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PubMed2003

[Factor Xa inhibitory assay to detect plasma heparin concentrations during and after cardiopulmonary bypass].

OBJECTIVE: For establishment and application of Xa Factor Inhibitory Assay to detect plasma heparin concentrations during and after cardiopulmonary bypass (CPB) and to evaluate the treatment with low-dose protamine reversal of heparin. METHODS: Venous blood samples were drawn from 25 patients with rheumatic valvular disease undergoing valvular replacement at different points (before CPB, after 5 min of heparinization, at termination of CPB, after 10 min, 1 hr and 5 hr of protamine reversal of heparin), and the plasma heparin concentrations were detected. RESULTS: The mean of plasma heparin concentration was the highest (13.16 U/ml) after 5 min of heparinization. At termination of CPB, it decreased (11.06 U/ml) and was significantly different from the concentrations at other points (P < 0.05). After 10 min of low-dose protamine reversal of heparin, the heparin concentration (0.45 U/ml) decreased to the same as that before CPB and continued to be stable at 1 hr and 5 hr; there was no significant difference in heparin concentration between these 4 points (P > 0.05). The highest batch CV in standard curves was 11.65%; the lowest was 0.37%. CONCLUSION: Factor Xa Inhibitory Assay is of good accuracy and repetition. The results from its clinical application demonstrated that the plasma heparin concentration of the patient at termination of CPB should be used for reference in determining the dose level of protamine for that every patient subjected to CPB.

Adult↗
PubMed1998

Factor Xa cleavage of tissue factor pathway inhibitor is associated with loss of anticoagulant activity.

Tissue factor:factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa:TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa:TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa:TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Argl99-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa:TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa:TFPI towards TF:factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF:factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.

Anticoagulants↗
PubMed1995

Effect of heparin on the inhibition of factor Xa by tissue factor pathway inhibitor: a segment, Gly212-Phe243, of the third Kunitz domain is a heparin-binding site.

Tissue factor pathway inhibitor (TFPI) inhibits the tissue factor--factor VIIa complex and factor Xa with its first and second Kunitz domains (K1 and K2), respectively. The inhibitory activity is enhanced by heparin, and the C-terminal basic part has been shown to be a heparin-binding site (HBS-1). To characterize and localize a second heparin-binding site (HBS-2), we studied the effect of heparin on the inhibitory activity of two forms of recombinant human TFPI, the full-length TFPI (rTFPI), and TFPI lacking the C-terminal basic part (rTFPI-C), by assaying the inhibition of human factor Xa. rTFPI-C inhibited factor Xa with an initial Ki of 6.79 nM in the absence of Ca2+ and 22.3 nM in the presence of 5 mM CaCl2. Heparin decreased the initial Ki to 1.79 nM in the absence of Ca2+ and 2.68 nM in the presence of 5 mM CaCl2, indicating the presence of HBS-2 in rTFPI-C. The dissociation constant for the binding of HBS-2 with heparin was determined to be 830 nM using fluorescein-labeled heparin and rTFPI-C. Heparin enhanced the inhibitory activity of a fragment consisting of the K2 and K3 domains, but it did not stimulate the inhibitory activity of the K2 domain. A synthetic peptide mimicking from Gly212 to Phe243 in the K3 domain reduced the effect of heparin on the inhibition by rTFPI-C and rTFPI. These results defined the location of HBS-2 in the basic region of the K3 domain between Gly212 and Phe243.

Amino Acid Sequence↗
PubMed1995

Characterization of des-(741-1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa.

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

Animals↗
PubMed1998

Human protein S cleavage and inactivation by coagulation factor Xa.

Human factor Xa specifically cleaves the anticoagulant protein S within the thrombin-sensitive domain. Amino-terminal amino acid sequencing of the heavy chain cleavage product indicates cleavage of protein S by factor Xa at Arg60, a site that is distinct from those utilized by alpha-thrombin. Cleavage by factor Xa is unaffected by the presence of hirudin and is completely blocked by tick-anticoagulant-peptide and D-Glu-Gly-Arg-chloromethyl ketone, the latter two being specific inhibitors of factor Xa. The cleavage requires the presence of phospholipid and Ca2+, and is markedly inhibited by the presence of factor Va. Factor Xa-cleaved protein S no longer possesses its activated protein C-dependent or -independent anticoagulant activity, as measured in a factor VIII-based activated partial thromboplastin time clot assay. The apparent binding constant for protein S binding to phospholipid (Kd approximately 4 nM +/- 1.0) is unaffected by factor Xa or thrombin cleavage, suggesting that the loss of anticoagulant activity resulting from cleavage is not primarily due to the loss of membrane binding ability. Cleavage and inactivation of protein S by factor Xa may be an additional way in which factor Xa exerts its procoagulant effect, after the initial stages of clot formation.

Amino Acid Sequence↗
PubMed1990

Interaction of factor Xa with heparin does not contribute to the inhibition of factor Xa by antithrombin III-heparin.

Factor Xa modified by reductive methylation (greater than 92%) loses the capacity to bind heparin as determined both by gel chromatography and by sedimentation equilibrium ultracentrifugation. The kinetic properties of methylated factor Xa differ, with respect to KM and Vmax for a synthetic tripeptide substrate and for antithrombin III inhibition rate constants, from those of the unmodified enzyme. The 10,000-fold rate enhancement elicited by the addition of heparin to the antithrombin III inhibition reaction, however, is the same. The observed second-order rate constants (k"obs) for antithrombin III inhibition of factor Xa and methylated factor Xa are 3000 and 340 M-1 s-1, respectively, whereas k"obs values for the inhibition of factor Xa or methylated factor Xa with antithrombin III-heparin are 4 X 10(7) and 3 X 10(6) M-1 s-1, respectively. These findings provide direct evidence that the interaction of factor Xa with heparin is not involved in the heparin-enhanced inhibition of this enzyme.

Animals↗
PubMed1998

Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor.

Tissue factor (TF) pathway inhibitor (TFPI) regulates factor X activation through the sequential inhibition of factor Xa and the VIIa.TF complex. Factor Xa formation was studied in a purified, reconstituted system, at plasma concentrations of factor X and TFPI, saturating concentrations of factor VIIa, and increasing concentrations of TF reconstituted into phosphatidylcholine:phosphatidylserine membranes (TF/PCPS) or PC membranes (TF/PC). The initial rate of factor Xa formation was equivalent in the presence or absence of 2.4 nM TFPI. However, reaction extent was small (<20%) relative to that observed in the absence of TFPI, implying the rapid inhibition of VIIa.TF during factor X activation. Initiation of factor Xa formation using increasing concentrations of TF/PCPS or TF/PC in the presence of TFPI yielded families of progress curves where both initial rate and reaction extent were linearly proportional to the concentration of VIIa.TF. These observations were consistent with a kinetic model in which the rate-limiting step represents the initial inhibition of newly formed factor Xa. Numerical analyses of progress curves yielded a rate constant for inhibition of VIIa.TF by Xa.TFPI (>10(8) M-1.s-1) that was substantially greater than the value (7.34 +/- 0.8 x 10(6) M-1.s-1) directly measured. Thus, VIIa.TF is inhibited at near diffusion-limited rates by Xa.TFPI formed during catalysis which cannot be explained by studies of the isolated reaction. We propose that the predominant inhibitory pathway during factor X activation may involve the initial inhibition of factor Xa either bound to or in the near vicinity of VIIa.TF on the membrane surface. As a result, VIIa.TF inhibition is unexpectedly rapid, and the concentration of active factor Xa that escapes regulation is linearly dependent on the availability of TF.

Animals↗
PubMed1993

A novel factor Xa inhibitor: structure-activity relationships and selectivity between factor Xa and thrombin.

A series of 3-amidinoaryl-2-[4-[ [(3S)-3-pyrrolidinyl]oxy]phenyl] propanoic acids have been investigated for development of a novel factor Xa inhibitor, possessing a potent inhibitory activity for factor Xa and a selectivity for factor Xa compared to thrombin. In order to study the structure-activity relationships and the selectivity, models of factors Xa complexes formed with the inhibitors were constructed on the basis of X-ray crystallographic data of a trypsin-inhibitor complex. The models showed that the binding mode of the inhibitors to the S1 pocket of the enzyme accounted for the structure-activity relationships and that the difference between Gln192 of factor Xa and Glu192 of thrombin had a key role in the selectivity.

Amidines↗
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↗
PubMed2005

Co-crystal structure and inhibition of factor Xa by PD0313052 identifies structurally stabilized active site residues of factor Xa and prothrombinase.

The enzyme complex prothrombinase plays a pivotal role in fibrin clot development through the production of thrombin, making this enzyme complex an attractive target for therapeutic regulation. This study both functionally and structurally characterizes a potent, highly selective, active site directed inhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residues in factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with human factor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecular association between factor Xa and PD0313052. This interaction was governed by association (k(on)) and dissociation (k(off)) rate constants of (1.0 +/- 0.1) x 10(7) M(-1) s(-1) and (1.9 +/- 0.5) x 10(-3) s(-1), respectively. The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character described by a Ki* = 0.29 +/- 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052 also identified a slow-onset mechanism with a Ki* = 0.17 +/- 0.03 nM and a k(on) and k(off) of (0.7 +/- 0.1) x 10(7) M(-1) s(-1) and (1.7 +/- 0.8) x 10(-3) s(-1), respectively. Crystals of factor Xa and PD0313052 demonstrated hydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216, as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate that the inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicate that active site residues of human factor Xa, including the catalytic Ser195, are effectively unaltered following assembly into prothrombinase.

Binding Sites↗
PubMed2000

Calcium enhances heparin catalysis of the antithrombin-factor Xa reaction by promoting the assembly of an intermediate heparin-antithrombin-factor Xa bridging complex. Demonstration by rapid kinetics studies.

Heparin catalyzes the inhibition of factor Xa by antithrombin mainly through an allosteric activation of the serpin inhibitor, but an alternative heparin bridging mechanism has been suggested to enhance the catalysis in the presence of physiologic calcium levels due to calcium interactions with the Gla domain exposing a heparin binding exosite in factor Xa. To provide direct evidence for this bridging mechanism, we studied the heparin-catalyzed reaction of antithrombin with factor Xa, Gla-domainless factor Xa (GDFXa), and a heparin binding exosite mutant of GDFXa in the absence and presence of calcium using rapid kinetic methods. The pseudo-first-order rate constant for factor Xa inhibition by antithrombin complexed with a long-chain approximately 70-saccharide heparin showed a saturable dependence on inhibitor concentration in the presence but not in the absence of 2.5 mM Ca(2+), indicating the formation of an intermediate heparin-serpin-proteinase encounter complex with a dissociation constant of approximately 90 nM prior to formation of the stable serpin-proteinase complex with a rate constant of approximately 20 s(-1). Similar saturation kinetics were observed for the inhibition of GDFXa by the antithrombin-heparin complex, except that Ca(2+) was not required for the effect. By contrast, no Ca(2+)-dependent saturation of the inhibition rate constant was detectable over the same range of inhibitor concentrations for reactions of either a short-chain approximately 26-saccharide high-affinity heparin-antithrombin complex with factor Xa or the long-chain heparin-antithrombin complex with the heparin binding exosite mutant, GDFXa R240A. These findings suggest that binding of full-length heparin chains to an exosite of factor Xa in the presence of Ca(2+) produces a chain-length-dependent lowering of the dissociation constant for assembly of the intermediate heparin-antithrombin-factor Xa encounter complex, resulting in a several 100-fold rate enhancement by a heparin bridging mechanism.

Antithrombins↗
PubMed2003

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

Amino Acid Sequence↗
PubMed1990

Cabbage seed protease inhibitor: a slow, tight-binding inhibitor of trypsin with activity toward thrombin, activated Stuart factor (factor Xa), activated Hageman factor (factor XIIa), and plasmin.

An inhibitor of procoagulant and fibrinolytic enzymes was derived from cabbage seeds by a procedure using acetone precipitation, ion-exchange chromatography, and gel filtration. The cabbage seed inhibitor was a 10-Kd monomeric protein with intrachain disulfide bonds. This preparation prevented clot formation in whole blood and blocked the ability of thrombin to induce clot formation in plasma and to induce platelet aggregation. A number of proteases were inhibited, as demonstrated by using purified enzymes in amidolytic assays. Tight-binding inhibition was observed for activated Stuart factor (factor Xa) and plasmin. Inhibition of thrombin and activated Hageman factor (factor XIIa) was observed with a molar excess of inhibitor. No inhibition was detected for activated plasma thromboplastin antecedent (factor XIa), plasma kallikrein, or C1 esterase. Reaction progress curves for trypsin indicated slow, tight-binding inhibition, with an apparent inhibition constant in the nanomolar range or less. The electrophoretic mobility of trypsin was altered by the inhibitor in nondenaturing polyacrylamide gel electrophoresis (PAGE) but not in sodium dodecyl sulfate (SDS)-PAGE, indicating noncovalent bonding. Only partial reversal of trypsin inhibition could be demonstrated by washing the inhibitor from enzyme immobilized on solid beads. A dot-blot technique with cabbage seed inhibitor was capable of detecting 10 ng nitrocellulose-bound trypsin. The dot-blot technique also appeared capable of detecting plasmin. These findings demonstrated the potential utility of this inhibitor as a probe for detection of tightly bound proteases. In summary, cabbage seed extracts contain an inhibitor with activity toward a broad range of proteases important to hemostasis. To our knowledge, this agent represents the first inhibitor isolated from a plant source that inhibits thrombin.

Brassica↗
PubMed1995

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin.

Crystal structures of DX9065a and a related bisamidino-aryl inhibitor specific for the blood-clotting factor Xa have been solved in complex with bovine beta-trypsin to a resolution of 1.9 A. Each inhibitor exhibits an extended conformation along the active site, in contrast to the compact folded structures observed for thrombin specific inhibitors. Few direct contacts (predominantly in the S1 pocket) are made between trypsin and the inhibitors. Transfer of the inhibitors to the active site of factor Xa suggests a three-site interaction: salt bridge formation at the base of the primary specificity pocket, extensive hydrophobic surface burial and a weak electrostatic interaction between the distal basic component of the inhibitor and an electronegative cavity of factor Xa formed by three backbone carbonyl oxygens. Additivity of these three interactions is the basis for the observed strong inhibition of factor Xa and provides a framework for the design of novel factor Xa inhibitors. A propionic acid group of the inhibitor would clash with the thrombin specific '60-insertion loop', thus conferring selectivity against thrombin.

Amino Acid Sequence↗
PubMed2000

Substitution of asparagine for arginine 347 of recombinant factor Xa markedly reduces factor Va binding.

Herein we describe a recombinant factor X (fX) with a single substitution at position 347 (fXR347N). Activated fXR347N had a reduced affinity for factor Va (fVa), although the catalytic impact of fVa binding remained intact. The mutation was selective as demonstrated by normal activation and inhibition, except in the presence of subsaturating heparin where the rate of inhibition by antithrombin III (ATIII) was 15% of normal. The reactivity of fXaR347N toward prothrombin was equivalent to wild-type fXa (fXaWT) in the absence of fVa and phospholipid. Addition (without phospholipid) of fVa dramatically increased the catalytic efficiency of fXaWT toward prothrombin but had a negligible effect on fXaR347N. On addition of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1) vesicles, fXaR347Ndisplayed an increased catalytic activity in response to fVa, but the apparent affinity for fVa on the phospholipid surface was 5-20-fold lower than that of fXaWT. On an activated platelet surface, however, fXaWT and fXaR347N activated prothrombin similarly. In a competitive binding assay that measures the displacement of radiolabeled fXa from fVa on a phospholipid surface, fXaR347N was approximately 10-fold less effective than fXaWT. Substitution of fXa at position 347 selectively attenuates the interaction between fXa and fVa without affecting its catalytic activity.

Amino Acid Substitution↗
PubMed2004

Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide.

We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv(59D8)). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clot-bound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications.TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for anti-fXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule. Two step affinity chromatography with Ni(2+) and beta(15-22)-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv(59D8)-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv(59D8)-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.

Amino Acid Sequence↗
PubMed1983

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X.

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.

Amino Acid Sequence↗