Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

Direct analysis reveals an absence of gamma-carboxyglutamic acid in cancer procoagulant from human tissues.

Additional carboxylation of glutamic acid by vitamin K-dependent gamma-carboxylase is a common posttranslational modification of many proteins, including some of blood clotting factors. Vitamin K-antagonists, such as warfarin, are often included in the therapy of malignant disease, decreasing the blood coagulation potential. Cancer procoagulant, a direct blood coagulation factor X activator from malignant tissue, is considered as a vitamin K-dependent protein, so it could serve as one of possible targets for the therapy with warfarin. However, there is still no experimental data demonstrating directly the presence of gamma-carboxyglutamic acid (Gla) in a cancer procoagulant molecule. The presence of Gla in cancer procoagulant isolated from human amnion-chorion membranes and from human malignant melanoma WM 115 cell line was analyzed directly, using specific anti-Gla monoclonal antibodies. There was no detectable amount of Gla in cancer procoagulant isolated from fetal or malignant tissue. Cancer procoagulant from human tissues does not contain Gla-rich domain. The finding indicates that cancer procoagulant is rather a poor target for warfarin therapy of malignant disease.

Detection of vitamin K-dependent proteins in venoms with a monoclonal antibody specific for gamma-carboxyglutamic acid.

gamma-Carboxyglutamic acid (Gla) is an unusual amino acid that is synthesized post-translationally from glutamate in a vitamin K-dependent reaction. The dicarboxylic side chain of Gla chelates Ca(2+), a property important for the biological activity of vitamin K-dependent proteins. To date, Gla-containing polypeptides have been identified in venom from two groups of organisms: elapid snakes, and snails of the genus Conus. In certain elapid snakes, a gamma-carboxylated coagulation factor Xa-like protein is a component of the venom whereas cone snails utilize Gla in a range of peptide neurotoxins. Using a monoclonal antibody that specifically recognizes Gla residues, venom samples from various organisms were screened by western blotting and immunofluorescence assays. Amino acid analyses were also performed on most samples. A survey of 21 snake species from 12 genera detected gamma-carboxylated polypeptides only in venom of snakes from the elapid subfamily Acanthophiinae. Gla-containing polypeptides were also observed in cone snail venom but not in venom or toxic salivary secretions from several other organisms. The Gla-specific antibody used here provides a simple immunochemical means to detect gamma-carboxylated polypeptides in venom and may allow new species to be identified that utilize Gla in the biosynthesis of toxic polypeptides.

Synthesis of gamma-carboxylated polypeptides by alpha-cells of the pancreatic islets.

gamma-Carboxylated proteins were detected in normal human pancreas by immunohistochemistry with a monoclonal antibody (M3B) specific for gamma-carboxyglutamyl residues. Staining appeared to be localized to the glucagon-secreting alpha-cells in the islets of Langerhans. Consistent with this, sections from a glucagonoma were stained much more intensely with the M3B antibody than those from an insulinoma. A murine alpha-cell line (alphaTC1 Clone 9) was cultured and gamma-carboxylated polypeptides, identified immunologically as prothrombin, protein S and (tentatively) Gas6, were isolated from the intracellular compartment by chromatography on an M3B-coupled resin. As in liver, prothrombin is synthesized by alpha-cells as a gamma-carboxylated zymogen that can be cleaved by ecarin to form an active serine protease that is inhibited by hirudin. The pancreas thus appears to be a novel site of synthesis for certain vitamin K-dependent proteins.

A human antibody that binds to the gamma-carboxyglutamic acid domain of factor IX is a potent antithrombotic in vivo.

10C12, a human antibody F(ab')2, which specifically binds to the Gla domain of factor IX, interfered with all known coagulation processes that involve factor IX/IXa. These include the function of the intrinsic Xase complex and the activation of zymogen factor IX by factor XIa and by the tissue factor:factor VIla complex. Furthermore, 10C12 potently inhibited activated partial thromboplastin clotting times (APTT) in plasma of guinea pig and rat, thus enabling in-vivo evaluation. In guinea pigs, a bolus administration of 10C12 (10 microg/kg) prevented cyclic flow variations in damaged carotid arteries without affecting coagulation or bleeding parameters. At a 100-fold higher dose, 10C12 had no effect on normal hemostasis as assessed by the cuticle bleeding time. At this dose, 10C12 was also efficacious in a rat arterial thrombosis model, substantially reducing clot weight and duration of vessel occlusion while prolonging ex-vivo APTT only 1.2-fold. The dose of heparin required to produce comparable antithrombotic effects prolonged the APTT by 12-fold and increased the tail bleeding time (TBT) by 8-fold. In contrast, 10C12 had no effect on TBT. However, rat tails showed a tendency for rebleeding which 10C12 exacerbated. In conclusion, the antithrombotic potency of the 10C12 antibody in two species provides evidence for an important role of F.IX, and its Gla domain in particular, during thrombogenesis under arterial flow conditions. The relative safety at effective doses of this fully human antibody suggests that it may have therapeutic value for treatment of thrombotic disorders.

Monoclonal antibody (VII-M31) to bovine factor VII: a specific epitope in the gamma-carboxyglutamic acid domain.

A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor VIIa in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Kd = 1.12 x 10(-10)M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2(+)-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a gamma-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII.

[Preparation of monoclonal antibodies against human bone gamma-carboxyglutamic acid containing protein (BGP) and its immunohistochemical application to various human tissues].

Bone gamma-carboxyglutamic acid containing protein (BGP) has been considered to play an important role in mineralization of bone, because this protein is capable of binding to calcium ions. However, the function and localization of BGP in normal bones have not yet been fully understood. In this study, we prepared monoclonal antibodies against human BGP, by immunizing BALB/c mice with human BGP. All five monoclonal antibodies obtained were shown to bind specifically to human BGP, but not to the other bone-derived proteins. In immunohistochemical analysis, these antibodies were shown to react with osteoblasts and osteocytes in normal human bone tissues, but not with chondrocytes, osteoclasts and osteoid tissues. These antibodies are expected to be useful for the diagnosis of human osteoblastic tumors and also for the analysis of the role of BGP in the ossification process in bone.

The quantification of gammacarboxyglutamic acid residues in plasma-osteocalcin.

In this paper we describe an assay procedure for determining the amount of gammacarboxyglutamic acid (Gla) residues in serum- or plasma-osteocalcin. The test includes removing by ethanol precipitation the majority of the proteins from the plasma (e.g., the Gla-containing coagulation factors) and the specific extraction of osteocalcin with the aid of immobilized immunopurified antibodies. It is demonstrated that the Gla-content of circulating osteocalcin from normal cows is similar to that of osteocalcin obtained from bone. Hence, the fact that part of the newly synthesized osteocalcin does not bind to the hydroxyapatite matrix in bone cannot be explained by an undercarboxylation of the molecule.

Immunolocalization of gamma-carboxyglutamic acid containing proteins in developing rat bones.

The localization of a gamma-carboxyglutamic acid (Gla)-containing protein, BGP (also called osteocalcin) was examined in developing calvaria, alveolar bones and long bones of newborn rats by immunostaining with the peroxidase-antiperoxidase method. In undemineralized tissues osteoblasts of intramembranous bones stained positive; osteoid was negative, whereas young mineralized bone stained weakly. After a mild demineralization all bones stained positive; no staining was found in cartilage, muscles or soft connective tissues. In addition to the osteoblasts, osteocytes were also immunoreactive. Osteoclasts, identified by a subsequent staining for acid phosphatase activity, demonstrated no immunostaining for BGP. These data support the hypothesis that BGP is synthesized by osteoblasts and osteocytes and is subsequently deposited in the mineralizing bone.

Measurements of gamma-carboxyglutamate and circulating osteocalcin in normal children and adults.

We have measured serum osteocalcin by radioimmunoassay, and urinary gamma-carboxyglutamic acid (Gla) by Dowex-1 chromatography in healthy adults and children. Circulating osteocalcin is 6.8 +/- 0.5 and 5.8 +/- 0.5 ng/ml in adult males (n = 25) and females (n = 26), respectively. Values are higher in children (n = 26), ranging from 25-30 ng/ml in a 1-year-old and declining to the adult level at puberty. The ratio of urinary Gla/creatinine as a function of age parallels the pattern of serum osteocalcin. Free Gla ranges from a high of 150 +/- 20 mumol Gla/g creatinine in infants (n = 17), decreasing until puberty at which time the excretion stabilizes at 44 +/- 4 mumol Gla/g creatinine (n = 27). The greater amounts of circulating osteocalcin and urinary Gla most likely reflect synthesis of osteocalcin during growth. With epiphyseal closure, levels decline and, in the adult, probably represent normal bone remodelling.