The mechanism(s) of thrombin-platelet interaction(s) will be studied with three long range objectives: i) to characterize and establish the function of the thrombin-induced inhibition of platelet adenylate cyclase (AC), ii) to use inhibition of AC as an assay of a thrombin receptor in soluble preparations, and iii) to isolate thrombin-binding proteins and to identify components of the putative thrombin """"""""receptor"""""""". Comparisons of thrombin-induced activation of control vs chymotrypsin-treated platelets and of activation of platelets by Alpha-thrombin vs Gamma-thrombin reveal that when the ability of thrombin to activate platelets quickly is impaired (pretreatment with chymotrypsin; activation by Gamma-thrombin), the ability to inhibit AC is blocked. We will test the hypotheses that i) inhibition of AC is necessary for normal platelet activation by thrombin and other agonists, and ii) thrombin regulates AC activity by either a receptor or an effector that is distinct from that for platelet activation, with only the AC system highly sensitive to proteolysis. Thrombin-induced inhibition of AC will be analyzed in membrane and soluble preparations, and the effects of proteases on regulation of cyclase by thrombin and other agonists will be correlated with the effects of protease pretreatment on the polypeptide composition of the cyclase preparation. The solubilized system will be used as an assay for receptor activity to establish an apparent mass for the receptor, to study dissociation and stability of the receptor and for following the course of purification. With similar experiments, the effect of collagen on the regulation of AC will be analyzed. To identify platelet thrombin-binding proteins, photoactivatable crosslinking reagents will be used in two ways: i) as a means for enrichment of the binding proteins, and ii) as a means for specifically attaching a radioactive label for screening for monoclonal antibodies against the binding proteins. For isolation of the binding proteins, a cleavable crosslinking reagent will be used. The derivatized thrombin will be crosslinked to the surface of labeled platelets, the complex will be solubilized and adsorbed to an immobilized antibody against thrombin, and the platelet protein will be released by cleavage of the crosslinking reagent. The product will be used as an immunogen for the preparation of monoclonal antibodies, using the immunogen and crosslinked thrombin-platelet complexes for clone selection. Antibody affinity columns will be used to isolate the binding proteins in high yield.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL016355-12
Application #
3335181
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1977-04-01
Project End
1990-09-29
Budget Start
1985-09-30
Budget End
1986-09-29
Support Year
12
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Suny Downstate Medical Center
Department
Type
Schools of Medicine
DUNS #
068552207
City
Brooklyn
State
NY
Country
United States
Zip Code
11203
Huang, Z; McGowan, E B; Detwiler, T C (1992) Ester and amide derivatives of E64c as inhibitors of platelet calpains. J Med Chem 35:2048-54
Huang, E M (1992) Different effects of A23187 and PMA on palmitoylated platelet proteins. Thromb Res 68:75-86
Speziale, M V; Detwiler, T C (1990) Free thiols of platelet thrombospondin. Evidence for disulfide isomerization. J Biol Chem 265:17859-67
McGowan, E B; Becker, E; Detwiler, T C (1989) Inhibition of calpain in intact platelets by the thiol protease inhibitor E-64d. Biochem Biophys Res Commun 158:432-5
Huang, E M (1989) Agonist-enhanced palmitoylation of platelet proteins. Biochim Biophys Acta 1011:134-9
Miller, J J; Browne, P C; Detwiler, T C (1988) Complexes of thrombin with secreted platelet proteins. Biochem Biophys Res Commun 151:9-15
Browne, P C; Miller, J J; Detwiler, T C (1988) Kinetics of the formation of thrombin-thrombospondin complexes: involvement of a 77-kDa intermediate. Arch Biochem Biophys 265:534-8
Huang, E M; Detwiler, T C (1987) Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization. Biochem J 242:11-8
Detwiler, T C; Turk, J L; Browne, P C (1987) Thiol-disulfide exchange by thrombospondin. Semin Thromb Hemost 13:276-80
McGowan, E B; Detwiler, T C (1986) Modified platelet responses to thrombin. Evidence for two types of receptors or coupling mechanisms. J Biol Chem 261:739-46

Showing the most recent 10 out of 13 publications