Phosphorylation of Human Fibrinogen In Vitro by Calcium-Activated Phospholipid-Dependent Protein Kinase from Pig Spleen1

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Abstract
Human plasma fibrinogen rapidly incorporated stoichiometric amounts of [32P]-phosphate when incubated with [32P]ATP and calcium-activated, phospholipid-dependent protein kinase purified from pig spleen. Half-maximal fibrinogen kinase activity was attained at less than 0.1 mM calcium acetate. The optimum concentration of phosphatidylserine was about 50 μg per ml. Diolein slightly potentiated the stimulatory effect of phosphatidylserine. The α-chain of fibrinogen, which is reported to contain endogenous phosphate (Blombäck, B., Blombäck, M., Edman, P., & Hessel, B. (1962) Nature193, 883–884 and Doolittle, R.F., Watt, K.W.K., Cottrell, B.A., Strong, D.D., & Riley, M. (1979) Nature280, 464–468) was phosphorylated by the protein kinase. The apparent Km value for the phosphorylation reaction (0.3–0.6μM fibrinogen) was comparable with the Km values reported for the hitherto most effective substrate proteins for protein kinase C. Up to 5 mol phosphate per mol fibrinogen could be incorporated, indicating at least three phosphorylatable sites per half molecule.