A practical guide to the evaluation and treatment of hemophilia.
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Factor VIII↗
Biomedical subjects
B Furie
Publications and source records attributed to B Furie.
A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion.
The selectin family of adhesion molecules mediates the initial interactions of leukocytes with endothelium. The extracellular region of each selectin contains an amino-terminal C-type lectin domain, followed by an EGF-like domain and multiple short consensus repeat units (SCR). Previous studies have indirectly suggested a role for each of the extracellular domains of the selectins in cell adhesion. In this study, a panel of chimeric selectins created by exchange of domains between L- and P-selectin was used to directly examine the role of the extracellular domains in cell adhesion. Exchange of only the lectin domains between L- and P-selectin conferred the adhesive and ligand recognition functions of the lectin domain of the parent molecule. However, chimeric selectins which contained both the lectin domain of L-selectin and the EGF-like domain of P-selectin exhibited dual ligand-binding specificity. These chimeric proteins supported adhesion both to myeloid cells and to high endothelial venules (HEV) of lymph nodes and mesenteric venules in vivo. Exchange of the SCR domains had no detectable effect on receptor function or specificity. Thus, the EGF-like domain of P-selectin may play a direct role in ligand recognition and leukocyte adhesion mediated by P-selectin, with the lectin plus EGF-like domains collectively forming a functional ligand recognition unit.
Animals↗
Expression cloning of a functional glycoprotein ligand for P-selectin.
The initial adhesive interactions between circulating leukocytes and endothelia are mediated, in part, by P-selectin. We now report the expression cloning of a functional ligand for P-selectin from an HL-60 cDNA library. The predicted amino acid sequence reveals a novel mucin-like transmembrane protein. Significant binding of transfected COS cells to P-selectin requires coexpression of both the protein ligand and a fucosyltransferase. This binding is calcium dependent and can be inhibited by a neutralizing monoclonal antibody to P-selectin. Cotransfected COS cells express the ligand as a homodimer of 220 kd. A soluble ligand construct, when coexpressed with fucosyltransferase in COS cells, also mediates P-selectin binding and is immunocrossreactive with the major HL-60 glycoprotein that specifically binds P-selectin.
Amino Acid Sequence↗
The importance of specific gamma-carboxyglutamic acid residues in prothrombin. Evaluation by site-specific mutagenesis.
Prothrombin has 10 gamma-carboxyglutamic acid residues which are essential for the metal ion binding properties and membrane binding function of the protein. To assess the importance of each gamma-carboxyglutamic acid residue we made, by site directed mutagenesis, a series of mutant human prothrombins each with a single glutamic acid to aspartic acid substitution at positions 6, 7, 14, 16, 19, 20, 25, 26, 29, or 32 which are gamma-carboxylated in native prothrombin. Along with wild-type prothrombin, the prothrombin mutants were expressed in Chinese hamster ovary cells, purified by immunoaffinity chromatography using polyclonal anti-prothrombin antibodies, and shown by amino acid analysis to contain the expected number of gamma-carboxyglutamic acid residues. Only substitution of gamma-carboxyglutamic acid 6 with aspartic acid yields a protein with procoagulant activity, affinity for phospholipid and KM(app) for prothrombinase indistinguishable from wild-type prothrombin. In contrast, the conservative gamma-carboxyglutamic acid to aspartic acid mutation at positions 16, 26, or 29 results in proteins with little or no procoagulant activity, Kd(app) for binding to phospholipid at least 200-fold higher than wild-type prothrombin and a KM(app) for interaction with the prothrombinase complex nearly 100-fold higher than wild-type prothrombin. The mutations at residues 7, 14, 19, 20, 25, or 32 yielded proteins with intermediate procoagulant activities, between 15 and 52% of wild-type prothrombin. These data have been interpreted to suggest that there are certain gamma-carboxyglutamic acid residues which are important to maintaining the basic structure of the calcium-liganded Gla domain while other gamma-carboxyglutamic acid residues subserve other functions including membrane binding and interdomain interactions.
1-Carboxyglutamic Acid↗
Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.
A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.
Animals↗
The interaction of prothrombin with phospholipid membranes is independent of either kringle domain.
Prothrombin contains two kringle domains, a structural motif common to other plasma proteins involved in hemostasis and fibrinolysis. To determine the role of the kringle domains of prothrombin, we prepared three recombinant human prothrombin forms lacking the first kringle domain (residues 63-144; PT/delta K1), the second kringle domain (residues 144-249; PT/delta K2), or both kringle domains (63-249; PT/delta K1,2). The isolated prothrombin proteins were greater than 95% pure by SDS-polyacrylamide gel electrophoresis and were well carboxylated. PT/delta K1 displayed 50% of the specific coagulant activity of plasma prothrombin, PT/delta K2 had 10% of the specific coagulant activity, and PT/delta K1,2 was inactive. Polyclonal antibodies directed against the Ca(II)-specific conformer of prothrombin bound PT/delta K1 and PT/delta K2 with the same affinity as prothrombin, indicating that the Ca(II)-induced conformational transition does not involve sites on the prothrombin kringle domains. Gel filtration studies demonstrated that radiolabeled plasma prothrombin and all of the prothrombin kringle deletion mutants bound to phospholipid vesicles in the presence of Ca(II) but not in the presence of Mg(II) or EDTA. Relative dissociation constants of 1.10 +/- 0.75 and 0.49 +/- 0.18 microM were obtained by quasielastic light scattering for the interaction of phospholipid vesicles with plasma prothrombin and PT/delta K1, respectively. These data indicate that neither the first nor the second kringle domain contain unique sites for the interaction of prothrombin with phospholipid vesicles and are not required for prothrombin-phospholipid binding.
1-Carboxyglutamic Acid↗
Rapid phosphorylation and selective dephosphorylation of P-selectin accompanies platelet activation.
P-selectin, a receptor for neutrophils and monocytes, is an adhesion molecule on the surface of activated platelets that resides in the alpha granule membrane of unstimulated platelets. To determine whether phosphorylation of P-selectin might accompany platelet activation, P-selectin in resting and thrombin-stimulated platelets labeled with o-[32P]phosphate was immunoprecipitated with the monoclonal antibody AC1.2 directed against P-selectin. SDS-gel electrophoresis of the immunoprecipitates indicated about 10-20-fold higher levels of 32P incorporated into P-selectin from thrombin-activated platelets than in resting platelets, although both sets of platelets contained equivalent amounts of P-selectin. The lower limits of the molar ratio of phosphate to P-selectin in activated platelets is about 0.52 +/- 0.08. Other platelet agonists, including the thrombin receptor peptide (SFLLR), epinephrine, ADP, and collagen, similarly stimulated phosphorylation of P-selectin. The kinetics of P-selectin phosphorylation following thrombin stimulation was rapid, with maximum phosphorylation observed at 15-30 s. Phosphoamino acid analysis of the phosphorylated P-selectin revealed the rapid synthesis of phosphoserine, phosphothreonine, and phosphotyrosine, but 80-90% of the phosphotyrosine and phosphothreonine disappeared within 5 min of platelet activation while the maximal level of phosphoserine remained stable. The rapid phosphorylation and selective dephosphorylation of specific amino acids in P-selectin following platelet activation may be important for P-selectin function and signal transduction within platelets.
Adenosine Diphosphate↗
In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells.
Coagulation factor IX is a serine protease for which high-level expression of biologically active protein in heterologous cells is limited due to inefficient proteolytic removal of the propeptide as well as vitamin K-dependent carboxylation of multiple amino-terminal glutamic acid residues. We have overexpressed the vitamin K-dependent gamma-carboxylase cDNA and monitored its ability to improve factor IX processing in Chinese hamster ovary (CHO) cells. From amino acid sequence analysis of bovine liver vitamin K-dependent gamma-carboxylase, degenerate oligonucleotides were used to isolate a 3.5-kbp bovine cDNA that encoded a 758-residue open reading frame. Expression of the cDNA in COS-1 and CHO cells yielded 17- and 16-fold increases in the in vitro gamma-carboxylase activity of microsomal preparations, respectively. Anti-serum raised against a predicted peptide sequence reacted with a 94-kDa polypeptide in the partially purified bovine liver preparation as well as in stably transfected CHO cells. The amount of antibody reactivity correlated with the increased ability to carboxylate a peptide substrate in vitro. These results strongly support the conclusion that the cDNA encodes the vitamin K-dependent gamma-carboxylase. Transient transfection of the gamma-carboxylase expression vector into factor IX-expressing CHO cells did not improve the specific procoagulant activity of secreted factor IX. In contrast, transfection of an expression vector encoding the propeptide processing enzyme PACE (paired basic amino acid cleaving enzyme) did improve the specific activity of secreted factor IX by 3-fold. These results demonstrate that the ability of CHO cells to modify glutamic acid residues to gamma-carboxyglutamic acid in secreted factor IX is not limited by the expression of the vitamin K-dependent gamma-carboxylase alone.
Amino Acid Sequence↗
Membrane binding kinetics of factor VIII indicate a complex binding process.
Factor VIII functions as a component of the tenase enzyme complex upon phospholipid membranes. Factor VIII binds to phosphatidylserine-containing membranes and apparently provides high affinity binding sites for factor IXa upon these membranes. We have characterized the binding kinetics of human factor VIII with phosphatidylserine-containing membranes and directly compared the measured properties with those of factor V. The initial phase of association was evaluated in a stopped-flow apparatus by fluorescence energy transfer from aromatic residues in the protein to dansyl-labeled phosphatidylethanolamine in the vesicles. Association proceeded at an apparent second-order rate of 0.12 microM-1 s-1 for extruded phospholipid vesicles and 0.42 microM-1 s-1 for sonicated vesicles under pseudo-first-order conditions in which the phospholipid concentration determined the rate. Increased temperature resulted in more rapid association, and the effect decreased in the order extruded vesicles > sonicated vesicles > extruded vesicles of dioleoylphospholipids, indicating that the structure of the phospholipid membrane contributes to the activation energy of binding. The binding of fluorescein-labeled factor VIII to membranes supported on glass microspheres (lipospheres) was monitored by flow cytometry. Under conditions in which the factor VIII concentration determined the rate there was rapid initial association at 6.9 microM-1 s-1, accounting for half of the bound factor VIII, and a slower component of 0.87 microM-1 s-1, accounting for the other half. Likewise, the dissociation of factor VIII from liposphere membranes was biphasic with a faster component of 0.010 s-1 and a slower component of 0.0012 s-1. Rates of association and dissociation for factor V were similar to those for factor VIII and were biphasic. These results allow estimation of the size of the phospholipid sites that interact with factors VIII and V and suggest that both proteins bind to membranes via a multistep process in which rapid association is followed by a slower step yielding higher affinity binding.
Animals↗
Propeptide processing during factor IX biosynthesis. Effect of point mutations adjacent to the propeptide cleavage site.
Factor IX is synthesized in a precursor form with a propeptide that contains the gamma-carboxylation recognition site, an element which directs the post-translational gamma-carboxylation of adjacent glutamic acid residues. After protein synthesis, the propeptide is cleaved to yield the mature Factor IX. To study propeptide processing, anti-proFactor IX antibodies were prepared using a synthetic peptide based upon the sequence of the Factor IX propeptide. Immunoaffinity-purified anti-proFactor IX antibodies were reactive with Factor IX Cambridge, a mutant form of Factor IX containing the propeptide, but were not reactive with Factor IX. These antibodies were used to examine the proteolytic processing of forms of Factor IX containing point mutations at P6, P3, P2, P1, P1', P2', and P3' adjacent to the propeptide cleavage site in order to determine the requirement of each of these amino acids for propeptide cleavage. Furthermore, the hierarchy of different pairs of basic residues at positions P1 and P2 was analyzed. The mutated cDNA constructs were expressed in Chinese hamster ovary cells. Propeptide processing was examined using intrinsically labeled Factor IX immunoprecipitated with either anti-pro-Factor IX antibodies or anti-Factor IX:total antibodies, and the Factor IX species were separated by SDS-gel electrophoresis. Under the expression conditions employed, the propeptide of wild type Factor IX was almost completely removed, whereas Factor IX mutated to threonine at P1 was not cleaved. The percentage of propeptide cleaved varied with the amino acid sequences of residues P2 and P1, respectively: Lys-Arg (93%), Arg-Arg (66%), Thr-Arg (33%), Arg-Lys (19%), Lys-Lys (10%), and Lys-Thr (< 1%). Apart from alterations of basic amino acids at P1 and P2, nonconservative mutations at P6 and P3 decreased propeptide cleavage, whereas conservative mutations at P3, P1', P2', or P3' resulted in cleavage efficiencies approximately equal to that for wild type Factor IX. These results indicate that the preference of paired basic residues at P1 and P2 is similar to other endopeptidases active toward proteins secreted through the constitutive pathway and that the propeptide residues NH2-terminal to these paired basic residues are important in defining enzyme-substrate binding.
Amino Acid Sequence↗
Role of propeptide in vitamin K-dependent gamma-carboxylation.
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Amino Acid Sequence↗
Recombinant blood clotting proteins for hemophilia therapy.
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Animals↗
Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets.
The glycoprotein P-selectin is a cell adhesion molecule of stimulated platelets and endothelial cells, which mediates the interaction of these cells with neutrophils and monocytes. It is a membrane component of cell storage granules, and is a member of the selectin family which includes E-selectin and L-selectin. P-selectin recognizes both lineage-specific carbohydrate ligands on monocytes and neutrophils, including the Lewis x antigen, sialic acid, and a protein component. In inflammation and thrombosis, P-selectin may mediate the interaction of leukocytes with platelets bound in the region of tissue injury and with stimulated endothelium. To evaluate the role of P-selectin in platelet-leukocyte adhesion in vivo, the accumulation of leukocytes within an experimental thrombus was explored in an arteriovenous shunt model in baboons. A Dacron graft implanted within an arteriovenous shunt is thrombogenic, accumulating platelets and fibrin within its lumen. These bound platelets express P-selectin. Here we show that antibody inhibition of leukocyte binding to P-selectin expressed on platelets immobilized on the graft blocks leukocyte accumulation and inhibits the deposition of fibrin within the thrombus. These results indicate that P-selectin is an important adhesion molecule on platelets, mediating platelet-leukocyte binding in vivo, that the presence of leukocytes in thrombi is mediated by P-selectin, and that these leukocytes promote fibrin deposition.
Animals↗
N-bromoacetyl-peptide substrate affinity labeling of vitamin K dependent carboxylase.
Vitamin K dependent carboxylase (carboxylase) is a membrane-associated endoplasmic reticular enzyme that catalyzes the conversion of certain glutamate residues of essential blood coagulation proteins to gamma-carboxyglutamyl (Gla) residues. A series of N-bromoacetyl-peptide substrate affinity labels based on the Gla domain of these blood-clotting proteins was synthesized, and the substrate and inactivator kinetic parameters were assessed. The most promising of these affinity peptides, N-bromoacetyl-FLEELY, was both substrate for carboxylase and an irreversible time-dependent inactivator of the enzyme, inactivating 80% of carboxylase under pseudo-first-order conditions. Addition of saturating amounts of a competing peptide substrate completely abolished the inhibitory properties of N-bromoacetyl-FLEELY, consistent with inactivation occurring at the active site. The partition ratio of inactivation/carboxylation was 1/30. The 94-kDa carboxylase was purified to 15-50% purity by a modification of a recent protocol [Wu, S.-M., Morris, D. P., & Stafford, D. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2236-2240] and covalently labeled with N-bromoacetyl-FLEEL[125I]Y. On silver-stained 10% sodium dodecyl sulfate-polyacrylamide gels, the predominant radiolabeled band was the 94,000 molecular weight species. This result independently validates that the 94-kDa protein is a carboxylase.
Acetates↗
Dioxygen transfer during vitamin K dependent carboxylase catalysis.
The vitamin K dependent carboxylase of liver microsomes is involved in the posttranslational modification of certain serine protease zymogens which are critical components of the blood clotting cascade. During coupled carboxylation/oxygenation this carboxylase converts glutamate residues, dihydrovitamin K, CO2, and O2 to a gamma-carboxyglutamyl (Gla) residue, vitamin K (2R,3S)-epoxide, and H2O with a stoichiometry of 1:1 for all substrates and products. In this paper we investigate the role of molecular oxygen in the reaction by following the course of the oxygen atoms using 18O2. Two different mass spectroscopic techniques, electron ionization positive ion mass spectrometry and supercritical fluid chromatography-negative ion chemical ionization mass spectrometry, were used to quantitate the amount of 18O incorporation into the various oxygens of the vitamin K epoxide product. We found that 0.95 mol atoms of oxygen were incorporated into the epoxide oxygen, 0.05 mol atoms of oxygen were incorporated into the quinone oxygen of vitamin K epoxide, and the remaining ca. 1.0 mol atoms of oxygen were incorporated into H2O. No incorporation of oxygen into vitamin K epoxide from 50% H2(18)O was observed. Thus, the carboxylase operates as a dioxygenase 5% of the time during carboxylation/oxygenation. The relevance of these findings with respect to the nonenzymic "basicity enhancement" model proposed by Ham and Dowd [(1990) J. Am. Chem. Soc. 112, 1660-1661] is discussed.
Animals↗
Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.
The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.
Amino Acid Sequence↗
P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities.
P-selectin on platelets and endothelial cells and E-selectin on endothelial cells are leukocyte receptors that recognize lineage-specific carbohydrates on neutrophils and monocytes. The proposed ligands for these receptors contain the Le(x) core and sialic acid. Since other investigators have shown that both E-selectin and P-selectin bind to sialylated Le(x), we evaluated whether E-selectin and P-selectin recognize the same counter-receptor on leukocytes. The interaction of HL60 cells with Chinese hamster ovary (CHO) cells expressing P-selectin or E-selectin was studied. To determine whether a protein component is required in addition to sialyl Le(x) for either P-selectin or E-selectin recognition, HL60 cells or neutrophils were digested with proteases, including chymotrypsin, elastase, proteinase Glu-C, ficin, papain, or thermolysin. Cells treated with these proteases bound E-selectin but not P-selectin. Fucosidase or neuraminidase treatment of HL60 cells markedly decreased binding to both E-selectin- and P-selectin-expressing CHO cells. Growth of HL60 cells in tunicamycin inhibited the ability of these cells to support P-selectin-mediated binding and, to a lesser extent, E-selectin-mediated binding. Purified P-selectin inhibited CHO:P-selectin binding to HL60 cells, but incompletely inhibited CHO:E-selectin binding to HL60 cells. However, purified soluble E-selectin inhibited CHO:P-selectin and CHO:E-selectin binding to HL60 cells equivalently and completely. COS cells, unable to bind to E-selectin or P-selectin, bound E-selectin but not P-selectin upon transfection with alpha-1,3-fucosyltransferase or alpha-1,3/1,4-fucosyltransferase. Similarly, LEC 11 cells expressing sialyl Le(x) bound E-selectin- but not P-selectin-expressing CHO cells. Sambucus nigra lectin, specific for the sialyl-2,6 beta Gal/GalNAc linkage, inhibited P-selectin but not E-selectin binding to HL60 cells. Although sialic acid and Le(x) are components of the P-selectin ligand and the E-selectin ligand, these results indicate that the ligands are related, having overlapping specificities, but are structurally distinct. A protein component containing sialyl Le(x) in proximity to sialyl-2,6 beta Gal structures on the P-selectin ligand may contribute to its specificity for P-selectin.
Animals↗
Molecular and cellular biology of blood coagulation.
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Blood Coagulation↗