Phosphorylation of factor Va and factor VIIIa by activated platelets.
Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II-like kinase may downregulate the activity of the two cofactors.
Kalafatis
M
M
Department of Biochemistry, University of Vermont, College of Medicine, Burlington 05405-0068.
Rand
M D
MD
Jenny
R J
RJ
Ehrlich
Y H
YH
Mann
K G
KG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D005169:Factor VIII / Q000302:isolation & purification / Q000378:metabolism*; D015944:Factor VIIIa / Q000302:isolation & purification / Q000378:metabolism*; D015943:Factor Va / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010710:Phosphates / Q000097:blood; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation; D011486:Protein C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D000596:Amino Acids; D046911:Macromolecular Substances; D010710:Phosphates; D010748:Phosphopeptides; D011486:Protein C; D011994:Recombinant Proteins; D015943:Factor Va; D015944:Factor VIIIa; D005169:Factor VIII; D013917:Thrombin