Activation of platelets by alpha-thrombin is a receptor-mediated event. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha-tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor.
Competition binding studies have been carried out to evaluate the antagonism of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated thrombin) with alpha-thrombin using computer-assisted analysis of the binding isotherms (LIGAND). alpha-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 alpha-thrombin (K1, 7 nM; R1, 20 sites/platelet; K2, 3 nM; R2, 1800 sites/platelet; K3, 510 nM; R3, 84,000 sites/platelet) as well as to a separate fourth site (Kx, 0.4 nM; Rx, 20 sites/platelet) for PPACK-thrombin that was not accessible to alpha-thrombin. In contrast, TLCK-thrombin did not bind to the high affinity site for alpha-thrombin but bound to the moderate and low affinity sites for alpha-thrombin with similar affinity (K2, 2 nM; R2, 890 sites/platelet; K3, 900 nM; R3, 100,000 sites/platelet) and to another site (Ky, 0.03 nM; Ry, 10 sites/platelet) which was not accessible to alpha-thrombin. As predicted from these binding studies, TLCK-thrombin did not compete with alpha-thrombin for platelet activation at concentrations as high as 1000 nM (500-fold excess). In contrast a 300-fold excess of PPACK-thrombin (670 nM) totally inhibited platelet activation by 2 nM thrombin. These results demonstrate 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; that TLCK-thrombin does not occupy this high affinity site and hence cannot inhibit platelet activation by alpha-thrombin; and that PPACK-thrombin does compete with alpha-thrombin at the high affinity site and is an antagonist of alpha-thrombin induced activation.
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D012701:Serotonin / Q000378:metabolism; D013917:Thrombin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D014107:Tosyllysine Chloromethyl Ketone / Q000031:analogs & derivatives / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D008024:Ligands; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D014107:Tosyllysine Chloromethyl Ketone; D012701:Serotonin; D013917:Thrombin; C050815:phenylalanyl-prolyl-arginine chloromethyl ketone-thrombin; C050816:tosyllysine chloromethyl ketone-thrombin