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Phosphorylation of factor Va and factor VIIIa by activated platelets
M Kalafatis, MD Rand, RJ Jenny, YH Ehrlich and KG Mann
Department of Biochemistry, University of Vermont, College of Medicine,
Burlington 05405-0068.
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.
Volume 81,
Issue 3,
pp. 704-719,
02/01/1993
Copyright © 1993 by The American Society of Hematology
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