Characterization of the EGF-like module pair 3-4 from vitamin K-dependent protein S using NMR spectroscopy reveals dynamics on three separate time scales and extensive effects from calcium binding

Protein S, a cofactor of anticoagulant activated protein C, exhibits three high-affinity Ca(2+)-binding sites in a region comprising four EGF modules. The EGF 3-4 module pair constitutes the smallest fragment that retains one high-affinity Ca(2+)-binding site and is therefore useful for investigation of the structural basis of the unusually high-affinity Ca(2+) binding compared to other EGF-containing proteins characterized so far. Extensive chemical shift effects caused by Ca(2+) binding to the EGF 3-4 module pair are observed, particularly from Ca(2+) binding to the high-affinity site in EGF 4. Ca(2+) binding to the high-affinity site in EGF 4 and the low-affinity site in EGF 3 is associated with slow and fast exchange on the NMR time-scale, respectively. We show the presence of two isoforms, characterized by a cis or trans Lys 167-Pro 168 peptide bond, that do not convert on time scales that were accessible to the experiments (k(ex) < 0.2 s(-1)). Both conformers have similar Ca(2+) affinities and backbone dynamics. Further, broadening of (1)H resonances involving residues in the major beta-sheet of EGF 3 and (15)N exchange terms, primarily in the N-terminal part of the protein, indicate the presence of slow exchange on a microsecond to millisecond time scale. (15)N spin relaxation data suggest that the module pair has a well-defined relative orientation between EGF modules 3 and 4 and has a significantly anisotropic rotational diffusion tensor in solution.     

High affinity interaction between C4b-binding protein and vitamin K-dependent protein S in the presence of calcium. Suggestion of a third component in blood regulating the interaction

The anticoagulant vitamin K-dependent protein S interacts with the complement regulatory protein C4b-binding protein (C4BP), both in purified systems and in plasma. The concentrations of these proteins in plasma are approximately equimolar (0.3 microM) and 30-40% of protein S in plasma is found in the noncomplexed state. Only the uncomplexed form of protein S displays anticoagulant activity and studies have shown that patients with a selective deficiency of free protein S have a high incidence of thrombosis. In this study, we report that the protein S-C4BP interaction is at least 100-fold tighter in the presence of Ca2+ than in EDTA. The KD in the presence of Ca2+ was estimated with a gel filtration technique to be less than 5 x 10(-10) M, whereas in the presence of EDTA, it was approximately 100-fold higher. Ca2+ titration experiments suggested that the Ca2+ sites which function in the protein S-C4BP interaction are of high affinity which, in turn, suggests that they may be independent of the gamma-carboxyglutamic acid region and may be present in the epidermal growth factor-like domains of protein S. The high affinity of the protein S-C4BP interaction in the presence of Ca2+ suggested that virtually all of the protein S in whole blood should be complexed with C4BP. However, even though the protein S-C4BP interaction in Ca2(+)-containing serum was shown to have the same high affinity as in purified systems, approximately 30-40% of the protein S in serum was free. These results appear best explained by the presence of a third component in whole blood which regulates the protein S-C4BP interaction, keeping approximately 30-40% of circulating protein S in its free, functionally anticoagulant form. It is speculated that persons with little free protein S may be deficient in this hypothetical third component.     

Solution structure of the Ca2+-Binding EGF3-4 pair from vitamin K-dependent protein S: identification of an unusual fold in EGF3

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.     

Regulation of vitamin K-dependent protein S. Inactivation by thrombin

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.     

Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S.

C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9 X 10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1 X 10(3) M-1 . S-1 and 1.8 X 10(-4)-4.5 X 10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7 X 10(-7) M was calculated for the C4b-binding protein-protein S interaction.     

A method for systematic purification from bovine plasma of six vitamin K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z

A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).     

Propeptide recognition by the vitamin K-dependent carboxylase in early processing of prothrombin and factor X.

Precursors of vitamin K-dependent proteins are synthesized with a propeptide that is believed to target these proteins for gamma-carboxylation by the vitamin K-dependent carboxylase. In this study synthetic propeptides were used to investigate gamma-carboxylation of the prothrombin and factor X precursors in rat liver microsomes. The extent of prothrombin processing by the carboxylase was also investigated. Antisera raised against the human prothrombin and factor X propeptides only recognized precursors with the respective propeptide regions. The data demonstrate structural differences in the propeptide region of the prothrombin and the factor X carboxylase substrates which raises questions about the hypothesis of a common propeptide binding site on the carboxylase for all precursors of vitamin K-dependent proteins. The hypothesis of separate binding sites is supported by data which demonstrate differences in binding of the prothrombin and factor X precursors to membrane fragments from rough and smooth microsomes. gamma-Carboxylation of the prothrombin precursors in vitro was investigated with conformational specific antibodies raised against a portion of the Gla (gamma-carboxyglutamic acid) region extending from residue 15 to 24. The synthetic peptide used as antigen contains three of the ten potential Gla sites in prothrombin. It is shown that these antibodies do not recognize mature prothrombin but recognize the decarboxylated protein. It is also demonstrated that the epitope is Ca2(+)-dependent. The antibodies were used to assess gamma-carboxylation of the prothrombin precursor in membrane fragments from microsomal membranes. The results suggest that microsomal gamma-carboxylation does not involve Glu residues 16, 19 and 20 of the Gla region.     

Purification of human vitamin K-dependent protein S and its limited proteolysis by thrombin

Vitamin K-dependent protein S exists in two forms in human plasma, namely as the free protein and in complex with C4b-binding protein [Dahlbäck & Stenflo (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2512-2516]. Now reported is a simple purification procedure for human protein S that includes barium citrate adsorption, DEAE-Sephacel chromatography and chromatography on Blue Sepharose. The yield was approx. 30% relative to the concentration of free protein S in plasma, which was found to be approx. 10 mg/l. Purified protein S migrated as a single-chain band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions and as a doublet of Mr approx. 85 000 and 75 000 on reduction. A third band of Mr 16 000 was observed after electrophoresis of 125I-labelled protein S and radioautography of reduced samples. This band appears to be disulphide-linked to the 75 000-Mr chain before reduction. Thrombin converted the 85 000-Mr chain of protein S into a 75 000-Mr chain and an 8000-Mr fragment, the latter again being detectable only by radioautography of reduced samples. The 16 000-Mr fragment was not observed, suggesting its degradation by thrombin. Under non-reducing conditions, no change in apparent molecular weight of thrombin-treated protein S was observed, indicating disulphide linkage of the fragments. Thrombin also affected the mobility of protein S on agarose-gel electrophoresis in the presence of Ca2+, suggesting a decreased affinity to Ca2+ of the cleaved form of protein S as compared with the undegraded molecule. After activation of the complement system in human serum, protein S was found to be a constituent part of the complex formed by C4b-binding protein and component C4b.     

Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S

Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.     

Proteolytic cleavage of vitamin K-dependent bovine plasma protein S by alpha-thrombin and plasma serine protease

It is known that protein S, a vitamin K-dependent plasma protein, isolated from a human source, gives a closely spaced doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and that this heterogeneity in molecular size results from a limited proteolysis of protein S mediated by alpha-thrombin in human species. We found here that alpha-thrombin also rapidly converted single-chain bovine protein S to a nicked form, which consisted of the NH2-terminal segment containing gamma-carboxyglutamic acid and the COOH-terminal large segment bridged by a disulfide linkage(s). These two segments were isolated and chemically characterized after S-alkylation of the nicked protein S. The results suggest that the alpha-thrombin-catalyzed hydrolysis of protein S probably occurs at a peptide linkage (Arg-Ser) located in the NH2-terminal portion. The conversion of single-chain protein S to the nicked form was also mediated by plasma kallikrein and plasmin, in addition to alpha-chymotrypsin and trypsin. However, the alpha-thrombin-catalyzed conversion did not occur when calcium ions were added to the reaction mixture.     

Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies.

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.     

Sex hormone-binding globulin, androgen-binding protein, and vitamin K-dependent protein S are homologous to laminin A, merosin, and Drosophila crumbs protein.

Androgen-binding protein (ABP) and sex hormone-binding globulin (SHBG) are extracellular steroid-binding proteins that are homologous to the COOH-terminal domain of vitamin K-dependent protein S, a protein important in blood clotting. We find that the sequences of ABP, SHBG, and protein S are also similar to two basement membrane proteins, laminin and merosin, and to an integral membrane protein, Drosophila crumbs protein. These latter three proteins have important roles in regulating differentiation and development. The sequence similarity corresponds to the G domain of laminin A chain, which binds heparin and type IV collagen. Analysis of a multiple alignment of these proteins reveals one well-conserved segment corresponding to the part of SHBG that binds to its membrane receptor and another corresponding to the part of protein S that binds to C4b-binding protein. The similarities suggest that ABP, SHBG, and protein S may also have functions related to that of laminin and merosin.     

Post-translational processing events in the secretion pathway of human protein C, a complex vitamin K-dependent antithrombotic factor.

Human protein C (HPC) undergoes several post-translational modifications, including gamma-carboxylation, N-linked glycosylation, and internal proteolytic processing. We have utilized a recombinant human kidney cell line (293) secreting correctly modified HPC (rHPC) to study the processing reactions for the modification of this complex protein. gamma-Carboxylation was shown to proceed via a vitamin K-dependent pathway and was required for both efficient secretion and anticoagulant activity. rHPC was rapidly secreted following the addition of vitamin K to depleted cells, and secretion was not inhibited by cyclohexamide indicating that non gamma-carboxylated rHPC accumulates as an intracellular releasable pool. However, in cells grown in the presence of vitamin K, the majority of intracellular rHPC was gamma-carboxylated, suggesting that this post-translational modification is not rate limiting for secretion under conditions optimal for vitamin K-dependent carboxylation. Nonglycosylated rHPC was found to be secreted inefficiently, and processing of the N-linked core in the endoplasmic reticulum, but not in the Golgi, was required for secretion. Further, the intracellular rHPC present in vitamin K-supplemented cells was core glycosylated, but not processed past the high mannose step. gamma-Carboxylation occurred after core glycosylation, indicating that this modification is not cotranslational. Further, glycosylation and gamma-carboxylation were not coupled and did not need to proceed sequentially. Proteolytic processing of the internal KR dipeptide was found to occur late in the secretion pathway, and the cleavage was calcium-dependent. The secretion rate of rHPC was also calcium-dependent but was independent of the calcium effect on internal KR dipeptide removal, indicating that cleavage is not required for efficient secretion. Our results define the sequence of processing events, the subcellular localization of the processing reactions, and the rate-limiting steps in the secretion pathway for this complex protein.     

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.     

Three-dimensional structure of the apo form of the N-terminal EGF-like module of blood coagulation factor X as determined by NMR spectroscopy and simulated folding.

The three-dimensional structure of a 42-residue fragment containing the N-terminal EGF-like module of blood coagulation factor X was determined by means of 2D NMR spectroscopy and computer simulation. The spectroscopic data consisted of 370 NOE distances and 27 dihedral angle constraints. These were used to generate peptide conformations by molecular dynamics simulation. The simulations used a novel functional form for the constraint potentials and were performed with two time steps to ensure rapid execution. Apart from preliminary runs to aid assignment of NOEs, 60 runs resulted in 13 accepted structures, which have two antiparallel beta sheets, no alpha helices, and five tight turns. There is no hydrophobic cluster. The root mean square deviation for the backbone of the 13 conformations is 0.65 +/- 0.11 A against their mean conformation. About half of the side chains have well-defined structure. The overall conformation is similar to that of murine EGF.     

Preparation of vitamin K-dependent proteins,such as clotting factors II,VII, IX and X and clotting inhibitor protein C

A review is given of preparative methods for the isolation of the vitamin K-dependent clotting factors II, VII, IX, X and clotting inhibitor protein C, all derived from human plasma. Factor II, activated factor VII and activated protein C are also obtained from recombinant animal cells. The methods for their purification are described. The problem of difference in posttranslational modifications between plasma derived and recombinant protein is discussed with regard to therapeutic proteins.     

Characterization of the metastasis-associated protein,S100A4. Roles of calcium binding and dimerization in cellular localization and interaction with myosin

Elevated S100A4 protein expression is associated with metastatic tumor progression and appears to be a strong molecular marker for clinical prognosis. S100A4 is a calcium-binding protein that is known to form homodimers and interacts with several proteins in a calcium-dependent manner. Here we show that S100A4 localizes to lamellipodia structures in a migrating breast cancer-derived cell line and colocalizes with a known S100A4-interacting protein, myosin heavy chain IIA, at the leading edge. We demonstrate that S100A4 mutants that are defective in either their ability to dimerize or in calcium binding are unable to interact with myosin heavy chain IIA. An S100A4 mutant that is deficient for calcium binding retains the ability to form homodimers, suggesting that S100A4 can exist as calcium-free or calcium-bound dimers in vivo. However, a calcium-bound S100A4 monomer only interacts with another calcium-bound monomer and not with an S100A4 mutant that does not bind calcium. Interestingly, despite the calcium dependence for interaction with known protein partners, calcium binding is not necessary for localization to lamellipodia. Both wild type and a mutant that is deficient for calcium binding colocalize with known markers of actively forming leading edges of lamellipodia, Arp3 and neuronal Wiskott-Aldrich syndrome protein. These data suggest that S100A4 localizes to the leading edge in a calcium-independent manner, and identification of the proteins that are involved in localizing S100A4 to the lamellipodial structures may provide novel insight into the mechanism by which S100A4 regulates metastasis.     

Interaction of vitamin K-dependent protein Z with thrombin. Consequences for the amidolytic activity of thrombin and the interaction of thrombin with phospholipid vesicles

Protein Z is a vitamin K-dependent protein of unknown function present in normal bovine plasma at a concentration of approximately 0.1 microM. Quantitative affinity chromatographic studies using diisopropylphosphoryl (DIP)-thrombin-Affi-Gel 10 as the affinity matrix and free DIP-thrombin as the competitor demonstrated that protein Z interacts with DIP-thrombin with a dissociation constant of 0.15 +/- 0.05 microM. Binding was independent of Ca2+. Protein C and factor IX, other vitamin K-dependent clotting proteins with the same domain structure as that of protein Z, did not interact with immobilized DIP-thrombin under these conditions; and factor X interacted with an affinity 20-fold lower than that for protein Z. The Michaelis constant, Km, for hydrolysis of pyro-Glu-Pro-Arg-p-nitroanilide by thrombin was increased 1.8-fold, from 130 to 230 microM, as a result of the binding of protein Z and the Km for H-Val-Leu-Arg-p-nitroanilide 1.4-fold, from 390 to 560 microM. From these kinetic studies, a dissociation constant of 0.11 +/- 0.04 microM was calculated for the binding of protein Z to alpha-thrombin. Protein Z bound to large phospholipid vesicles (25% phosphatidylserine, 75% phosphatidylcholine) with a dissociation constant of 0.39 +/- 0.16 microM at a phospholipid to protein ratio of 82 mol of phospholipid/mol of protein Z at saturation. In the presence of protein Z thrombin associated with phospholipid vesicles, whereas thrombin did not interact with phospholipid vesicles in the absence of protein Z. These studies, therefore, demonstrate a physiologically relevant interaction between protein Z and thrombin. They also suggest a mechanism whereby thrombin is localized to an injury site by virtue of its interaction with protein Z bound to phospholipid surfaces.     

Immunochemical accessibility of ribosomal protein S4 in the 30 S ribosome. The interaction of S4 with S5 and S12

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.     

Solution-phase equilibrium binding interaction of human protein S with C4b-binding protein.

Solution-phase equilibrium binding studies of human protein S (HPS) and C4b-binding protein (C4BP) were undertaken using purified components. Free C4BP was measured in solutions at equilibrium by using HPS immobilized on a solid phase, coupled with an antibody detection system. Disruption of the solution-phase equilibrium was minimized by using a brief (15 min) exposure to the solid-phase HPS. These studies yielded an equilibrium dissociation constant (Kd) approximately 6 x 10(-10) M and a stoichiometry of approximately 1.7 molecules of HPS bound to each molecule of C4BP. This Kd is between 27-fold and 930-fold lower than previously published values obtained by using solid-phase and nonequilibrium methods. Equilibrium was achieved in solutions containing low nanomolar concentrations of both HPS and C4BP in less than or equal to 1 h at 37 degrees C, suggesting a rapid association rate constant for the interaction. Thrombin cleavage of HPS had no effect on the observed binding parameters. The binding interaction between HPS and C4BP appears to be partly calcium dependent, since in the presence of EDTA the Kd was increased to about 6 x 10(-9) M, with no change in the stoichiometry. This high-affinity binding interaction between HPS and C4BP, whose Kd is more than 500-fold lower than the proteins' plasma concentrations, heightens the apparent physiologic importance of complex formation.