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Vitamin K-dependent protein S is an anticoagulant plasma protein serving as cofactor to activated protein C in degradation of coagulation factors Va and VIIIa on membrane surfaces. In addition, it forms a noncovalent complex with complement regulatory protein C4b-binding protein (C4BP), a reaction which inhibits its anticoagulant function. Both forms of protein S have affinity for negatively charged phospholipids, and the purpose of the present study was to elucidate whether they bind to the surface of activated platelets or to platelet-derived microparticles. Binding of protein S to human platelets stimulated with various agonists was examined with FITC-labeled monoclonal antibodies and fluorescence-gated flow cytometry. Protein S was found to bind to membrane microparticles which formed during platelet activation but not to the remnant activated platelets. Binding to microparticles was saturable and maximum binding was seen at approximately 0.4 microM protein S. It was calcium-dependent and reversed after the addition of EDTA. Inhibition experiments with monoclonal antibodies suggested the gamma-carboxyglutamic acid containing module of protein S to be involved in the binding reaction. An intact thrombin-sensitive region of protein S was not required for binding. The protein S-C4BP complex did not bind to microparticles or activated platelets even though it bound to negatively charged phospholipid vesicles. Intact protein S supported binding of both protein C and activated protein C to microparticles. Protein S-dependent binding of protein C/activated protein C was blocked by those monoclonal antibodies against protein S that inhibited its cofactor function. In conclusion, we have found that free protein S binds to platelet-derived microparticles and stimulates binding of protein C/activated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles. 146 47

Vitamin K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. To determine which regions in protein S are important for its cofactor activity, we have raised and characterized a large panel of monoclonal antibodies against human protein S. Several of the antibodies were directed against Ca2(+)-dependent epitopes, and they were found to be located either in the domain containing gamma-carboxyglutamic acid (Gla), the thrombin-sensitive region, or in the first epidermal growth factor (EGF)-like domain. The first two types of epitopes were exposed at approximately 1 mM Ca2+, whereas the epitope(s) in the EGF-like domains required less than 1 microM Ca2+, suggesting the presence of one or more high affinity Ca2(+)-binding site(s). The antibodies, as well as their Fab' fragments, against all three types of Ca2(+)-dependent epitopes efficiently inhibited the activated protein C cofactor function of protein S, but through different mechanisms. The antibodies against the Gla domain exerted their effects through inhibition of protein S binding to negatively charged phospholipid. Fab'-fragments of antibodies against the thrombin-sensitive region and the first EGF-like domain were the most potent inhibitors of the activated protein C cofactor function but did not inhibit phospholipid binding of protein S. In conclusion, we have identified the domains in protein S that are important for the activated protein C cofactor activity. The Gla domain is instrumental in the binding of protein S to phospholipid, whereas the thrombin-sensitive region and the first EGF-like domain may be directly involved in protein-protein interactions on the phospholipid surface.
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PMID:Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies. 169 22

C4b-binding protein (C4BP) is a multimeric glycoprotein in plasma with important regulatory functions in the complement system. It occurs in two forms, as free protein and in a non-covalent bimolecular complex with the vitamin K-dependent protein S. Protein S is an important anticoagulant and enhances the rate of inactivation by activated protein C of blood coagulation factors, Va and VIIIa. Protein S bound to C4BP is inactive as an anticoagulant, indicating C4BP to have a regulatory function in the blood coagulation process. Approximately 50% of C4BP in plasma circulates in complex with protein S, but little has been known about as to how these proteins interact. This report describes the structure of C4BP and its relation to protein S binding. A novel C4BP subunit, designated the beta-chain, which in all likelihood contains the protein S binding site, has been identified, isolated and characterized. The major form of C4BP is composed of seven alpha-chains and one beta-chain, and the subunits are covalently linked by their carboxy-terminal regions giving the molecule a spider-like quaternary structure. A subpopulation of C4BP, which does not bind protein S, was found to lack the beta-chain. This provides support for the concept that the single protein S binding site is located on the beta-chain. The beta-chain is structurally related to the alpha-chain of C4BP, and both subunits belong to the superfamily of C3b/C4b-binding proteins. The genes coding for the alpha- and beta-chains of C4BP were found to be closely linked within a cluster of genes, coding for structurally related proteins, on the long arm of human chromosome 1.
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PMID:Molecular analysis of the beta-chain of human C4b-binding protein. 204 26

We recently developed an enzyme-linked immunosorbent assay (ELISA) for total protein S (PS) antigen using the monoclonal antibody S-12. During the screening of thrombophilic patients we identified a patient, who was using marcoumar, with 0% PS by monoclonal ELISA and 23% PS by polyclonal ELISA. Further analysis of this patient and his family showed that the patient was a compound heterozygote for type 1 PS deficiency and for an abnormal PS molecule (PS-Heerlen) that was not recognized by the S-12 antibody. Similar observations were made in two sisters from an unrelated Dutch family. Subsequent studies showed that PS Heerlen has a slightly lower molecular weight (71,000) than normal PS (73,000), binds normally to C4b-binding protein, and retains full activated protein C cofactor activity. The alteration in the PS Heerlen molecule was identified as a substitution of Ser460 by Pro, which is due to a unique T---C transition in exon 13 of the active PS-alpha gene. The substitution occurs in the consensus sequence for the potential N-linked glycosylation of Asn458. Digestion with N-glycanase showed that normal PS probably contains three N-linked oligosaccharide side chains, while PS Heerlen contains only two (Asn458 not glycosylated?). Segregation analysis in the two original families showed that the presence of the genetic abnormality was always associated with the PS-Heerlen phenotype. The frequency of the PS-Heerlen allele was found to be 0.52% in the general population and 0.67% in a population of patients with unexplained thrombophilia. There is no evidence that the PS Heerlen allele is associated with an increased risk for thrombosis.
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PMID:Heerlen polymorphism of protein S, an immunologic polymorphism due to dimorphism of residue 460. 214 91

Vitamin K-dependent protein S exists in two forms in plasma, as free protein and in a bimolecular, noncovalent complex with the regulatory complement protein C4b-binding protein (C4BP). The effects of C4BP on the protein Ca cofactor activity of protein S were studied in a plasma system and in a system using purified components from both human and bovine origin. Bovine protein S was found to interact with human C4BP with a 5-fold higher affinity than that observed for the interaction between human protein S and human C4BP. The binding of protein S, from either species, to human C4BP results in the loss of the protein Ca cofactor function. In bovine plasma, protein S could be totally complexed by the addition of human C4BP, with a concomitant total loss of protein Ca cofactor activity. The addition of purified human C4BP to human plasma resulted in only partial loss of protein Ca cofactor activity and the plasma protein S was not completely complexed. Human protein S functioned as a cofactor to human protein Ca, but not to bovine protein Ca, whereas bovine protein S demonstrated very little species specificity and functioned as a cofactor both with human and bovine protein Ca. The species specificity of the protein Ca-protein S interaction was useful in elucidating the effect of C4BP in the plasma system. In the system with purified bovine components, protein S was required for the degradation of factor Va by low concentrations of protein Ca, whereas in the system with human components protein Ca alone, even when added at very low concentrations, exhibited potential to degrade factor Va, and the presence of protein S only enhanced the reaction rate approximately 5-fold. In both these systems, the stimulating effect of protein S on factor Va degradation by protein Ca was completely lost when protein S bound to C4BP.
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PMID:Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. 294 33

Vitamin K-dependent protein S belongs to the family of clotting factors (e.g. Factors IX and X, and protein C). Unlike the other clotting factors, the C-terminal half (residues 250-634) of protein S is not a serine proteinase. In fact, the function of residues 250-634 of protein S is unknown. By using computer programs designed to detect evolutionary relationships between proteins, we find that this part of protein S is similar to rat androgen-binding protein, a protein produced and secreted by testicular Sertoli cells. The homology between protein S and androgen-binding protein suggests new approaches for elucidating their functions.
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PMID:Vitamin K-dependent protein S is similar to rat androgen-binding protein. 295 82

Thrombin treatment of the vitamin K-dependent protein S resulted in the loss of the activated protein C cofactor activity associated with protein S. The addition of phospholipid vesicles inhibited the inactivation. Thrombin treatment did not alter the molecular weight of the native protein. However, upon reduction, a peptide of approximately 3000 daltons was released from the treated protein. The interaction between calcium and protein S was reduced by thrombin treatment. When the calcium interaction was determined by the quenching of the intrinsic fluorescence of protein S, thrombin treatment appeared to inhibit the interaction between calcium and the protein. When the calcium interaction was observed by measuring the effect on the electrophoretic mobility of the protein, thrombin treatment reduced the interaction between calcium and protein S. However, the effect of thrombin treatment on the interaction between calcium and protein S was less than observed by the fluorescent method. This observation suggests that fluorescence quenching may be a result of a structural change induced by calcium binding. Thrombin treatment of protein S appears to uncouple the calcium binding from the structural change. In addition, the interaction between protein S and phospholipid vesicles was reduced by thrombin treatment. These results suggest that the thrombin conversion of protein S into a two-chain protein causes the loss of a calcium-induced change in protein structure, loss of the lipid-binding properties, and the loss of cofactor activity.
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PMID:Regulation of vitamin K-dependent protein S. Inactivation by thrombin. 623 14

C4b-binding protein (C4BP), a regulatory component in the complement system, binds to an anticoagulant vitamin K-dependent plasma protein S (PS) which acts as a cofactor of activated protein C. We raised monoclonal antibodies against C4BP and PS, and developed two different one-step sandwich enzyme immunoassay (EIA) systems for human total C4BP (assay A) and PS-C4BP complex (assay B) by using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The reaction time of the assay was 45 min in both EIA systems: 30 min for the immunoreaction and 15 min for the color reaction. The sensitivities were 12 and 20 mg/l in assays A and B, respectively. Linearity was obtained between 31 and 500 mg/l in both EIA systems. Assay A could detect both uncomplexed C4BP and PS-C4BP complex with equal efficiency so that total C4BP level was not affected by PS. The levels of total C4BP and PS-C4BP complex were found to significantly increase in sera from patients with membranous nephropathy and decrease with liver cirrhosis in comparison with the levels in normal subjects. On the other hand, a difference in the total C4BP and PS-C4BP complex levels was not shown between IgA nephropathy and normal subjects. Affinity column analysis and difference of total C4BP and PS-C4BP complex levels showed that most of C4BP in sera exists as PS-C4BP complex.
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PMID:One-step sandwich enzyme immunoassays for human C4b-binding protein (C4BP) and protein S-C4BP complex using monoclonal antibodies. 775 11

Protein C is a vitamin K-dependent protein circulating in plasma as a zymogen to an anticoagulant serine protease. After its activation, protein C cleaves and inactivates coagulation factors Va and VIIIa. Human protein C is synthesized in liver and undergoes extensive post-translational modification during its synthesis. Recently, the protein C inhibitor was demonstrated to be synthesized in several organs of the human male reproductive tract. Moreover, vitamin K-dependent protein S, which functions as a co-factor to activated protein C, was found to be synthesized in the Leydig cells of human testis. The aim of this study was to elucidate whether the protein C gene is also expressed in the male reproductive system. Specific immunostaining of protein C was found in Leydig cells of human testis, in the excretory epithelium of epididymis, and in some epithelial glands of the prostate, whereas no immunostaining was detected in seminal vesicles. Northern blotting and non-radioactive in situ hybridization demonstrated protein C mRNA in Leydig cells, in the excretory epithelium of epididymis, and in some of the epithelial glands of the prostate. The mRNA was distributed perinuclearly and the localization was in accordance with the specific immunostaining for protein C. The epithelium of epididymis was also found to contain both protein S mRNA and immunoreactivity. The demonstration of both protein C and protein S immunoreactivities, as well as their mRNAs, in male reproductive tissues suggests as yet unknown local functions for these proteins.
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PMID:The gene encoding vitamin K-dependent anticoagulant protein C is expressed in human male reproductive tissues. 776 27

Vitamin K-dependent protein S is an anticoagulant plasma protein that functions as a co-factor to activated protein C in the degradation of coagulation factors Va and VIIIa. We investigated the tissue/cellular distribution of protein S synthesis by Northern blotting, in situ hybridization, and immunohistochemistry. Northern blotting together with in situ hybridization, using specific oligodeoxynucleotide probes, demonstrated protein S mRNA in liver, lung, testis, epididymis, ovary, uterus, and brain. In the reproductive system, protein S mRNA was present in the cytoplasm of Leydig cells, interstitial cells of the ovary, epithelial cells of the epididymis, and in the endometrium, including endometrial mucous glandular membrane in the myometrium. Bronchial epithelial cells and alveolar macrophages were positive in the respiratory system. In the central nervous system, pyramidal neurons in the cerebral cortex and in the hippocampal region, and dentate fascia neurons gave strongly positive signals. Immunohistochemistry with monoclonal antibodies yielded a staining pattern that correlated well with results of in situ hybridization. In conclusion, results from Northern blotting, in situ hybridization, and immunohistochemistry suggested that rabbit protein S is expressed in several extrahepatic tissues. The presence of protein S transcripts in these fully differentiated cells suggests a cell type-specific gene expression which may be related to local anticoagulation or to other as yet unknown protein S functions.
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PMID:The gene encoding vitamin K-dependent anticoagulant protein S is expressed in multiple rabbit organs as demonstrated by northern blotting, in situ hybridization, and immunohistochemistry. 782 69


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