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PubMed1989

Free factor Xa is on the main pathway of thrombin generation in clotting plasma.

The effect of a synthetic pentasaccharide that specifically causes the inactivation of factor Xa on the development of prothrombinase activity in human plasma was monitored using four triggers of coagulation: (a) human brain thromboplastin; (b) contact activation; (c) factor X activating enzyme complex; (d) prothrombin activating enzyme complex. Inhibition was similar with the triggers a, b and c. With prothrombinase (d), the inhibition strongly decreased with increasing amounts of factor Va present. This indicates that only free factor Xa is inhibited. Because both the intrinsic pathway (b) and the extrinsic pathway (a) are inhibited by the pentasaccharide, we conclude that free factor Xa plays a rate-limiting role in the pathways, so that there is no reason to postulate the existence of 'supercomplexes' consisting of factors IXa, VIIIa, X(a), Va and prothrombin adsorbed on the same phospholipid particle (intrinsic system) or factor VII(a), X(a), Va and prothrombin adsorbed on tissue thromboplastin (extrinsic system).

Blood Coagulation↗
PubMed1989

Three point mutations in the factor IX genes of five hemophilia B patients. Identification strategy using localization by altered epitopes in their hemophilic proteins.

In five patients with hemophilia B and detectable Factor IX antigen, altered reactivity to a specific polyclonal antibody fraction or monoclonal anti-Factor IX antibodies was noted. Amplification of selected portions of their Factor IX genes by polymerase chain reaction allowed rapid identification of a single base transition in each of the five families tested. In a patient with severe hemophilia and an altered calcium binding domain, a G to A transition in exon II changed the codon for Glu-27 to Lys (Factor IXSeattle 3). Patients from two families with mild hemophilia with decreased reactivity to a MAb that binds to a site within the sequence coded for by exon IV had a G to A transition changing the codon for Gly-60 to Ser (Factor IXDurham). Two unrelated patients with moderately severe hemophilia lacked reactivity to another murine monoclonal anti-Factor IX which binds to an epitope in the amino-terminal half of the heavy chain of Factor IXa. In these patients, exon VIII contained a G to A transition changing Arg-248 to Gln (Factor IXSeattle 4).

Adult↗
PubMed1995

Binding of blood coagulation factor VIII and its light chain to phosphatidylserine/phosphatidylcholine bilayers as measured by ellipsometry.

Factor VIII is a plasma protein which plays an essential role in the coagulation system. When assembled with the enzyme Factor IXa on a phospholipid membrane, it functions as a cofactor in the enzyme complex that cleaves the zymogen Factor X to Factor Xa. We studied the binding of both Factor VIII and the Factor VIII light chain to planar phospholipid bilayers consisting of 25% dioleoylphosphatidylserine and 75% dioleoylphosphatidylcholine (PSPC) by ellipsometry. Equilibrium-binding studies revealed that both Factor VIII and its light chain bind with high affinity to PSPC bilayers. The binding affinity of Factor VIII, with a dissociation constant Kd of 0.24 nM, was comparable with that of the Factor VIII light chain (Kd 0.49 nM). Maximal binding was 2.3 mmol of protein per mol of PSPC for Factor VIII and 7.1 mmol of protein per mol of PSPC for the Factor VIII light chain. Adsorption kinetics of both Factor VIII and its light chain conformed to the classical Langmuir adsorption model yielding dissociation constants calculated from the rates of adsorption that were similar to those obtained by equilibrium-binding studies. In contrast, measurements of rates of desorption revealed a deviation from those expected for a single class of binding sites. The desorption rate of Factor VIII increased with increasing residence time on the lipid membrane. This indicates transition of Factor VIII to a configuration with a lower binding affinity. As this time-dependent change in affinity could affect the validity of the measurement of binding parameters, in particular equilibrium-binding determinations carried out on a long timescale, binding affinity was also estimated from adsorption kinetics at half-maximal surface coverage, a relatively rapid procedure for the determination of the affinity. A Kd of 0.087 nM was obtained under these conditions. Measurement of equilibrium binding to small PSPC vesicles, a system in which equilibrium is rapidly attained, resulted in similar binding parameters (Kd = 0.13 nM and a maximal binding of 2.8 mmol of protein per mol of PSPC). These data confirm the results of equilibrium binding to planar bilayers. Taken together, our results indicate that Factor VIII, by means of its 80 kDa light chain, binds to PSPC bilayers with a dissociation constant below the concentration of Factor VIII in plasma and therefore may readily bind to exposed phospholipid membranes under physiological conditions.

Adsorption↗
PubMed1984

Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX.

The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX by a modification of a method described for bovine Factor IX activation by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707). Initial rates of trichloroacetic acid-soluble 3H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl2 (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of 3H preceded the appearance of Factor IXa activity, and the percentage of 3H released remained constant when the mole fraction of 3H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of 3H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include Km (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (kcat) (7.7/s) is approximately 20-50 times higher than that reported by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707) for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant.

Factor IX↗
PubMed2001

Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B.

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the gamma-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor-like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.

Antibody Specificity↗
PubMed1990

Binding of human factor VIII to phospholipid vesicles.

Factor VIII, a protein cofactor involved in blood coagulation, functions in vitro on a phospholipid membrane surface to greatly increase the rate of factor X activation by factor IXa. Using gel filtration, rapid sedimentation, and resonance energy transfer we have studied the interaction of recombinant-derived human factor VIII with small and large unilamellar phospholipid vesicles composed of phosphatidylserine and phosphatidylcholine. Resonance energy transfer, from intrinsic fluorophores in factor VIII to dansyl-phosphatidylethanolamine incorporated into vesicles, has been adapted for quantitative equilibrium measurements. Factor VIII binds rapidly and reversibly to small and large vesicles. At 8 degrees C the interaction of factor VIII with small vesicles fits a simple bimolecular model with a KD of 2 nM and a phospholipid binding site defined by 180 phospholipid monomers. At 25 degrees C the binding of factor VIII to small vesicles containing 20% phosphatidylserine can be described by an apparent KD of 4 nM; the phospholipid/protein ratio at saturation was 170. Binding to large vesicles was demonstrated with a KD of 2 nM and a phospholipid/protein ratio at saturation of 385. Binding was dependent upon the phosphatidylserine mole fraction and was nonlinear from 0 to 30% phosphatidylserine content. A direct comparison of factor VIII and factor V binding indicated that the affinity of factor V to phospholipid vesicles was equivalent to that of factor VIII and that the phosphatidylserine requirement was lower. A model is proposed to explain the nonlinear phosphatidylserine dependence of binding for factor VIII.

Chromatography, Gel↗
PubMed1991

The site of activation of factor X by cancer procoagulant.

Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1980

The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin.

The kinetics of inhibition of four hemostatic system enzymes by antithrombin were examined as a function of heparin concentration. Plots of the initial velocity of factor Xa-antithrombin or plasmin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb and subsequent plateau regions. In each case, the kinetic profile is closely correlated with the concentration of the heparin . antithrombin complex formed within the reaction mixture. A decrease in the velocity of inhibition is not observed at high levels of added mucopolysaccharide despite the generation of significant quantities of heparin-enzyme interaction products. The second-order rate constants for the neutralization of factor Xa or plasmin by the mucopolysaccharide . inhibitor complex are 2.4 x 10(8) M-1 min-1 and 4.0 x 10(6) M-1 min-1, respectively. These parameters must be contrasted with the similarly designated constants obtained in the absence of heparin which are 1.88 x 10(5) M-1 min-1 and 4.0 x 10(4) M-1 min-1, respectively. Plots of the initial velocity of the factor IXa-antithrombin or the thrombin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb, pseudoplateau, descending limb, and final plateau regions. In each case, the ascending limb and pseudoplateau are closely correlated with the concentration of heparin c antithrombin complex formed within the reaction mixture. Furthermore, the descending limb and final plateau of these two processes coincide with the generation of increasing amounts of the respective mucopolysaccharide-enzyme interaction products. The second-order rate constants for the neutralization of factor IXa or thrombin by the heparin . antithrombin complex are 3.0 x 10(8) M-1 min-1 and 1.7 x 10(9) M-1 min-1, respectively. The second-order rate constants for the inhibition of mucopolysaccharide-factor IXa or mucopolysaccharide-thrombin interaction products by the heparin . antithrombin complex are 2.0 x 10(7) M-1 min-1 and 3.0 x 10(8) M-1 min-1, respectively. These kinetic parameters must be contrasted with similarly designated constants obtained in the absence of mucopolysaccharide which are 2.94 x 10(4) M-1 min-1 and 4.25 x 10(5) M-1 min-1, respectively. Thus, our data demonstrate that binding of heparin to antithrombin is required for the mucopolysaccharide-dependent enhancement in the rates of neutralization of thrombin, factor IXa, factor Xa, or plasmin by the protease inhibitor. Furthermore, a careful comparison of the various constants suggests that the direct interaction between heparin and antithrombin may be largely responsible for the kinetic effect of this mucopolysaccharide.

Antithrombins↗
PubMed1996

Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor.

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.

Amino Acid Sequence↗
PubMed1993

Flow cytometric analysis of surface membrane proteins on activated platelets and platelet-derived microparticles from healthy and thrombasthenic individuals.

We used flow cytometry to investigate surface membrane protein expression by platelets and platelet-derived microparticles from normal individuals and a patient with Glanzmann's thrombasthenia. Microparticles were detected by both forward scatter and side scatter using FACScan. The binding of coagulation factors on microparticles was investigated by using monoclonal anti-Factor IX (IXa) and anti-Factor X (Xa) antibodies. Furthermore, the procoagulant activity of microparticles was measured with a chromogenic substrate (S-2222) using a microtiter enzyme-linked immunosorbent assay. Both types of platelets showed similar release of microparticles. Microparticles released from platelets after activation with the calcium ionophore A23187 did not bind factors IXa and Xa, but when purified factors Va and Xa were added to the incubation buffer, factor Xa binding increased markedly in both normal and thrombasthenic platelets. Both normal and thrombasthenic platelets showed a similar time-dependent release of microparticles when activated with A23187. However, the binding of an antibody to granule membrane protein-140 also increased time-dependently in normal microparticles, but was little increased in thrombasthenic microparticles. These findings suggest that glycoprotein IIb/IIIa does not participate in the expression of prothrombinase activity on the surface of activated platelets and microparticles, whereas this glycoprotein appears to have an important role in the movement of granule membrane protein-140 from platelets to microparticles.

Adult↗
PubMed2004

Substrate-dependent modulation of the mechanism of factor XIa inhibition.

Factor XIa is a serine protease which participates in both the extrinsic and intrinsic pathways of blood coagulation. In this work we used active site directed inhibitors to study the mechanism of factor IX activation by factor XIa. To this end, we developed a new sensitive method for the detection of factor IXa based on its affinity to antithrombin III. Using this assay, we found that the peptidic inhibitors, leupeptin and aprotinin, exhibited similar potencies in inhibiting factor IX activation and the cleavage of a tripeptidic chromogenic substrate by factor XIa. As expected, leupeptin and aprotinin were competitive with respect to the tripeptidic chromogenic substrate. However, the inhibition of factor IX activation was best described by mixed-type inhibition with the affinity of leupeptin and aprotinin to the factor XIa-factor IX complex only approximately 10-fold lower than their affinity toward factor XIa. These results, consistent with previous factor XI domain analyses, suggest that the active site of factor XIa does not contribute significantly to the affinity of factor XIa toward factor IX. The competitive component of the inhibition of factor IX activation suggests that binding of factor IX to factor XIa heavy chain affects the interactions of leupeptin and aprotinin with the active site.

Aprotinin↗
PubMedDate not supplied

[Blood protein technological industrial developments as a mirror of fundamental studies bgy the Institute of Biochemistry of the Ukrainian National Academy of Sciences].

We have examined the technology for an industrial chromatographic production highly purified factor VIII concentrate intended for therapy of the hemophilia A and characterized this factor VIII. The final product has been prepared from cryoprecipitate of pooled human plasma using a large-scale procedure combining three conventional chromatographic steps based on AEM and CEM ion exchange and SPG or SHR gel filtration chromatography. The specific activity of the product was 459 +/- 19 IU factor VIII/mg protein (n = 10), corresponding to a purification factor of about 15,000. The concentrate was free of the fibrinogen, alpha-2-macroglobulin, alpha-1-acidglycoprotein, haptoglobin. Only three contaminants could be detected: fibronectin, immunoglobulins A and G (about 0.020, 0.004 and 0.034 microgram/IU factor VIII, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Another examination was concern to the technology for an industrial chromatographic production highly purified factor IX concentrate intended for therapy of the hemophilia B and characterized this factor IX. The final product has been prepared from pooled human plasma using a large-scale procedure combining four conventional chromatographic steps based on AEM ion exchange, AFM affinity and SGS gel filtration chromatography. The specific activity of the product was 149 +/- 10 IU factor IX/mg protein (n = 10), corresponding to a purification factor of about 9000. The concentrate was free of the vitamin K-dependent clotting factors II, VII and X and of proteins C and S. Most of possible contaminants were absent in this new product. High-molecular-weight kininogen, factor VIII, XI, XII or prekallikrein were not detected. There were no activated factors, such as factors IXa and Xa, no thrombin and no phospholipids. Only two contaminants could be detected: C4 and inter-alpha-trypsin inhibitor (about 0.8 and 1.2 mg/IU factor IX, respectively). The purity of the final product was confirmed by SDS polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Thrombogenicity tests in rabbits revealed that the high purified factor IX by Institute of Biochemistry technology tested had a lower thrombogenic power than the commercial factors IX tested. The concentrate has been subjected to a special solvent--detergent treatment for definite time and temperature during its production to virus inactivation (it will be describe in following special examination). These data demonstrate that a highly purified therapeutic clotting factor VIII and IX concentrates can be prepared from human plasma by conventional chromatographic methods developed by Institute of Biochemistry of NAS of Ukraine and Combio Ltd.

Academies and Institutes↗
PubMed1997

Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation.

The C-terminal fibronectin-type-III-like module of the tissue factor (TF) extracellular domain plays a requisite role in the activation of macromolecular substrates by factor VIIa (VIIa) in complex with TF. Unlike the mutations Lys165-->Ala, Lys166-->Ala in TF, which prevent efficient proteolysis of factor X, we found that the coagulant defect of a site-specific Trp158-->Arg, Ser160-->Gly replacement mutant of TF is largely attributable to the inability of TF to efficiently support the activation of the bound zymogen VII to the active protease VIIa. Binding studies demonstrated comparable affinity of binding of VIIa or VII by wild-type TF and TF(R158G160). In comparison with wild-type TF, the catalytic efficiency of factor X activation was reduced 56-fold with TF(A165A166) as the cofactor, but only 3.5-fold with TF(R165G160). The activation of VII bound to TF by factor Xa or VIIa was reduced 2-fold in the presence of TF(R158G160) and 7-8-fold with TF(A165A166). This suggests that the molecular recognition of VII in complex with TF by the enzymes TF-VIIa and factor Xa are similar. Generation of factor IXa by TF(R158G160)-VIIa was unaltered, but reduced 2-fold with TF(A165A166). In addition, the mutations affected the cleavage of the two scissile bonds of factor IX differently, providing further support for the idea that the cofactor, TF, influences the fine specificity of activation of macromolecular substrates by the TF-VIIa complex.

Blood Coagulation↗
PubMed1993

Kinetics of factor Xa inhibition by tissue factor pathway inhibitor.

Tissue factor pathway inhibitor is a multivalent, Kunitz-type proteinase inhibitor. It directly inhibits factor Xa and, in a factor Xa-dependent fashion, produces feedback inhibition of the factor VIIa/tissue factor catalytic complex which is responsible for the initiation of coagulation. Human recombinant TFPI (rTFPI) produced in Escherichia coli was used to define the kinetic constants describing the human factor Xa:TFPI interaction. The inactivation of factor Xa by E. coli-rTFPI is indistinguishable from that of rTFPI produced in mammalian SK-hepatoma cells, suggesting that post-translational modifications such as glycosylation and phosphorylation do not play a major role in the inhibitory process. The slow, tight-binding inhibition of factor Xa follows the scheme: [formula: see text] Where the enzyme (E) and inhibitor (I) form an initial, immediate collision complex (EI) that then isomerizes slowly to a tightened final EI* complex. In the absence of other additions, the initial Ki (=k2/k1) and final Ki* for the inhibition of factor Xa by E. coli-rTFPI are 1.24 nM and 26.4 pM, respectively. In the presence of calcium ions (5 mM) the interaction between factor Xa and rTFPI is substantially weaker, with a Ki of 42.7 nM and Ki* of 85.2 pM. The addition of other components of the prothrombinase complex produces enhanced factor Xa inhibition predominantly through an effect on the initial Ki. In the presence of calcium ions and saturating concentrations of phospholipids and factor Va, the Ki and Ki* for factor Xa inactivation are 2.04 nM and 52.3 pM. The enhancing effect of heparin on the inhibitory process is concentration dependent and exhibits an optimum, reminiscent of the "template" model for heparin's acceleration of thrombin and factor IXa inhibition by antithrombin III. At optimal concentrations, the major mechanism of heparin action is also a reduction in the Ki of the initial encounter complex between factor Xa and rTFPI.

Amino Acid Sequence↗
PubMed1989

Properties of a highly purified human plasma factor IX:c therapeutic concentrate prepared by conventional chromatography.

We have characterized a highly purified (HP) factor IX concentrate intended for therapy of hemophilia B. The product has been prepared from pooled human plasma using a large-scale procedure combining three conventional chromatographic steps based on DEAE ion exchange and affinity on immobilized heparin. The specific activity of the product was 119 +/- 10 IU factor IX:c/mg protein (n = 15), corresponding to a purification factor of about 9,000. The concentrate was free of the vitamin K-dependent clotting factors II, VII and X and of proteins C and S. Most of the contaminants found in factor IX complex concentrate (PCC) were absent in this new product. High-molecular-weight kininogen, factors VIII, XI, XII or prekallikrein were not detected. There were no activated factors, such as factors IXa, and Xa, no thrombin and no phospholipids. Only two contaminants could be detected: C4 and inter-alpha-trypsin inhibitor (about 0.8 and 1.2 mg/1,000 IU factor IX:c, respectively). The purity of the product, as compared to PCC, was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, Grabar-Williams immunoelectrophoresis, and bidimensional immunoelectrophoresis. Thrombogenicity tests in rabbits revealed that the HP factor IX tested had a lower thrombogenic power than the PCC tested. The concentrate has been subjected to a 0.3% tri(n-butyl) phosphate-1% Tween 80 treatment for 6h at 25 degrees C during its production to reduce or eliminate the risk of transmission of plasma-borne lipid-enveloped viruses. These conditions inactivated more than 3.8 log10 of vesicular stomatitis virus and more than 4.3 log10 of sindbis virus within 1 and 2 h of treatment, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation Factors↗
PubMed1989

Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin.

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated fVIII to function as a cofactor in the activation of factor X by factor IXa, we found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

Animals↗
PubMed2004

Update on the mechanism of action and future of activated prothrombin complex concentrates.

Activated prothrombin complex concentrates (aPCCs) are an established treatment for bleeding in patients with inhibitors. These products are derived from prothrombin complex concentrates, purified from human plasma with dedicated activation steps included in their manufacturing process. Despite these activation steps, the majority of the prothrombin complex proteins remain as zymogens, with only a relatively small content of activated coagulation enzymes. Among these, the order of concentration based on activity units is the following: factor VIIa to factor Xa to thrombin to factor IXa. Studies in various in vitro and in vivo model systems indicate that the mechanism of action of aPCCs is primarily based on an enzyme-substrate complex consisting of factor Xa and prothrombin. These findings are complemented by others showing that prothrombin is a major procoagulant that is capable of triggering hemostasis under physiologic and pathophysiologic conditions. Despite the findings of the mechanism of action, aPCCs have a long history of successful clinical use, with established dosing regimens, and a relatively low risk of thromboembolic complications compared with other treatment options for patients with inhibitors.

Blood Coagulation Factors↗
PubMed1995

Active site-blocked factors VIIa and IXa differentially inhibit fibrin formation in a human ex vivo thrombosis model.

The role of tissue factor/factor VIIa (FVIIa) and factor VIIIa/factor IXa (FVIIIa/FIXa) complexes in thrombus formation was examined in a human ex vivo blood flow system by use of active site-blocked FVIIa (FVIIai) and FIXa (FIXai) as selective inhibitors. Blood was drawn directly from the veins of volunteers into a mixing device where FVIIai and FIXai were mixed with flowing blood. The blood then entered parallel-plate chambers containing coverslips coated with human fibrillar collagen or tissue factor-expressing cell layers of tumor necrosis factor-alpha-stimulated human endothelial cells, human smooth muscle cells, and J82 cells. Exposure of stimulated endothelial cells to blood flowing at a venous shear rate of 65/s led to fibrin deposition, which was inhibited by infusion of FVIIai (IC50, 3 nmol/L), as quantified by micro-densitometry of fibrin-stained coverslips. Whereas FIXai (600 nmol/L) was only a weak inhibitor, FVIIai (60 nmol/L) reduced fibrinopeptide A (FPA) plasma levels from 504 +/- 79 to 171 +/- 27 ng/mL and concomitantly inhibited platelet thrombus deposition. Similarly, experiments with smooth muscle cells and J82 cells showed that FVIIai but not FIXai efficiently reduced FPA levels. Conversely, with tissue factor-free collagen, ,hich induces platelet-dependent fibrin formation, infusion of FIXai but not of FVIIai inhibited fibrin deposition (IC50, 8 nmol/L) and reduced FPA levels from 55 +/- 8 to 9 +/- 5 ng/mL. However, FIXai did not affect the number of platelet thrombi deposited on collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗