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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL015335-14
Application #
3334927
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1979-04-01
Project End
1990-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
14
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Boston University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Huang, R S; Sorisky, A; Church, W R et al. (1991) ""Thrombin"" receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides. J Biol Chem 266:18435-8
Davies, T A; Weil, G J; Simons, E R (1990) Simultaneous flow cytometric measurements of thrombin-induced cytosolic pH and Ca2+ fluxes in human platelets. J Biol Chem 265:11522-6
Davies, T A; Drotts, D L; Weil, G J et al. (1989) Cytoplasmic Ca2+ is necessary for thrombin-induced platelet activation. J Biol Chem 264:19600-6
Davies, T A; Drotts, D; Weil, G J et al. (1988) Flow cytometric measurements of cytoplasmic calcium changes in human platelets. Cytometry 9:138-42
Simons, E R; Davies, T A; Greenberg, S M et al. (1988) Platelet membrane potentials and their significance in monitoring stimulus response coupling. Soc Gen Physiol Ser 43:265-79
Davies, T A; Dunn, J M; Simons, E R (1987) Evaluation of changes in cytoplasmic pH in thrombin-stimulated human platelets. Anal Biochem 167:118-23
Davies, T A; Katona, E; Vasilescu, V et al. (1987) Sequential sodium-proton exchange in thrombin-induced human platelets. Biochim Biophys Acta 903:381-7
Pagonis, C; Tauber, A I; Pavlotsky, N et al. (1986) Flavonoid impairment of neutrophil response. Biochem Pharmacol 35:237-45
Banga, H S; Simons, E R; Brass, L F et al. (1986) Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci U S A 83:9197-201
Greenberg-Sepersky, S M; Simons, E R; White, J G (1985) Studies of platelets from patients with the grey platelet syndrome. Br J Haematol 59:603-9

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