACK-thrombin does compete with a-thrombin at the high affinity site and is an antagonist of a-thrombin induced activation.