Interaction of vitamin K dependent proteins with membranes

Nelsestuen, Gary L.; Kisiel, Walter; Scipio, Richard G. Di

doi:10.1021/bi00604a017

Cited by 206 publications

(137 citation statements)

References 18 publications

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“…In all cases, an apparent K d of approximately 4 nM was derived. This low value, relative to other vitamin K-dependent plasma proteins, is in good agreement with those reported by others (7-70 nM) for human protein S from different sources and using various purification procedures (43,45,46) and initially observed semiquantitatively by Nelsestuen et al (47). Our results suggest that cleav- FIG.…”

Section: Resultssupporting

confidence: 93%

Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

Long

Lu²,

Xie³

et al. 1998

Journal of Biological Chemistry

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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...

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Section: Resultssupporting

confidence: 93%

Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

Long

Lu²,

Xie³

et al. 1998

Journal of Biological Chemistry

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“…Membrane binding of vitamin K-dependent coagulation factors have previously been determined by ellipsometry, 43,44 light scattering, 6,45,46 and fluorescence polarization. 47 Recently, the utility of BIAcore technique for evaluation of membrane-binding ability of vitamin K-dependent proteins has been demonstrated.…”

Section: Discussionmentioning

confidence: 99%

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

Sun

Shen

Dahlbäck

2003

Blood

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Protein C is a member of the vitamin Kdependent protein family. Proteins in this family have similar ␥-carboxyglutamic acid (Gla)-rich domains, but their affinities for negatively charged phospholipid membranes vary more than 1000-fold. We have shown that it is possible to enhance anticoagulant activity and membrane affinity of protein C by selective mutagenesis of the Gla domain. In this study, 3 new mutants, Q10G11N12 ( IntroductionNegatively charged phospholipid membranes play an important role in blood coagulation, providing a suitable surface for the assembly of enzyme-cofactor complexes, which efficiently convert circulating zymogens to active enzymes. Phosphatidylserine (PS) is a key component in the negatively charged membrane that supports the clotting reactions. [1][2][3] In most cells, PS is normally mainly present in the inner leaflet of the cell membrane, but certain cellular processes such as platelet activation result in the exposure of PS on the cell surface. The vitamin K-dependent coagulation proteins in plasma bind to the surface of negatively charged phospholipid with a common mechanism involving the ␥-carboxyglutamic acid (Gla)-rich domains of these proteins. 4 Vitamin K is required in the posttranslational carboxylation of glutamic acid residues to Gla. 5 The Gla domains comprise approximately 45 amino acid residues, and they are structurally very similar in the different vitamin K-dependent proteins. 6 Despite the extensive structural similarity between different Gla domains, they bind negatively charged phospholipid with very different affinity, with dissociation constants (K d 's) ranging more than 1000-fold. 6 This variation in phospholipid binding ability is related to amino acid sequence differences at relatively few positions. This has made it possible to modulate the phospholipid binding abilities of this group of proteins by site-directed mutagenesis. [7][8][9] The nonvitamin K-dependent cofactors of blood coagulation also interact with the phospholipid membranes, certain proteins being membrane spanning, whereas others, such as factor V (FV) and factor VIII (FVIII), contain binding sites for negatively charged phospholipid membranes. 2,10 FV circulates as a procofactor to activated FV (FVa), which functions as a cofactor to activated FX (FXa) in the activation of prothrombin to thrombin. Activated FVIII (FVIIIa) serves a similar function in the FIXa-mediated activation of FX. 2,3 The protein C anticoagulant system, which includes the vitamin K-dependent proteins protein C and protein S, regulates the activities of FVa and FVIIIa by limited proteolysis (reviewed by Dahlbäck et al 11 and Esmon et al 12 ). Protein C is activated on the endothelial cell surface by the thrombin-thrombomodulin complex, and the serine protease activated protein C (APC) down-regulates coagulation by cleaving a limited number of peptide bonds in each of FVa and FVIIIa. Protein S serves as a cofactor to APC by increasing the affinity of APC for the membrane 13 and also by changing the distance of the activ...

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“…The acceleration of APC-catalyzed factor Va inactivation by negatively charged membranes is presumably a reflection of the fact that both APC (Nelsestuen et al, 1978) and factor Va (Bloom et al, 1979) bind to such membranes. This allows the inactivation of membrane-bound factor Va by membranebound APC.…”

Section: Dlscusslonmentioning

confidence: 99%

The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C

Bakker

Tans

Janssen-Claessen

et al. 1992

European Journal of Biochemistry

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Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x lo5 M -l s-'. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 pM phospholipid vesicles composed of 20 moly" dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of OlezGroPSer some inhibition of APC-catalyzed factor Va inactivation was observed.Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine < oleic acid < phosphatidic acid < phosphatidylglycerol < phosphatidylmethanol < phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the K , for factor Va and the V,,, of factor Va inactivation were a function of the phospholipid concentration. The K , increased from 1 nM at 2.5 pM phospholipid (Ole2GroPSer/Ole2GroPCho 20: 80, mol/mol) to 65 nM at 250 pM phospholipid. The V,,, increased from 20 mol factor Va inactivated * min-' . mol APC-' at 2.5 pM phospholipid to 62 mol factor Va inactivated . min-' . mol APC-' at 10 pM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation, Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.

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Interaction of vitamin K dependent proteins with membranes

Cited by 206 publications

References 18 publications

Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

Human Protein S Cleavage and Inactivation by Coagulation Factor Xa

Gla domain–mutated human protein C exhibiting enhanced anticoagulant activity and increased phospholipid binding

The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C

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