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Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [(1)H]-(15)N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.

Serum amyloid P (SAP) and C-reactive protein (CRP) are two members of the pentraxin family of proteins. These proteins associate with a variety of other materials that are found in serum under normal or pathological circumstances. This study showed that carboxylated compounds, especially lactic acid, were capable of dissociating pentraxins from several macromolecular binding sites. When measured by sucrose density gradient ultracentrifugation, complete dissociation of the complex of hSAP (human SAP) with C4b-binding protein (C4BP) occurred at > or = 5 mM lactate. Lactate dissociated the hSAP-membrane complex and prevented hSAP self-association. The only interaction that was not dissociated by 10 mM lactate was the hSAP-heparin complex. The relative efficacies of several dissociating agents were O-phosphorylethanolamine > lactate > succinate > carbonate > epsilon-amino-n-caproic acid. This suggested that the carboxyl group plus a hydrogen-bonding site on the hydrocarbon chain was important, but a charged amino group was not a contributor to function when the anion was provided by a carboxyl group. The concentration of lactic acid needed to dissociate hSAP from C4BP was dependent on protein concentration in a manner suggesting the cooperative binding of lactate (coefficient = 2) to hSAP. Pure proteins, at concentrations found in normal serum, required about 12 mM lactate for half-dissociation of the hSAP-C4BP complex. Other pentraxins also interacted with lactic acid, but with lower affinities. An important observation was that lactic acid was capable of dissociating rat CRP from lipoproteins in rat serum. Human CRP bound very weakly to lactate, so that lactate probably is not a significant regulator of this pentraxin.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody (mAb 6F6) directed against the beta-chain of C4b-binding protein (C4BP) was previously shown to inhibit the binding of protein S to C4BP. To localize the epitope of this antibody, 23 overlapping synthetic peptides (15-mers) covering the entire sequence (1-235) of the beta-chain of C4BP were used. When the immobilized peptides were screened for their ability to bind mAb 6F6, only peptide beta(51-65) showed high-affinity binding. The apparent affinity of mAb 6F6 for immobilized peptide beta(51-65) was somewhat similar to that for native C4BP with Kd approximately 1 nM for C4BP and approximately 9 nM for peptide beta(51-65). Peptide beta(51-65) inhibited the binding of the mAb 6F6 to immobilized C4BP with half-maximal inhibition at 30 microM peptide. Clotting assays of protein S anticoagulant cofactor activity using a factor Xa-1-stage assay with activated protein C allow measurement of free protein S in solution since only free protein S is active. Studies using such clotting assays showed that preincubation of C4BP with either mAb 6F6 or polyclonal anti-beta(31-45) antibodies inhibited the formation of the complex between C4BP and protein S. Previous studies showed that, although peptide beta(51-65) itself does not inhibit complex formation, peptide beta(31-45) does bind directly to protein S and does inhibit protein S binding to C4BP. The three-dimensional structure of the first SCR (residues 2-60) of the C4BP beta-chain was made on the basis of homology modeling.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M(-)(1) s(-)(1), k(306) = 2.7 x 10(7) M(-)(1) s(-)(1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubation of protein S with C4BP resulted in a total abrogation of protein S cofactor activity. C4BP also protected FVa from inactivation by APC in the absence of protein S. Control experiments with CLB-PS13, a monoclonal anti-protein S antibody, indicated that inhibition of FVa inactivation by C4BP was not mediated through contaminating traces of protein S in our reaction systems. Protection of FVa was prevented by a monoclonal antibody directed against the C4BP alpha-chain. Recombinant rC4BPalpha comprised of only alpha-chains also protected FVa, but in the presence of protein S, the level of protection was decreased, since rC4BPalpha lacks the beta-chain responsible for C4BP binding to protein S. A truncated C4BP beta-chain (SCR-1+2) inhibited protein S cofactor activity, but had no effect on FVa inactivation by APC in the absence of protein S. In conclusion, C4BP protects FVa from APC-catalyzed cleavage in a protein S-independent way through direct interactions of the alpha-chaims of C4BP with FVa and/or APC.

The interaction between vitamin K-dependent protein S and the C4b-binding protein (C4BP) was studied using surface plasmon resonance and genetic engineering. The affinity, as well as association and dissociation rates of the complex, was measured for human and bovine protein S at five different calcium concentrations. The binding to C4BP of six protein hybrids containing different parts of coagulation factor IX and protein S was studied in the absence and presence of calcium. The results show that dissociation of the human protein S-C4BP complex is extremely slow in the presence of > or = 10 microM calcium (k(off) = 7 x 10(-6) s(-1)) and the association rate constant is k(on) = 7 x 10(4) M(-1) s(-1). Human and bovine protein S were found to bind to human C4BP with the same affinity, K(D) = 0.1 nM, but the rates of association and dissociation were higher for the bovine protein S (k(on) = 2 x 10(5) M(-1) s(-1), k(off) = 2 x 10(-5) s(-1)). In the absence of calcium, the affinity for C4BP was reduced by a factor of 65 for human protein S and by a factor of 40 for bovine protein S. The decreased affinity could be mainly attributed to an increased off-rate (12-17-fold), while the on-rate decreased 3-4-fold. The studies using chimeric proteins show that the portion of protein S that is responsible for binding to C4BP is fully contained in the two laminin-G-type repeats, which are homologous to the sex hormone binding globulin (SHBG). All hybrids that contain the laminin-G-type repeats bind to C4BP with the same affinity as recombinant protein S, whereas hybrids lacking these repeats show no detectable binding to C4BP. The present data also suggest that the effect of calcium on the C4BP-binding properties is mediated by calcium binding site(s) in the laminin-G-type repeats.

Several plasma and membrane proteins belong to a superfamily of structurally related proteins that contain internal homology of a variable number (2-30) of repeating units. Each SCR (short consensus repeat) unit is approximately 60 amino acid residues in length, with the positions of 1 Trp, 2 Pro, and 4 Cys residues being conserved. The aim of this study was to provide experimental evidence that each SCR may exist as an independent structural domain maintained by disulfide bonds. The well-characterized C4b-binding protein (C4BP) with eight SCR units in each of its seven identical chains was chosen for this study. Analysis of the disulfide-bonding pattern indicated that intrachain disulfide bonds may be localized within each SCR unit, with the first and third and the second and fourth half-cystines in each unit being linked. This pattern of disulfides may confer to C4BP (and to other structurally related proteins) a conformation which apparently allows the assembly of the SCR units (4-30) in a tandem fashion. Such an arrangement of the polypeptide chain(s) may explain, in part, the elongated shape of these protein molecules. The structural motif of the SCR units of C4BP is discussed in relation to those previously described for the type II domain of fibronectin and the kringle structure present in various proteins of the coagulation system.

Protein S is a vitamin K dependent protein of unknown function, which is present in mammalian plasma. It was isolated from bovine plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, and column chromatography on DEAE-Sephadex, heparin-agarose, and polyhomoarginine-Sepharose. Bovine Protein S (Mr 64,200) is a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Thr-Leu-Leu-. It contains 7.0% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. Human Protein S (Mr 69,000) is also a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Ser-Leu-Leu-. It contains 7.8% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. These results indicate that Protein S from bovine or human plasma shows many similarities to the other vitamin K dependent proteins present in plasma.