Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection

• Published in: The Journal of biological chemistry 1988-10, Vol.263 (29), p.14884-14890
• Main Authors: Solymoss, S, Tucker, M M, Tracy, P B
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Blood coagulation. Blood cells; Blood Platelets - metabolism; Cell Membrane - metabolism; Coagulation factors; cofactors; Enzyme Activation; Factor V - antagonists & inhibitors; Factor V - isolation & purification; Factor Va; Factor Xa; Fundamental and applied biological sciences. Psychology; Glycoproteins - isolation & purification; Glycoproteins - metabolism; Humans; Kinetics; man; Molecular and cellular biology; plasma proteins; platelets; Protein C - isolation & purification; Protein C - metabolism; Protein S; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)68121-9

Kinetic analyses were done to determine what effect factor Xa and protein S had on the activated protein C (APC)-catalyzed inactivation of factor Va bound to phospholipid vesicles or human platelets. In the presence of optimal concentrations of phospholipid vesicles and Ca2+, a Km of 19.7 +/- 0.6 nM factor Va and a kcat of 23.7 +/- 10 mol of factor Va inactivated/mol of APC/min were obtained. Added purified plasma protein S increased the maximal rate of factor Va inactivation only 2-fold without effect on the Km. Protein S effect was unaltered when the phospholipid concentration was varied by 2 orders of magnitude. The reaction on unactivated human platelets yielded a Km = 12.5 +/- 2.6 nM and kcat = 6.2 +/- 0.6 mol of factor Va inactivated/mol of APC/min. Added purified plasma protein S or release of platelet protein S by platelet activation doubled the kcat value without affecting the Km. Addition of a neutralizing anti-protein S antibody abrogated the effect of plasma protein S or platelet-released protein S, but was without effect in the absence of plasma protein S or platelet activation. Studies with factor Xa indicated that factor Xa protects factor Va from APC-catalyzed inactivation by lowering the effective concentration of factor Va available to interact with APC. From these data a dissociation constant of less than 0.5 nM was calculated for the interaction of factor Xa with membrane-bound factor Va. Protein S abrogated the ability of factor Xa to protect factor Va from inactivation by APC without affecting the interaction of factor Xa with factor Va. These combined data suggest that one physiological function of protein S is to allow the APC-catalyzed inactivation of factor Va in the presence of factor Xa.