The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p'-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the "tenase" complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.

Annals of the New York Academy of SciencesVolume 714, Issue 1 p. 32-40 Macromolecular Complex Assembly of Prothrombinase Is a Central Process of Thrombosis UMA SINHA, UMA SINHA COR Therapeutics, Inc. 256 East Grand Avenue South San Francisco, California 94080Search for more papers by this authorSTAN HOLLENBACH, STAN HOLLENBACH COR Therapeutics, Inc. 256 East Grand Avenue South San Francisco, California 94080Search for more papers by this authorDAVID L. WOLF, Corresponding Author DAVID L. WOLF COR Therapeutics, Inc. 256 East Grand Avenue South San Francisco, California 94080Corresponding author.Search for more papers by this author UMA SINHA, UMA SINHA COR Therapeutics, Inc. 256 East Grand Avenue South San Francisco, California 94080Search for more papers by this authorSTAN HOLLENBACH, STAN HOLLENBACH COR Therapeutics, Inc. 256 East Grand Avenue South San Francisco, California 94080Search for more papers by this authorDAVID L. WOLF, Corresponding Author DAVID L. WOLF COR Therapeutics, Inc. 256 East Grand Avenue South San Francisco, California 94080Corresponding author.Search for more papers by this author First published: April 1994 https://doi.org/10.1111/j.1749-6632.1994.tb12028.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1 Mann, K. G., R. J. Jenny & S. Krishnaswamy. 1988. Co-factor proteins in the assembly and expression of blood clotting enzyme complexes. Annu. Rev. Biochem. 57: 915–956. 2 Suttie, J. W. & C. M. Jackson. 1977. Prothrombin structure, activation and biosynthesis. Physiol. Rev. 52: 1. 3 Mann, K. G., M. E. Nesheim, W. R. Church, P. E. Haley & S. Krishnaswamy. 1990. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 76: 1–16. 4 Nesheim, M. E., C. Kettner, E. Shaw & K. G. Mann. 1981. Co-factor dependence of factor Xa incorporation into the prothrombinase complex. J. Biol. Chem. 256: 6537–6540. 5 Skogen, W. F., C. T. Esmon & A. C. Cox. 1984. Comparison of coagulation factor Xa and des(1–44) factor Xa in the assembly of prothrombinase. J. Biol. Chem. 259: 2306–2310. 6 Wolf, D. L., U. Sinha, T. E. Hancock, P. H. Lin, T. L. Messier, C. T. Esmon & W. R. Church. 1991. Design of constructs for the expression of biologically active recombinant human factors X and Xa. Kinetic analysis of the expressed proteins. J. Biol. Chem. 266: 13726–13730. 7 Sinha, U., T. Hancock, P. H. Lin, S. Hollenbach & D. Wolf. 1992. Expression, purification and characterization of inactive human coagulation factor Xa(Asn322 Ala419). Protein Exp. Purif. 3: 518–524. 8 Owen, M., S. O. Brennan, J. H. Lewis & R. W. Carrell. 1983. Mutation of antitrypsin to antithrombin. N. Engl. J. Med. 309: 694–698. 9 Haley, P. E., M. F. Doyle & K. S. Mann. 1989. The activation of bovine protein C by factor Xa. J. Biol. Chem. 264: 16303–16310. 10 Discipio, R. G., M. A. Hermodson & E. W. Davie. 1977. Activation of human factor X (Stuart factor) by a protease from Russell's viper venom. Biochemistry 16: 5253–5260. 11 Sprang, S. R., R. J. Fletterick, L. Graf, W. Rutter & C. S. Craik. 1988. Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants. Crit. Rev. Biotechnol. 8: 225–236. 12 Knight, C. G. 1986. Characterization of enzyme inhibition. In Proteinase Inhibitors. A. J. Barrett & G. Salvesen, Eds.: 23–51. Elsevier. Amsterdam . 13 Collen, D., J. M. Stassen & M. Verstraete. 1983. 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In Hemostasis and Thrombosis. R. W. Colman, J. Hirsch, V. J. Marder & E. W. Salzman, Eds.: 1063–1072. J. B. Lippincott. Philadelphia , PA. 18 Hirsh, J. 1991. Rationale for development of low-molecular-weight heparins and their clinical potential in the prevention of postoperative venous thrombosis. Am. J. Surg. 161: 512–518. 19 Benedict, C. R., J. Ryan, B. Wolitzky, R. Ramos, M. Gerlach, P. Tijburg & D. Stern. 1991. Active site-blocked factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting extravascular coagulation in a canine thrombosis model. J. Clin. Invest. 88: 1760–1765. 20 Sitko, G. R., D. R. Ramjit, I. I. Stabilito, D. Lehman, J. J. Lynch & G. P. Vlasuk. 1992. Conjunctive enhancement of enzymatic thrombolysis and prevention of thrombotic reocclusion with the selective factor Xa inhibitor, tick anticoagulant peptide. Circulation 85: 805–815. Citing Literature Volume714, Issue1Platelet–dependent Vascular OcclusionApril 1994Pages 32-40 ReferencesRelatedInformation

The thrombin receptor has been shown to be a novel member of the family of G-protein coupled receptors (Vu, T.-K. H., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. (1991) Cell 64, 1057-1068). This receptor appears to be activated through a thrombin-mediated proteolytic mechanism which exposes a "tethered ligand" responsible for receptor activation. In order to investigate the initial interactions of thrombin with this receptor, we have constructed cell lines which express high levels of the human thrombin receptor and studied the binding of various forms of thrombin to the cell surface. Analysis of transfected cells with thrombin receptor monoclonal antibodies identified a particular cell line (clone #5-18) which displayed > 150,000 thrombin receptors per cell. Clone #5-18 appeared to express functional receptors since treatment with thrombin resulted in both a 15-20 fold increase of cytoplasmic phosphoinositide levels and a comparable shift in the EC50 of thrombin-mediated calcium mobilization when compared to non-transfected CHO cells. Binding of 125I-alpha-thrombin to clone #5-18 did not reach equilibrium at 37 degrees C. However, direct binding studies of 125I-alpha-, 125I-diisopropylphospho (DIP)-alpha-, and 125I-beta-thrombin to clone #5-18 demonstrated that binding at 4 degrees C was saturable and reversible for each ligand. Analysis of the binding data revealed Kd's of 0.8 nM, 0.7 nM and 9.7 nM for 125I-alpha-, 125I-DIP-alpha- and 125I-beta-thrombin respectively. Association of 125I-alpha-, DIP-alpha, and beta-thrombin could be competed by unlabelled alpha- and DIP-alpha-thrombin. Unlabelled beta-thrombin, which has a modified anion-binding exosite, was a poor competitor for 125I-alpha- and 125I-DIP-alpha-thrombin, but did compete for 125I-beta-thrombin. In addition, the hirudin54-65 peptide competed at submicromolar concentrations for the binding of alpha- and DIP-alpha-thrombin, but not for beta-thrombin. This peptide binds specifically at the anion-binding exosite of alpha-thrombin and has been shown to have a lower affinity for beta-thrombin. These results demonstrate directly a high affinity interaction between thrombin and its receptor, and suggest that an important component is the high affinity association of the thrombin receptor with the anion-binding exosite of thrombin.

Recombinant catalytically inactive factor Xa (factor rXai) is capable of assembly into inactive prothrombinase complexes, thus serving as a competitive inhibitor (Ki = 0.3nM) of active factor Xa. In order to study the role of gamma carboxylation in prothrombinase complex assembly, we have prepared differentially gamma carboxylated factor rXai and have measured the activities of these proteins in prothrombinase complex inhibition and in extension of plasma clotting. A factor rXai preparation containing 8 out of a possible maximum of 11 g carboxyglutamic acid (GLA) residues was found to be as active as chemically inactivated plasma factor Xa which was fully gamma carboxylated. Loss of a single additional g carboxyglutamic acid in the recombinant protein, however lead to a marked loss in activity. Factor rXai preparation with 8 GLA residues is also detected by a monoclonal antibody specific for a GLA dependent epitope. Thus assembly of the factor Va/Xa complex on phospholipid membranes does not require the presence of all of the g carboxyglutamic acid residues present in the plasma protein.

Factor X is a plasma protein involved in both the intrinsic and extrinsic pathways of blood coagulation. Post-translational modifications of the protein involve gamma-carboxylation of specific glutamic acid residues, beta-hydroxylation of one aspartic acid residue, and N- and O-linked glycosylation. Even though it is known that gamma-carboxylation is instrumental in regulating biological activity, the role of glycosylation in the function and properties of factor X has not been previously investigated. We utilized lectin binding and glycosidase treatment to investigate the functional role of carbohydrates on the activation peptide of factor X. Sambucus nigra agglutinin, a lectin that binds to sialic acid terminally linked alpha(2-6) to galactose or N-acetyl-galactosamine inhibits activation of human factor X in a dose-dependent manner. Inhibition of activation was observed for both intrinsic (factor IXa/VIIIa) and extrinsic (factor VIIa/tissue factor) pathway complexes. In accordance with this, selective removal of sialic acid residues on the activation peptide of factor X by neuraminidase also results in a drastic reduction of activation of the zymogen by these complexes. Corresponding reduction of activity in classical clotting assays (activated partial thromboplastin time and prothrombin time) also agrees with this observation. These results suggest a possible role of N-linked carbohydrates in the activation of factor X.

The biological effects of platelet-derived growth factor (PDGF) are mediated by cell surface alpha and beta PDGF receptors, which, as a result of ligand binding, undergo dimerization in a manner consistent with PDGF being bivalent. In order to directly demonstrate PDGF bivalency and to define the binding of PDGF AB to isolated beta receptor, we developed solid-phase binding assays using purified recombinant extracellular domain of human PDGF receptors. PDGF AA, AB, and BB were prepared from the monomeric chains expressed in Escherichia coli, and each was purified to homogeneity; PDGF AB contained < 0.5% of either homodimer. The interactions of these isoforms with immobilized PDGF receptors were examined by several approaches. Scatchard analysis revealed high affinity binding (Kd = 0.5-1.0 nM) of radiolabeled PDGF AA and AB to alpha receptor and of PDGF BB to both receptor subtypes. Contrary to previous reports, PDGF AB also bound beta receptor with high affinity (Kd = 0.9 nM). When a B-chain-specific monoclonal antibody that recognizes the putative binding domain of PDGF BB was used for ligand detection, we found that PDGF AB binding to beta receptor occurred exclusively through the B-chain subunit, whereas binding to alpha receptor occurred through either subunit. In addition, site-directed mutagenesis was used to specifically inactivate the B chain of PDGF AB, which eliminated binding to the beta receptor without affecting alpha receptor binding. These results establish that PDGF is bivalent and that monovalent ligand retains high affinity receptor binding.

Platelet derived growth factor (PDGF) induces activation of the protein tyrosine kinase domain of the PDGF receptor, resulting in receptor dimerization and the initiation of mitogenesis in responsive cells. In order to identify domains of the receptor involved in these processes, a panel of monoclonal antibodies (MAbs) against the extracellular region of the human PDGF receptor was developed and screened to identify which of these specifically block PDGF binding. One of these, MAb 2A1E2, binds PDGF beta receptor with high affinity and blocks PDGF BB binding in a whole cell binding assay with an IC 50 of 0.1 nM. Inhibition of binding results in the inhibition of ligand-induced receptor phosphorylation, dimerization and mitogenesis in cells expressing the PDGF beta receptor. MAb 2A1E2 has been mapped to the fifth Ig domain of the PDGF beta receptor, implying that this domain is important for ligand binding, dimerization and/or activation. The potency of MAb 2A1E2 for inhibiting PDGF BB binding indicates that this antibody is ideally suited to identify and characterize PDGF BB-induced biological responses.

We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and gamma-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes (Ki = 3 x 10(-10) M). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.

Activation of vitamin K-dependent plasma proteases occurs by specific interaction with components of the blood coagulation cascade. In this report, we describe the direct expression and enzymatic characterization of the human coagulation zymogen factor X and its activated form, factor Xa, from transformed Chinese hamster ovary fibroblast cell lines. Expression was achieved using either a full-length factor X cDNA or a unique mutant factor Xa cDNA. The functional factor Xa precursor contained a novel tripeptide bridge in place of the native 52-amino acid activation peptide. This mutation allowed for intracellular processing and secretion of the activated form of factor X. Secreted recombinant factors X (rX) and Xa (rXa) were purified by sequential anion-exchange and immunoaffinity chromatography. The enzymatic activities of factors rX and rXa were compared with those of plasma factors X and Xa in three independent assay systems. In comparison to human plasma factor X, the amidolytic, prothrombinase complex, and plasma clotting activities of factor rX were 50, 85, and 43%, respectively. The corresponding comparative activities for factor rXa were 32, 64, and 48%, respectively. The ability to directly express mutant forms of biologically active human factor X will facilitate the structure/function analysis of this important blood coagulation protein and may lead to the development of novel coagulation inhibitors.

Luteinizing hormone (LH) is a member of a glycoprotein hormone family which includes the pituitary-derived follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH), as well as the placental derived chorionic gonadotropin (CG) (1). The hormone family is characterized by a noncovalent complex of two unique subunits, a and g. While the a subunit is shared, the 6 subunit differs and confers receptor specificity (1). In this report, we present the sequence of the dog 1LH cDNA isolated from a XgtlO dog pituitary cDNA library (5) and compare its amino acid (aa) and nucleic acid homologies to that of rat (2), cow (3), and human (4) OLH. The mature canine OLH protein (1-121 aa) shows an 89% homology with rat, 84% homology with cow and 74% homology with human OLH. Similarities at the nucleic acid level across the same coding region are 84% (rat), 88% (cow), and 82% (human).