In human platelets, activation of the ubiquitous intracellular serine- threonine kinase protein kinase C stimulates platelet aggregation and release of storage granule contents. PKC activation also prevents the ability of a subsequent agonist, such as thrombin, to induce phosphoinositidase C activity or Ca2+ mobilization in platelets. The recent discovery that protein kinase C is a gene family with multiple isozymes raises the possibility that these apparently disparate effects are mediated by different isozymes that are selectively regulated, which is the principal hypothesis to be tested in this proposal. PKC isozymes in platelets and megakaryocyte-like human erythroleukemia (HEL) cells will be identified and characterized by amplifying mRNA by the polymerase chain reaction, followed by determination of their nucleotide sequences. The distribution of protein kinase C isozymes in HEL cells will be compared to that of megakaryocytes by in situ hybridization. Partial cDNA clones obtained by polymerase chain reaction will be used to probe cDNA libraries of megakaryocytes, HEL cells, and platelets. Tissue distribution of mRNA encoding novel or highly expressed isozymes will be determined and the cDNA will be transiently expressed in COS cells. Preliminary evidence has shown that protein kinase C-alpha, beta, and the newly-described delta are present in platelets, while only the latter two are found in HEL cells. In this proposal, separate lines of HEL cells with defined PKC isozyme phenotypes will be created by stable transfection (PKC-alpha) or antisense oligonucleotides (beta, delta) and will serve as models for platelet function. These cell lines will be compared for their ability to bind activation-dependent monoclonal antibodies that recognize platelet-specific epitopes, as well as for their accumulation of cAMP, mobilization of Ca2+, turnover of phospholipase D, phosphorylation of tyrosine kinase/phosphatase substrates and expression of mRNA coding for the protooncogenes c-fos, c- myc, c-myb and serine/threonine phosphatases. As preliminary evidence suggests that PKC-delta is a predominant isozyme in both platelets and HEL cells, the recently cloned and sequenced cDNA encoding human PKC-delta will be used to obtain a human genomic clone, the fine structure of which will be characterized, including the 5' regulatory region. These projects should yield novel information regarding the physiological role and genetic regulation of the protein kinase C gene family in platelets and their precursor cells, and may provide details critical to our understanding of the intracellular regulation of platelet function and thrombus formation.
