A hypothesis is proposed for the activation of platelets by thrombin that involves its simultaneous interaction with both high affinity (Kd 0.3nM) and moderate affinity (Kd 10 nM) receptors. The high affinity receptor is thought to be platelet glycoprotein I(GPI) that interacts with phospholipase C and adenylate cyclase through the inhibitory nucleotide regulatory component (Ni or Gi): alternatively phospholipase C may be coupled directly to GPI. The moderate affinity receptor has not been identified but may be coupled to phsopholipase A2 or may be phospholipase A2 itself. The relative contribution of the phospholipase C and A2 pathways in platelet activation is considered to depend on the relative degrees of occupancy of the high and moderate affinity sites at differing thrombin concentrations. The following studies are proposed in order to test this hypothesis: (i) stimulus-response coupling will be evaluated by measuring the relative contributions of the phospholipase C and A2 pathways at differing thrombin concentrations corresponding to receptor occupancies for high affinity sites of 30-98% and for moderate affinity sites of 1-80%; (ii) the effect of the absence of the high affinity site on the two pathways will be examined in protease-treated platelets, in Bernard-Soulier platelets and in platelets in which monoclonal antibodies block the high affinity thrombin-binding site on GPI; (iii) the effect of the absence of moderate affinity binding will be examined in Hermansky-Pudlak platelets, in platelets showing abnormalities in arachidonate metabolism and in platelets treated with natural inhibitors of phospholipase A2 activity; (iv) following solubilization in Triton X-100 or other detergents, the multimolecular complexes modulating the two pathways will be separated and their individual components characterized; (v) the various components of the high and moderate affinity pathways will be reconstituted into liposomes as a test of their functional significance. Important techniques to be used in these studies include radiation inactivation for the measurement of the functional sizes of receptors and enzymes in membranes and complex mixtures, computer analysis (LIGAND) for the determination of steady state binding parameters, phospholipid analyses and monoclonal antibody production for the identification and purification of platelet components necessary to elicit a thrombin response by the high affinity and moderate affinity pathways.
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