Endogenous cleavage of the Arg-379-Ala-380 bond in vitronectin results in a distinct conformational change which 'buries' Ser-378, its site of phosphorylation by protein kinase A.

Activation of blood platelets by thrombin was previously shown to specifically release protein kinase A, which in human plasma singles out and phosphorylates one protein, identified as vitronectin. This protein is known to be involved in processes that follow platelet stimulation, specifically, in the binding of heparin (interfering with the heparin-mediated inhibition of thrombin and Factor Xa by antithrombin III), in the growth of endothelial cells and in fibrinolysis. This paper shows that phosphorylation of vitronectin by protein kinase A is stoichiometric (approx. 1 mol/mol), that it is targeted to one site (Ser-378) at the C-terminal edge of the heparin-binding domain, and that it distinguishes between the two physiologically occurring forms of vitronectin: the one-chain (75 kDa) form, and the nicked two-chain (65 + 10 kDa) form, held together by an interchain disulphide bridge. Protein kinase A phosphorylates the one-chain form but not the two-chain form, although Ser-378 and the complete recognition sequence of the kinase are still present in the clipped 65 kDa chain. Cleavage of the Arg-379-Ala-380 bond results therefore in a conformationally distinct form of vitronectin in which Ser-378 is 'buried'. This is demonstrated by our finding that Ser-378 is present in the 65 kDa chain of clipped vitronectin but inaccessible to phosphorylation at physiological pH. Upon binding heparin, the phosphorylation site becomes exposed and able to undergo a stoichiometric phosphorylation at physiological pH.