Casein kinase II (CKII) and protein kinase C (PKC) are ubiquitous kinases that can phosphorylate a number of proteins. The presence of CKII in platelets was recently confirmed using immunoelectron microscopy whereas several isoforms of PKC are found in platelets. CKII as well as PKC may influence coagulation since it has been demonstrated that both kinases phosphorylate several important proteins involved in the blood clotting process. Factor Va, the cofactor for prothrombinase, was found to be differentially phosphorylated on both the heavy and light chains by two platelet kinases which share similarities with CKII and PKC. Phosphorylation on Ser/692 of the heavy chain of the cofactor by a CKII- like kinase is stoichiometric and increases the susceptibility of the cofactor to inactivation by activated protein C (APC). Phosphorylation on the light chain of the cofactor by a PKC-like occurs at two sites and the significance of this phosphorylation is still unknown. However, upon stimulation of platelets by alpha-thrombin, platelet factor Va light chain is the major secreted platelet phosphoprotein. Preliminary experiments demonstrated that the platelet CKII-like kinase shares little similarity with the known CKII molecules with respect to subunit composition whereas the PKC-like kinase is most likely a PKC isoform present in platelets. The general objective of this grant proposal is to identify and characterize the kinases involved in the differential phosphorylation of factor Va. Once identified, the kinetic and biochemical properties of each kinase will be studied separately. Partial amino acid sequence obtained from peptides following limited enzymatic digestion of the platelet CKII-like kinase will be used to produce degenerate oligonucleotides that will be utilized for the screening of a human megakaryocyte cDNA library. The cDNA for the CKII-like kinase will be isolated and sequenced. The cDNA for the CKII-like kinase will also be used to clone the kinase and thus produce large amounts of kinase necessary to study the significance of phosphorylation of various blood clotting proteins. Phosphorylated platelet or plasma factor V as well as factor V/R506Q (factor V Leiden) which is found in 20% of patients with deep venous thrombosis, will be studied for their ability to interact with the components of prothrombinase, to be inactivated by APC as well as for their ability to interact with the platelet membrane. The use of the phosphorylated cofactor, will allow us to determine the role of phosphorylation in the generation of thrombosis.
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