Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

Abstract

Human factor Xa specifically cleaves the anticoagulant protein S within the thrombin-sensitive domain. Amino-terminal amino acid sequencing of the heavy chain cleavage product indicates cleavage of protein S by factor Xa at Arg 60 , a site that is distinct from those utilized by ␣-thrombin. Cleavage by factor Xa is unaffected by the presence of hirudin and is completely blocked by tick-anticoagulant-peptide and D-Glu-GlyArg-chloromethyl ketone, the latter two being specific inhibitors of factor Xa. The cleavage requires the presence of phospholipid and Ca 2؉ , and is markedly inhibited by the presence of factor Va. Factor Xa-cleaved protein S no longer possesses its activated protein C-dependent or -independent anticoagulant activity, as measured in a factor VIII-based activated partial thromboplastin time clot assay. The apparent binding constant for protein S binding to phospholipid (K d Ӎ 4 nM ؎ 1.0) is unaffected by factor Xa or thrombin cleavage, suggesting that the loss of anticoagulant activity resulting from cleavage is not primarily due to the loss of membrane binding ability. Cleavage and inactivation of protein S by factor Xa may be an additional way in which factor Xa exerts its procoagulant effect, after the initial stages of clot formation.Protein S is a vitamin K-dependent, 635-amino acid, nonenzymatic glycoprotein (M r Х 78,000; Ref. 1) that acts as an anticoagulant in blood (2-4). The domain organization of protein S is similar to other plasma vitamin K-dependent proteins in that it contains an amino-terminal ␥-carboxyglutamate-rich domain and several (four) epidermal growth factor-like domains (5, 6). Unique to protein S, however, is a 29-amino acid thrombin-sensitive domain, located between the ␥-carboxyglutamate and first epidermal growth factor-like domains. The thrombin-sensitive domain corresponds to exon IV of the protein S gene (7). Protein S also contains a sex steroid binding protein-like domain (8) at the carboxyl-terminal end of the protein, in place of the serine protease domain found in the vitamin K-dependent plasma proteases. The sex steroid binding protein-like domain has been identified as being composed of two tandem repeat units homologous to the Cys-poor laminin A globular (G) domain found in a number of extracellular ligand binding basement membrane proteins involved in cellular growth and differentiation (9). Schneider and co-workers have described a cultured cell growth arrest-specific protein (Gas6) from NIH 3T3 mouse cells (10) and human IMR90 fibroblasts (11), which, except for the absence of a thrombinsensitive domain, is homologous throughout to protein S.Thrombin cleavage converts protein S into a two-chain, disulfide-linked protein that no longer possesses anticoagulant activity (12). The thrombin cleavage sites were first established for bovine protein S (at Arg 52 and Arg 70 ) by Dahlback et al. (13) and recently at the corresponding residues (Arg 49 and Arg 70 ) for human protein S (1).The defined mechanism(s) by which protein S exhibits its anticoagulant acti...

The Thrombin-sensitive Region of Protein S Mediates Phospholipid-dependent Interaction with Factor Xa

“…These recent findings are in accordance with our observations here that protein S binds to fXa but not to fVIIa. It is likely that protein S-fXa interactions play a part in these functions of protein S. Our results are also compatible with observations of Long et al (7), who showed that fXa cleaves protein S at Arg 60 of the TSR and that this reaction requires negatively charged phospholipids and calcium ions.…”

Cleaved Protein S (PS), Total PS, Free PS, and Activated Protein C Cofactor Activity as Risk Factors for Venous Thromboembolism

“…Surprisingly, we found a positive correlation between cleaved PS concentrations and APC cofactor activity (r ϭ 0.51). Indeed, cleaved PS was devoid of APC cofactor activity in a FVIII proteolysis assay (12 ) and in a global coagulation test (13 ), and the lack of such activity was confirmed in this assay. This correlation could reflect a shift in total and free PS.…”

“…PS also contains a protease-sensitive loop, called the thrombin-sensitive region, between the ␥-carboxyglutamic acid-containing domain and the first epidermal growth factor-like domain. Under physiologic conditions, the thrombin-sensitive region can be cleaved after Arg 60 (11 ), and this cleavage can be reproduced in vitro by factor Xa (FXa) (12 ). The resulting two-chain disulfidelinked protein can no longer potentiate APC activity (13 ).…”

Vitamin K-dependent proteins in Ciona intestinalis , a basal chordate lacking a blood coagulation cascade

“…The Gla domain of Ci-Gla4 is separated from the first EGF domain by a short putative disulfide loop that occupies a position analogous to the proteolytically sensitive loop of Protein S (27,28), as well as to a disulfide loop in Gas6 that is not known to be cleaved by proteases. The common evolutionary origin of Ci-Gla4, Protein S, and Gas6 is further suggested by the observation that this disulfide loop is encoded by a single exon for all three proteins.…”