Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL040901-06
Application #
2219779
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1990-01-01
Project End
1998-01-31
Budget Start
1995-02-01
Budget End
1996-01-31
Support Year
6
Fiscal Year
1995
Total Cost
Indirect Cost

  Institution

Name
Saint Louis University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63103

  Publications

Li, Jianjun; Hu, Weimin; Baldassare, Joseph J et al. (2003) The ethanol metabolite, linolenic acid ethyl ester, stimulates mitogen-activated protein kinase and cyclin signaling in hepatic stellate cells. Life Sci 73:1083-96
Baldassare, J J; Bi, Y; Bellone, C J (1999) The role of p38 mitogen-activated protein kinase in IL-1 beta transcription. J Immunol 162:5367-73
Cheng, J; Baldassare, J J; Raben, D M (1999) Dual coupling of the alpha-thrombin receptor to signal-transduction pathways involving phosphatidylinositol and phosphatidylcholine metabolism. Biochem J 337 ( Pt 1):97-104
Weber, J D; Cheng, J; Raben, D M et al. (1997) Ablation of Goalpha overrides G1 restriction point control through Ras/ERK/cyclin D1-CDK activities. J Biol Chem 272:17320-6
Weber, J D; Hu, W; Jefcoat Jr, S C et al. (1997) Ras-stimulated extracellular signal-related kinase 1 and RhoA activities coordinate platelet-derived growth factor-induced G1 progression through the independent regulation of cyclin D1 and p27. J Biol Chem 272:32966-71
Baldassare, J J; Jarpe, M B; Alferes, L et al. (1997) Nuclear translocation of RhoA mediates the mitogen-induced activation of phospholipase D involved in nuclear envelope signal transduction. J Biol Chem 272:4911-4
Cheng, J; Weber, J D; Baldassare, J J et al. (1997) Ablation of Go alpha-subunit results in a transformed phenotype and constitutively active phosphatidylcholine-specific phospholipase C. J Biol Chem 272:17312-9
Poulos, J E; Weber, J D; Bellezzo, J M et al. (1997) Fibronectin and cytokines increase JNK, ERK, AP-1 activity, and transin gene expression in rat hepatic stellate cells. Am J Physiol 273:G804-11
Padfield, P J; Panesar, N; Henderson, P et al. (1996) Differential effects of G-protein activators on 5-hydroxytryptamine and platelet-derived growth factor release from streptolysin-O-permeabilized human platelets. Biochem J 314 ( Pt 1):123-8
Reynolds, N J; Baldassare, J J; Henderson, P A et al. (1994) Translocation and downregulation of protein kinase C isoenzymes-alpha and -epsilon by phorbol ester and bryostatin-1 in human keratinocytes and fibroblasts. J Invest Dermatol 103:364-9

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