A number of physiologically relevant agonists of human platelets are known to activate the phosphoinositide signalling pathway(s) by stimulating PtdIns-PLC activity. We have recently shown that platelets express several of the family of PtdIns-PLC isoforms namely, PtdIns-PLC- Beta1, gamma1, gamma2, delta, and alpha for studying agonist-induced PIP2 hydrolysis in isolated platelet (HEL cell) membranes, the thromboxane A2 receptor has been shown to activate PtdIns-PLC Beta 1. Resent data from our laboratory suggests that the TXA2, thrombin and collagen receptors each activate distinct PtdIns-PLC isozymes. Preliminary studies also indicate that the G-protein coupling for these receptors to their specific PtdIns-PLC isoforms are quite dissimilar. Although PtdIns-PLC gamma1 does not reconstitute thrombin mediated PIP2 hydrolysis in the reconstitution assay, studies with intact platelets demonstrate thrombin- induced translocation of PtdIns-PLC gamma 1 to the cytoskeleton/skeleton and its association with gelsolin. The primary goals of this proposal are: 1) identification of the PtdIns-PLC isozymes that reconstitute thrombin- and collagen-stimulated PIP2 hydrolysis in platelet membranes and the characterization of their G-protein regulation. 2) cloning of the PtdIns-PLC isoform that is G-protein coupled to the thrombin receptor. 3) characterization of thrombin-induced translocation of PtdIns-PLC gamma1, its association with gelsolin and possible activation. Studies also will focused on understanding the mechanism involved in PtdIns-PLC gamma 1, translocation, association with gelsolin and possible activation. In addition, these agonist-induced changes in the subcellular distribution, associations and activation will be correlated with changes in cytoskeleton/skeleton G-actin and diacylglycerol levels. 4) examination of the role of the multiple PtdIns-PLC isoforms in PIP 2 hydrolysis by overexpression and """"""""knock out"""""""" of specific PtdIns-PLC isoforms in HEL cells. Platelets play a major role in the pathogenesis both of the vasculature and the perivasculature cells and the administration of antiplatelet treatments are efficacious in only some patients, therefore, a detailed understanding of the mechanisms of agonist-induced platelet activation may provide the basis for future therapeutic interventions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL040901-08
Application #
2332483
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1990-01-01
Project End
1999-01-31
Budget Start
1997-02-01
Budget End
1999-01-31
Support Year
8
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Saint Louis University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
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