A tetrapeptide, residues 6 to 9 in normal prothrombin, was isolated from the NH2-terminal, Ca2+-binding part of normal prothrombin. The electrophoretic mobility of the peptide was too high to be explained entirely by its amino-acid composition. According to 1H nuclear magnetic resonance spectroscopy and mass spectrometry, the peptide contained two residues of modified glutamic acid, -y-carboxyglutamic acid (3-amino-1,1,3-propanetricarboxylic acid), a hitherto unidentified amino acid. This amino acid gives normal prothrombin the Ca2 +_ binding ability that is necessary for its activation. Observations indicate that abnormal prothrombin, induced by the vitamin K antagonist, dicoumarol, lacks these modified glutamic acid residues and that this is the reason why abnormal prothrombin does not bind Ca2+ and is nonfunctioning in blood coagulation.Prothrombin is a plasma glycoprotein that is activated during the process of blood coagulation to the proteolytic enzyme thrombin. The biosynthesis of prothrombin is vitamin K dependent, and deficiency of this vitamin or administration of the vitamin K antagonist, dicoumarol, gives rise to an abnormal prothrombin which does not function in blood coagulation (1-6). The activation of prothrombin in vivo requires the binding of Ca2+ (7); abnormal prothrombin does not bind Ca2+ (2, 8, 9). During the activation of normal prothrombin an NH2-terminal fragment (molecular weight approximately 25,000) is split off; the difference between abnormal and normal prothrombin has been localized to this part of the molecule. Evidence has been produced that the difference is due to the lack of certain prosthetic groups in abnormal prothrombin (10-13).In an endeavour to define the difference between normal and abnormal prothrombin, the NH2-terminal fragments from both proteins were isolated and degraded further. A heptapeptide from normal prothrombin (residues 4 to 10) and a corresponding heptapeptide from abnormal prothrombin were isolated by BrCN degradation and trypsin digestion. The heptapeptide from normal prothrombin differed from the corresponding peptide in abnormal prothrombin in that it had a higher anodal electrophoretic mobility at pH 6.5 (13).By extensive proteolytic digestion, the heptapeptide from normal prothrombin was degraded to a tetrapeptide. This tetrapeptide, containing residues 6 to 9, still had an abnormally high anodal electrophoretic mobility at pH 6.5. This paper reports evidence that each of the two glutamic acid residues of this peptide are modified by replacement of one hydrogen on the -y carbon atom by a carboxyl group. This work will be described in greater detail elsewhere. MATERIALS AND METHODSIsolation of Tetrapeptide. The heptapeptide from normal prothrombin (residues 4 to 10) (ref. 13) was first thoroughly digested with aminopeptidase M (Sigma) and afterwards with carboxypeptidase B (Sigma). A tetrapeptide was isolated from the digest by gel chromatography on Sephadex G-25 superfine and obtained in pure form as judged by high voltage electrophoresis at...

Reversible membrane binding of gamma-carboxyglutamic acid (Gla)-containing coagulation factors requires Ca(2+)-binding to 10-12 Gla residues. Here we describe the solution structure of the Ca(2+)-free Gla-EGF domain pair of factor x which reveals a striking difference between the Ca(2+)-free and Ca(2+)-loaded forms. In the Ca(2+)-free form Gla residues are exposed to solvent and Phe 4, Leu 5 and Val 8 form a hydrophobic cluster in the interior of the domain. In the Ca(2+)-loaded form Gla residues ligate Ca2+ in the core of the domain pushing the side-chains of the three hydrophobic residues into the solvent. We propose that the Ca(2+)-induced exposure of hydrophobic side chains is crucial for membrane binding of Gla-containing coagulation proteins.

Protein S, a recently described vitamin K-dependent plasma protein, is shown to exist in two forms in plasmafree protein andlin complex with C4b-binding protein. C4b-binding protein is involved in the regulation of the rate of complement activation. A major proportion of C4b-binding protein in plasma is in complexwith protein S. The complex is a major and previously unrecognized component of the group of plasma proteins that adsorbs to barium citrate. The complex dissociates in the presence of NaDodSO4, indicating that C4b-binding protein and protein S are held together by noncovalent bonds. Uncomplexed C4b-binding protein waspurified from the supernatant after barium citrate adsorption. On NaDodSO4/polyacrylamide gels without reduction, it appeared to have a slightly faster migration rate than the C4b-binding protein dissociated from the complex with protein S. After reduction, the subunits-of-the two forms ofC4b-binding protein appeared to have identical molecular weights. Furthermore, there is an equilibrium between free and bound protein S in plasma. METHODS AND MATERULS Protein S was isolated from 4 liters of freshly frozen normal human plasma by using a modification of existing techniques '(3). C4bp-S was purified from human plasma by barium citrate adsorption, and chromatography on DEAE-Sephacel and on Sepharose CL-4B (to be published). Uncomplexed C4bp was purified from the supernatant after barium citrate adsorption essentially as described (5). The purified proteins were labeled with, "2I by the lactoperoxidase method (10).Electrophoretic and Immunological Techniques. Antibodies against protein S and C4bp were raised in rabbits. Two other antisera against C4bp were also available, one kindly provided by Anders Sjoholm (Inst. of Medical Bacteriology, University ofLund, Lund, Sweden) and the other by A. R. Bradwell (Dept. of Immunology, University ofBirmingham, Birmingham, England). NaDodSOpolyacrylamide disc gel electrophoresis, agarose gel electrophoresis, electroimmunoassay, crossed immunoelectrophoresis, and double immunodiffusion were performed by standard methods (for references see ref. 11).Human plasma protein S was analyzed by radioimmunoassay.Fifty jud of "II-labeled protein S (125I-protein S; approximately 25 ng), 50 uld of rabbit anti-protein S diluted 1:3200 in assay buffer (10 mM Tris/10 mM EDTA/0. 15 M NaCl, pH 8.0, containing S mg ofbovine serum albumin per ml), 50 Al of sample, and 350 tJ of assay buffer also containing normal rabbit serum diluted 1:320 were added to plastic tubes (11 x 75 mm). The tests were run in triplicate. The reaction mixtures were incubated overnight at 40C, and then 500 ,1 of goat anti-rabbit IgG antiserum diluted 1:40 in the assay buffer containing 5% (wt/ vol) polyethylene glycol 6000 were added. The tubes were incubated for 1 hr at room temperature and -then centrifuged at 2500 X g for 15 min. The supernatants were decanted, and the radioactivity in the precipitates was measured in a gamma counter. Standard curves were prepared with dilutions of purif...

Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation. A human liver cDNA library constructed in bacteriophage Xgtll was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S. Several cDNA clones were isolated and sequenced. The combined cDNA sequences encoded the mature protein and 15 residues ofthe leader sequence when compared to bovine protein S. Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S. After an NH2-terminal y carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.Protein S is a single-chain plasma glycoprotein that undergoes vitamin K-dependent y-carboxylation during its biosynthesis (1-4). The concentration of protein S in human blood plasma is -25 mg/liter (4). Both human and bovine protein S have been purified from plasma, and recently the amino acid sequence of the bovine protein was established (5). Unlike the vitamin K-dependent clotting factors, protein S is not a proenzyme to serine protease but functions as a cofactor to activated protein C (6, 7). Patients with hereditary protein S deficiency, as well as those with protein C deficiency, suffer from a predisposition to venous thrombosis (8, 9). In addition to its established role as a cofactor to activated protein C, a regulatory role for protein S in the complement system has been suggested based on the observation that half of protein S in plasma is in a 1:1 complex with complement component C4b-binding protein (10,11).Bovine protein S has 11 y-carboxyglutamic acid (Gla in sequences) residues (12). In addition, acid hydrolysates ofthe bovine protein have been found to contain 03-hydroxyaspartic acid (Hya in sequences) (13-15), which is located in regions homologous to the epidermal growth factor (EGF) precursor (16) in all vitamin K-dependent plasma proteins except prothrombin (13-15, 17, 18); its function is unknown. We now report the isolation and sequence of human cDNA clones that code for mature human protein S. MATERIALS AND METHODSA human fetal liver cDNA library in phage Xgtll was prepared by a modification of the procedure of Gubler and Hoffman (19) similar to that described by Lapeyre and Amalric (20). The library contained >6 x 107 recombinants with inserts averaging 1800 nucleotides in length. Approximately 2 x 107 plaques from the amplified library were screened by standard techniques (21,22). RNA blot-hybridization analysis was conducted by standard methods (23). Nick-translated DNA fragments from a bovine protein S cDNA clone, pBLS-2400 (5), and a human protein C cDNA clone (N. Capalucci and R.W., unpublished data) were used as probes.For sequence analysis, 200...

An assay was developed for the measurement of human protein C inhibitor antigen (PCI) in blood plasma and other biological fluids. Both native PCI, modified inhibitor, and complexes of inhibitor with activated protein C or plasma kallikrein could be measured with the assay. Inhibitor antigen concentrations were found to be very high in seminal plasma (> 200 mg/liter), more than 40 times the concentration of PCI found in blood plasma. The inhibitor in seminal plasma was unable to form complexes with activated protein C. Gel filtration and immunoblotting findings indicated that the inhibitor in seminal plasma is present in a high molecular mass complex or cleaved to its modified form. As PCI antigen was absent from seminal plasma of patients with dysfunctional seminal vesicles, the seminal vesicle glands would appear to be the major source of seminal plasma PCI, a conclusion supported by immunohistochemical demonstration of the presence of PCI epitopes in the secretory epithelium of the seminal vesicles. Specific PCI immunoreactivity was also shown to be present in the testes, the epididymis glands, and the prostate, suggesting the inhibitor to have a complex or multiple function in the male reproductive system. Conclusive evidence ofa local synthesis of PCI in the four male sex glands was provided by Northern blot analysis of RNA from these organs.

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