Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling.

Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane.

Enhancement of human protein C function by site-directed mutagenesis of the gamma-carboxyglutamic acid domain.

This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine.

Activated protein C resistance in patients with peripheral vascular disease.

Purpose: The frequency of activated protein C (APC) resistance, caused by factor V R506Q gene mutation and abnormal APC ratio, in patients with peripheral vascular diseases was analyzed.

Methods: All patients electively admitted to the vascular ward unit of our tertiary care academic medical center from January 1995 through October 1996 (n = 679) were prospectively analyzed using an APC-resistance screening test to determine the frequency of abnormal APC ratio (< or =2.6).

The C4b-binding protein-protein S interaction is hydrophobic in nature.

C4b-binding protein (C4BP) is a major regulatory molecule of the complement system. By forming a non covalent complex with the anticoagulant cofactor protein S (PS), it also plays an important role in blood coagulation. C4BP is composed of one beta-chain and seven alpha-chains that are essentially built from complement control protein (CCP)-modules.

Amino acid residues in thrombin-sensitive region and first epidermal growth factor domain of vitamin K-dependent protein S determining specificity of the activated protein C cofactor function.

Human protein S (PS) potentiates the anticoagulant activity of human but not bovine activated protein C (APC), whereas bovine PS is a cofactor to APC from both species. The structural requirements for the specificity of the APC cofactor function of human PS are located in its thrombin-sensitive region (TSR) and the first epidermal growth factor (EGF1)-like module. To elucidate which residues in these two modules determine the specificity of the APC cofactor activity, 41 human PS mutants were expressed.

The SHBG-like region of protein S is crucial for factor V-dependent APC-cofactor function.

Activated protein C (APC) regulates blood coagulation by degrading factor Va (FVa) and factor VIIIa (FVIIIa). Protein S is a cofactor to APC in the FVa degradation, whereas FVIIIa degradation is potentiated by the synergistic APC-cofactor activity of protein S and factor V (FV). To elucidate the importance of the sex-hormone-binding globulin (SHBG)-like region in protein S for expression of anticoagulant activity, a recombinant protein S/Gas6 chimera was constructed.

Cleavage requirements of factor V in tissue-factor induced thrombin generation.

Factor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components.

Structural investigation of the A domains of human blood coagulation factor V by molecular modeling.

Factor V (FV) is a large (2,196 amino acids) nonenzymatic cofactor in the coagulation cascade with a domain organization (A1-A2-B-A3-C1-C2) similar to the one of factor VIII (FVIII). FV is activated to factor Va (FVa) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1-A2) and a light chain (A3-C1-C2). Activated protein C (APC), together with its cofactor protein S (PS), inhibits the coagulation cascade via limited proteolysis of FVa and FVIIIa (APC cleaves FVa at residues R306, R506, and R679).

The C-terminal region of the factor V B-domain is crucial for the anticoagulant activity of factor V.

Factor V (FV) is recently shown to express anticoagulant activity. It functions as a synergistic cofactor with protein S to activated protein C (APC) in the degradation of factor VIIIa (FVIIIa). FV is composed of multiple domains, A1-A2-B-A3-C1-C2.

Commentary: Involvement of Amino Acid Residues 423-429 of Human Protein S in Binding to C4b-Binding Protein.

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Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain.

A common 4G allele in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene as a risk factor for pulmonary embolism and arterial thrombosis in hereditary protein S deficiency.

Reduced fibrinolytic capacity due to increased plasminogen activator inhibitor-1 (PAI-1) activity in plasma is a common finding in patients with coronary heart disease or venous thromboembolism, although its clinical significance is debated. Recently, a dimorphism in the PAI-1 promoter (4G-5G) has been reported and homozygosity for the 4G allele is associated with increased transcription and higher PAI-1 levels. Homozygous 4G genotype has been suggested to be a risk factor for myocardial infarction.

A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen.

A new method to determine the concentration of free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.

Activation of the contact-phase system on bacterial surfaces--a clue to serious complications in infectious diseases.

Fever, hypotension and bleeding disorders are common symptoms of sepsis and septic shock. The activation of the contact-phase system is thought to contribute to the development of these severe disease states by triggering proinflammatory and procoagulatory cascades; however, the underlying molecular mechanisms are obscure. Here we report that the components of the contact-phase system are assembled on the surface of Escherichia coli and Salmonella through their specific interactions with fibrous bacterial surface proteins, curli and fimbriae.

Characterization of recombinant epidermal growth factor (EGF)-like modules from vitamin-K-dependent protein S expressed in Spodoptera cells--the cofactor activity depends on the N-terminal EGF module in human protein S.

Epidermal growth factor (EGF)-like modules in protein S, a physiological anticoagulant protein that functions as a cofactor to activated protein C, have been expressed in Spodoptera cells using baculovirus. EGF modules 1-3, 1-4, 2-3 and 2-4 were produced on a preparative scale. The isolated modules were more than 95% homogeneous, as judged by sequence determination.

Factor V Q506 mutation (activated protein C resistance) associated with reduced intrapartum blood loss--a possible evolutionary selection mechanism.

Objectives: To ascertain whether relationship exists between the presence of APC resistance [a hypercoagulable state due to a mutation (R506Q) in the factor V gene] and the occurrence of pre-eclampsia (PE), intrauterine growth retardation (IUGR), and pregnancy bleeding complications.

Design: A retrospective study.

Subjects: A study group of 122 women with PE and/or IUGR during a recent pregnancy and a control group of 465 healthy pregnant women.

Association of vitamin K-dependent coagulation proteins and C4b binding protein with triglyceride-rich lipoproteins of human plasma.

The triglyceride (TG) concentration in plasma is an independent risk factor for coronary heart disease. There is evidence that TG-rich lipoprotein (TGRLP), ie, chylomicrons (CMs), chylomicron remnants (CMRs), and VLDLs associate with factor VII and prothrombin and that the association enhances a platelet factor Xa-mediated prothrombin activation when the CM-prothrombin complex is exposed to platelets. In this study, we examined the association of the vitamin K-dependent coagulation factors VII, IX, X, and prothrombin, as well as the anticoagulation protein C and its cofactor protein S, in plasma lipoproteins obtained from human fasting and postprandial plasma.

Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: studies related to proline-10.

Bovine and human protein C show high homology in the amino acids of their GLA domains (amino-terminal 44 residues), despite the about 10-fold higher membrane affinity of the human protein. A proposed membrane contact site and mechanism suggested that this difference was largely due to the presence of proline at position 10 of bovine protein C versus histidine at position 10 of human protein C [McDonald, J.F.

Clarification of the risk for venous thrombosis associated with hereditary protein S deficiency by investigation of a large kindred with a characterized gene defect.

Background: Protein S is an important regulatory protein of the coagulation cascade. The risk for venous thrombosis associated with protein S deficiency has been uncertain because all previous risk estimates used phenotypic evaluation alone, which can be ambiguous.

Objective: To quantitate the risk for thrombosis associated with a characterized protein S gene mutation that causes a Gly295-->Val substitution and protein S deficiency.

SHBG region of the anticoagulant cofactor protein S: secondary structure prediction, circular dichroism spectroscopy, and analysis of naturally occurring mutations.

Protein S (PS) and growth arrest specific factor 6 (GAS6) are vitamin K-dependent proteins with similar structures. They are mosaic proteins possessing a carboxyl-terminal region presenting sequence similarity with plasma sex hormone binding globulin (plasma SHBG), although apparently not involved in steroid binding. The SHBG-like modules have sequence similarity with the G repeats of the chain A of laminin.

Synergistic cofactor function of factor V and protein S to activated protein C in the inactivation of the factor VIIIa - factor IXa complex -- species specific interactions of components of the protein C anticoagulant system.

Human factor V has been shown not only to be a precursor to procoagulant factor Va but also to express anticoagulant properties. Thus, factor V was recently found to potentiate the effect of protein S as cofactor to activated protein C (APC) in the inactivation of the factor VIIIa-factor IXa complex. The purpose of this study was to determine whether the APC-cofactor function of factor V was also expressed in the bovine protein C system and to elucidate the molecular background for the species specificity of APC.

Female gender and resistance to activated protein C (FV:Q506) as potential risk factors for thrombosis after elective hip arthroplasty.

Resistance to activated protein C (APC) caused by the R506Q mutation in factor V is the most common inherited risk factor for venous thrombosis. To elucidate whether APC-resistance is a risk factor for venous thrombosis after elective total hip replacement, the association between APC-resistance (presence of FV:Q506 allele) and postoperative thrombosis was investigated in patients (n = 198) randomised to received short (during hospitalisation, n = 100) or prolonged prophylaxis (three weeks after hospitalisation, n = 98) with low molecular weight heparin (LMWH). Among APC-resistant individuals receiving short prophylaxis, 7/10 developed thrombosis as compared to 2/12 receiving long prophylaxis (p <0.

The 20210 A allele of the prothrombin gene is a common risk factor among Swedish outpatients with verified deep venous thrombosis.

A dimorphism in the 3'-untranslated region of the prothrombin gene (G to A transition at position 20210) has recently been reported to be associated with increases in plasma prothrombin levels and in the risk of venous thrombosis. We have examined the prothrombin dimorphism among 99 unselected outpatients with phlebography verified deep venous thrombosis, and in 282 healthy controls. The prevalence of the 20210 A allele was 7.