Biomedical subjects

W H Kane

Publications and source records attributed to W H Kane.

At least 19 recordsLinked to original sources

PubMed2005

Mutation of hydrophobic residues in the factor Va C1 and C2 domains blocks membrane-dependent prothrombin activation.

The binding of factor (FVa) to phosphatidylserine (PS) membranes regulates assembly of the prothrombinase complex. Two pairs of solvent-exposed amino acids, Tyr(1956)/Leu(1957) in the C1 domain and Trp(2063)/Trp(2064) in the C2 domain, each make significant contributions to the affinity of FVa for PS membranes, but individually neither pair of amino acids is required for prothrombinase assembly on 25% PS membranes. In this study we characterize a FVa mutant with alanine substitutions in both the C1 and C2 domains: (Y1956,L1957,W2063,W2064)A. We conclude that: (i) prothrombinase assembly on PS membranes requires Trp(2063)/Trp(2064) and/or Tyr(1956)/Leu(1957); (ii) combined mutation of Trp(2063)/Trp(2064) and Tyr(1956)/Leu(1957) results in only a modest 4-fold decrease in the rate of thrombin generation in the absence of membranes; (iii) the present data provide experimental support for the joint participation of the C1 and C2 domains in the binding of FVa to phospholipid membranes as suggested by the recently solved structure for FVai (A1/A3-C1-C2).

Cell Membrane↗

PubMed2001

Soluble phosphatidylserine binds to a single identified site in the C2 domain of human factor Va.

Factor V(a) (FV(a)) is a cofactor for the serine protease factor X(a) that activates prothrombin to thrombin in the presence of Ca(2+) and a membrane surface. FV(a) is a heterodimer composed of one heavy chain (A1 and A2 domains) and one light chain (A3, C1, and C2 domains). We use fluorescence, circular dichroism, and equilibrium dialysis to demonstrate that (1) the FV C2 domain expressed in Sf9 cells binds one molecule of C6PS with a k(d) of approximately 2 microM, (2) stabilizing changes occur in the FV C2 domain upon C6PS binding, (3) the C6PS binding site in the FV C2 domain is located near residue Cys(2113), which reacts with DTNB, and (4) binding to a PS-containing membrane is an order of magnitude tighter than that to soluble C6PS. Coupled with a recently published crystal structure of the C2 domain, these results support a model for the mechanism of C2-membrane interaction.

Amino Acid Sequence↗

PubMed2001

Fine mapping of inhibitory anti-factor V antibodies using factor V C2 domain mutants. Identification of two antigenic epitopes involved in phospholipid binding.

Hemorrhagic factor V inhibitors frequently bind to the second C-type (C2) domain of factor V and interfere with phospholipid binding. To define specific residues recognized by inhibitors from four patients (one bovine thrombin-induced and three spontaneous antibodies), epitope mapping was performed using recombinant human factor V lacking most of the B-type domain (FV des B) and alanine-substituted mutants within the C2 domain (FV des B C2 mutants). FV des B C2 mutants located in the region between Lys2060 and Glu2069 were resistant to inhibition by three IgG preparations including the bovine thrombin-induced antibody in both prothrombinase and phospholipid-binding assays. In contrast, mutations at Lys2087 and Lys2092/Glu2096 were significantly resistant to inhibition by the fourth IgG preparation in both prothrombinase and phospholipid-binding assays. These results confirm interference of phospholipid binding by hemorrhagic factor V inhibitors and support the role(s) of these residues in phospholipid binding.

Aged↗

PubMed2000

Identification of functionally important amino acid residues within the C2-domain of human factor V using alanine-scanning mutagenesis.

We have previously determined that the C2-domain of human factor V (residues 2037-2196) is required for expression of cofactor activity and binding to phosphatidylserine (PS)-containing membranes. Naturally occurring factor V inhibitors and a monoclonal antibody (HV-1) recognized epitopes in the amino terminus of the C2-domain (residues 2037-2087) and blocked PS binding. We have now investigated the function of individual amino acids within the C2-domain using charge to alanine mutagenesis. Charged residues located within the C2-domain were changed to alanine in clusters of 1-3 mutations per construct. In addition, mutants W2063A, W2064A, (W2063, W2064)A, and L2116A were constructed as well. The resultant 30 mutants were expressed in COS cells using a B-domain deleted factor V construct (rHFV des B). All mutants were expressed efficiently based on the polyclonal antibody ELISA. The charged residues, Arg(2074), Asp(2098), Arg(2171), Arg(2174), and Glu(2189) are required for maintaining the structural integrity of the C2-domain of factor V. Four of these residues (Arg(2074), Asp(2098), Arg(2171), and Arg(2174)) correspond to positions in the factor VIII C-type domains that have been identified as point mutations in patients with hemophilia A. The epitope for the inhibitory monoclonal antibody HV-1 has been localized to Lys(2060) through Glu(2069) in the factor V C2-domain. The epitope for the inhibitory monoclonal antibody 6A5 is composed of amino acids His(2128) through Lys(2137). The PS-binding site in the factor V C2-domain includes amino acid residues Trp(2063) and Trp(2064). This site overlaps with the epitope for monoclonal antibody HV-1. These factor V C2-domain mutants should provide valuable tools for further defining the molecular interactions responsible for factor V binding to phospholipid membranes.

Alanine↗

PubMed1999

Crystal structures of the membrane-binding C2 domain of human coagulation factor V.

Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved beta-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.

Amino Acid Sequence↗

PubMed1999

Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex.

Thrombin-activated factor Va exists as two isoforms, factor Va(1) and factor Va(2), which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained following incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site at Asn-2181, was partially glycosylated when expressed in COS cells. To confirm the structural basis for factor Va(1) and factor Va(2), we mutated Asn-2181 to glutamine (N2181Q) and expressed this mutant using a B domain deletion construct (rHFV des B) in COS cells. Thrombin activation of N2181Q released a light chain with mobility identical to that of factor Va(2) on SDS-PAGE. The functional properties of purified N2181Q were similar to those of factor Va(2) in prothrombinase assays carried out in the presence of limiting concentrations of phosphatidylserine. The binding of human factor Va(1) and factor Va(2) to 75:25 POPC/POPS vesicles was also investigated in equilibrium binding assays using proteins containing a fluorescein-labeled heavy chain. The affinity of human factor Va(2) binding to POPC/POPS vesicles was approximately 3-fold higher than that of factor Va(1). These results indicate that partial glycosylation of factor V at asparagine-2181 is the structural basis of the light chain doublet and that the presence of this oligosaccharide reduces the affinity of factor Va for biological membranes.

Animals↗

PubMed1998

Inhibitory anti-factor V antibodies bind to the factor V C2 domain and are associated with hemorrhagic manifestations.

Factor V inhibitors may develop as spontaneous autoantibodies, as alloantibodies after exposure to bovine thrombin preparations, or in factor V-deficient patients after plasma therapy. Clinical manifestations range from asymptomatic laboratory abnormalities to life-threatening hemorrhage. We have characterized the anti-factor V antibodies from 12 patients diagnosed with factor V inhibitors. In 8 patients, hemorrhagic complications (5 autoantibodies and 3 bovine thrombin-induced alloantibodies) developed, and 4 were asymptomatic (2 autoantibodies and 2 alloantibodies). The IgG fractions from all 12 patients immunoprecipitated the factor Va light chain, but only the 8 IgG fractions associated with hemorrhage inhibited factor V activity in a prothrombinase assay. Nine IgG fractions, including the 8 patients with hemorrhage, immunoprecipitated the isolated second C-type domain (C2). The 8 IgG fractions from the symptomatic patients also immunoprecipitated recombinant chimeras containing only the N-terminal third of the factor V C2 domain, and isolated recombinant C2 domain abrogated the inhibitory effect of the antibodies. Five of the inhibitory IgG fractions blocked binding of factor V to phosphatidylserine. These results suggest that inhibitory anti-factor V antibodies are associated with hemorrhagic manifestations and frequently bind to a common region within the C2 domain, whether originating spontaneously or after exposure to bovine thrombin.

Adolescent↗

PubMed1997

Studies of a second family with the Quebec platelet disorder: evidence that the degradation of the alpha-granule membrane and its soluble contents are not secondary to a defect in targeting proteins to alpha-granules.

We recently described a Quebec family with an autosomal dominant bleeding disorder characterized by mildly reduced-low normal platelet counts, an epinephrine aggregation defect, multimerin deficiency, and proteolytic degradation of several, soluble alpha-granular proteins. Similar clinical features led us to investigate a second family with an unexplained, autosomal dominant bleeding disorder. The affected individuals had reduced to normal platelet counts, absent platelet aggregation with epinephrine, and multimerin deficiency. Their platelet alpha-granular proteins factor V, thrombospondin, von Willebrand factor, fibrinogen, fibronectin, osteonectin, and P-selectin were proteolyzed and comigrated with the degradation products found in patients from the other family. However, their platelet albumin, IgG, external membrane glycoproteins, CD63 (a lysosomal and dense granular protein), calpain, and plasma von Willebrand factor were normal, indicating restriction in the proteins proteolyzed. Electron microscopy studies indicated preserved alpha-granular ultrastructure, despite degradation of soluble and membrane alpha-granular proteins. Immunoelectron microscopy studies of the patients' platelets indicated that fibrinogen, von Willebrand factor, P-selectin, multimerin, and factor V were within alpha-granules, with normal to reduced labeling for these proteins. Pathologic proteolysis of alpha-granular contents, rather than a defect in targeting proteins to alpha-granules, may be the cause of the protein degradation in the Quebec platelet disorder.

Adult↗

PubMed1996

An autosomal dominant, qualitative platelet disorder associated with multimerin deficiency, abnormalities in platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen and an epinephrine aggregation defect.

Multimerin is a massive soluble, multimeric protein found in platelets and endothelial cells. Recent studies identified multimerin as a specific coagulation factor V binding protein, complexed with platelet, but not plasma, factor V. These findings led us to investigate individuals with inherited factor V deficiencies for possible multimerin abnormalities. Platelet proteins were evaluated using immunoassays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunoprecipitation, and direct binding studies. Patients with factor V Quebec, a disorder with abnormal platelet factor V, had a quantitative deficiency in multimerin (n = 11 tested; mean, 12.5%; range, 5% to 27% of the normal pool; normal range, 45% to 214%) with a normal multimer pattern. Quantitative and qualitative abnormalities were detected in their platelet factor V. An unrelated patient who was deficient in platelet and plasma factor V had normal platelet multimerin. The levels of platelet beta-thromboglobulin, von Willebrand factor, thrombospondin, and fibrinogen antigen were normal in the factor V Quebec patients. However, proteins with abnormal mobility were detected in their platelet lysate and releasate, and their platelet thrombospondin, von Willebrand factor, and fibrinogen showed evidence of proteolytic degradation. Platelet counts of the factor V Quebec patients ranged from mildly thrombocytopenic to low normal (mean, 159 x 10(9)/L; range, 104 to 198 x 10(9)/L). In addition, their platelets failed to aggregate in response to 6 to 10 micromol/L epinephrine despite normal numbers of platelet alpha 2-adrenergic receptors. These data indicate that patients with factor V Quebec have an inherited bleeding disorder distinct from other platelet disorders and associated with multiple abnormalities, including multimerin deficiency, abnormal platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen, and an epinephrine aggregation defect.

Afibrinogenemia↗

PubMed1996

Effect of heterologous factor V heavy chain sequences on the secretion of recombinant human factor VIII.

Factor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIII/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIII A1 domain dramatically reduced secretion of factor V approximately 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII approximately 3-fold, whereas substitution of the factor V A2 domain actually reduced secretion approximately 4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.

Amino Acid Sequence↗

PubMed1995

Thrombin-catalyzed activation of recombinant human factor V.

Proteolytic activation of human factor V by thrombin results from the cleavage of three peptide bonds at Arg709, Arg1018, and Arg1545. In order to define the functional importance of these sites, mutants with isoleucine substitutions blocking thrombin cleavage at one, two, or all three activation sites were expressed in COS-7 cells. The wild type protein is activated approximately 10-fold by thrombin or Russell's viper venom (RVV-V). Thrombin cleavage at Arg709 alone did not result in an increase in procoagulant activity. Cleavage at both Arg709 and Arg1018 resulted in an approximately 3.4-fold increase in activity. Cleavage at these sites was required for rapid cleavage by thrombin at Arg1545, however, which resulted in maximal activation of the factor V molecule. In contrast, isolated cleavage at Arg1545 by RVV-V was sufficient for efficient and complete activation of factor V. The effect of isoleucine substitutions at one or both thrombin cleavage sites in a B-domain deletion mutant lacking amino acids 811-1491 was also investigated. The specific activity of all four mutants was approximately 30% compared to thrombin activated factor V, indicating that these isoleucine substitutions do not drastically alter the structure of the protein and that cleavage at these sites is not required for the expression of partial procoagulant activity.

Amino Acid Sequence↗

PubMed1994

Localization of functionally important epitopes within the second C-type domain of coagulation factor V using recombinant chimeras.

Coagulation factor V, an integral component of the prothrombinase complex, possesses two C-type domains at the carboxyl-terminal end of the molecule. Homologous C-type domains are present in factor VIII as well as several non-coagulation proteins. Deletion of the second C-type domain of factor V results in the loss of procoagulant activity and the ability to bind phosphatidylserine. We now report the effect of substitution of all or a portion of the C2 domain of factor V with the corresponding regions of factor VIII or the human breast carcinoma protein BA46. Substitution of the entire domain with a heterologous C2 domain does not restore significant procoagulant activity, although smaller, exon-size substitutions do result in chimeras with partial activity (approximately 10% of factor Va). Using chimeras with partial substitutions, we determined that the amino-terminal region of the domain is involved in binding to phosphatidylserine. In contrast, the central region of the domain is not involved in phosphatidylserine binding, but an antibody binding at or near this site inhibits procoagulant activity, suggesting that this region is involved in a separate function. Lastly, the molecular basis for the light chain doublet, which is important in the expression of full procoagulant activity, is located within the carboxyl-terminal region of the C2 domain.

Animals↗

PubMed1994

Topical thrombin and acquired coagulation factor inhibitors: clinical spectrum and laboratory diagnosis.

Topical bovine thrombin preparations are used extensively in cardiovascular, neurosurgical, and otolaryngologic procedures. Patients who are treated with these topical thrombin preparations may develop antibodies to bovine coagulation factors that may cross-react with the endogenous human clotting proteins. We have identified four patients with acquired factor inhibitors following exposure to topical thrombin at Duke University Medical Center and summarize these cases in addition to 13 patients previously reported in the literature. In most cases, the inhibitor developed following a second (or subsequent) exposure to topical thrombin. The clinical course was extremely variable, ranging from totally asymptomatic to life-threatening hemorrhage. The most consistent laboratory abnormality was a prolonged bovine thrombin clotting time, which corrected, at least partially, when human thrombin was substituted for bovine thrombin. Some of these patients also developed factor V inhibitors with prolonged prothrombin and activated partial thromboplastin times. Although these patients have prolonged clotting times, they should not be considered "autoanticoagulated," since thromboembolic complications can still occur. Therapeutic intervention is largely empirical and depends on the clinical manifestations of the individual patient.

Administration, Topical↗

PubMed1994

Evaluation of preanalytical variables associated with measurement of prothrombin fragment 1.2.

We have used a monoclonal antibody-based ELISA for plasma prothrombin fragment 1.2 (F1.2) to establish appropriate sample collection and storage conditions for this biomarker of thrombin generation. F1.2 concentrations were not altered by exogenous factor Xa, thrombin, or thromboplastin if blood was collected by routine venipuncture into tubes containing heparin as anticoagulant (but not citrate, acid-citrate-dextrose, EDTA, or oxalate) and if plasma antithrombin III concentration was > or = 30% of normal. Heparinized plasma F1.2 was stable for > or = 8 h at 20-25 degrees C, and if premixed with a stabilizing reagent, for > or = 4 years at -70 degrees C. Mean values for heparinized plasma F1.2 collected and stored by recommended procedures were increased in patients with thrombosis and conditions of increased thrombotic risk, and were sensitive to heparin and oral anticoagulant therapies. We conclude that plasma obtained by routine venipuncture into tubes with heparin as anticoagulant is an appropriate specimen for F1.2 measurements for most patients.

Adult↗

PubMed1993

Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane-binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b-9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9-treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

Adult↗

PubMed1993

DDAVP in acquired hemophilia A: case report and review of the literature.

One patient with an acquired factor VIII inhibitor is reported in which an acute lower intestinal hemorrhage was successfully managed using Desmopressin (DDAVP). The patient initially had a factor VIII level of 10% with a inhibitor titer of 1.9 Bethesda units. Following administration of DDAVP the factor VIII level rose to 86% and there was a decrease in the number and volume of bloody stools. The inhibitor disappeared following treatment with corticosteroids, however the patient ultimately expired due to complications of ischemic colitis. This case and 21 previously reported cases of acquired hemophilia treated with DDAVP are reviewed. The data support a role for DDAVP in the treatment of non life threatening hemorrhage in patients with acquired hemophilia and low titer factor VIII inhibitors (< 5 Bethesda units or factor VIII > or = 5%).

Adolescent↗

PubMed1992

Structure of the gene for human coagulation factor V.

Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain.

Human coagulation factor V is an integral component of the prothrombinase complex. Rapid activation of prothrombin is dependent on the interactions of this nonenzymatic cofactor with factor Xa and prothrombin in the presence of calcium ions and a phospholipid or platelet surface. Factor V is similar structurally and functionally to the homologous cofactor, factor VIII, which interacts with factor IXa to accelerate factor X activation in the presence of calcium and phospholipids. Both of these cofactors, when activated, possess homologous heavy and light chains. Binding to anionic phospholipids is mediated by the light chains of these two cofactors. In bovine factor Va, a phosphatidylserine-specific binding site has been localized to the amino-terminal A3 domain of the light chain. In human factor VIII, on the other hand, a region within the carboxyl-terminal C2 domain of the light chain has been shown to interact with anionic phospholipids. We have constructed a series of recombinant deletion mutants lacking domain-size fragments of the light chain of human factor V (rHFV). These mutants are expressed and secreted as single-chain proteins by COS cells. Thrombin and the factor V activator from Russell's viper venom process these deletion mutants as expected. The light chain deletion mutants possess essentially no procoagulant activity, nor are they activated by treatment with factor V activator from Russell's viper venom. Deletion of the second C-type domain results in essentially complete loss of phosphatidylserine-specific binding whereas the presence of the C2 domain alone (rHFV des-A3C1, which lacks the A3 and C1 domains of the light chain) results in significant phosphatidylserine-specific binding. The presence of the A3 domain alone (rHFV des-C1C2) does not mediate binding to immobilized phosphatidylserine. Increasing calcium ion concentrations result in decreased binding of recombinant human factor V and the mutant rHFV des-A3C1 to phosphatidylserine, similar to previous studies with purified plasma factor V and phospholipid vesicles. These results indicate that human factor V, similar to human factor VIII, possesses a phosphatidylserine-specific binding site within the C2 domain of the light chain.

Base Sequence↗