We plan to pursue our studies of platelet receptors and of the mechanism of platelet activation by several approaches - some developed in this laboratory as part of our earlier investigations. By using fully biologically active photoreactive derivatives of thrombin and of active site blocked thrombin (TLCK-thrombin), high intensity light, and short (30-60sec.) incubation times, we have identified one high and two low affinity thrombin receptors. With multiply (rather than singly) derivatized thrombins having photoreactive groups at different spatial locations, light-induced covalent binding is to several platelet membrane proteins, implying that the receptor may be an assembly of such proteins. This will be investigated as will their size, composition, and apparent function. Threshold amounts of non-activating TLCK-thrombin evoke an enhanced subsequent response to yieldthrombin -i.e. faster depolarization and serotonin secretion, and a net increase in the number of bound sites. Since the time period involved is very short and equilibrium is neither desired nor attained, this up-regulation may correspond either to a net increase in the number of accessible binding sites or to an increase in the rate of binding to an unaltered number of these sites. We hope to resolve this question. We shall also continue our investigations of the transduction process. We have demonstrated a thrombin-induced rapid increase in intraplatelet Na+ and pH. Now the effect of these cation gradient changes on the internal mobilization of Ca++, the activation of the metabolic system, and the movement of granules to the plasma membrane must be studied. We have already determined that lysosomal granule release is a later event (starts only 30-60 sec. after exposure to thrombin) under K+ gradient control. The role of these monovalent and divalent cations needs to be explored further, a study we plan to undertake using clean isolated plasma membrane vesicles or granules prepared by cavitation using a modification of our successful neutrophil procedure. We also plan to continue our interest in the reported multiple stimulatability of platelets. The fluorescence activated cell sorter will be useful in all these studies as it can separate platelets according to membrane potential, to internal pH, to relative number of covalently bound receptors, and to extent of granule retention as well as to size. These investigations should help to clarify the complex platelet stimulus response.
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