Determination of the site-specific and isoform-specific glycosylation in human plasma-derived antithrombin by IEF and capillary HPLC-ESI-MS/MS

The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP-HPLC-ESI-MS equipped with a quadrupole ion-trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of alpha-AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF-band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn(135) and Asn(155), alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main-isoform of alpha-AT but missing one terminal SA. There was no site specificity found for the mono-sialo structure. The isoform at pI 5.31 is the major isoform of beta-AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than beta-AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance.     

Separation of latent, prelatent, and native forms of human antithrombin by heparin affinity high-performance liquid chromatography

Latent antithrombin (LAT) is a partially denatured form of human antithrombin (AT). LAT does not inhibit clotting of the blood, but has previously been shown to inhibit angiogenesis and carcinogenesis. Another probably partially denatured form is the so-called prelatent AT (P-LAT), described by Larsson et al. [J. Biol. Chem. 276 (2001) 11996]. In the present work, an analytical heparin affinity chromatography method is described that separates an AT form, which is formed during the pasteurization process and which we believe to be identical to the previously described P-LAT, from native AT and LAT. Non-pasteurized AT was shown to contain no P-LAT, while four, heat-treated commercial AT products all contained P-LAT (1-6%, mean=4%). P-LAT has a slightly lower affinity to heparin than does native AT, but exhibits a much stronger heparin affinity when compared to LAT. P-LAT and native AT were shown to have very similar thrombin inhibiting activity, while LAT lacks such activity.     

Mechanisms responsible for catalysis of the inhibition of factor Xa or thrombin by antithrombin using a covalent antithrombin-heparin complex

Covalent antithrombin-heparin (ATH) complexes, formed spontaneously between antithrombin (AT) and unfractionated standard heparin (H), have a potent ability to catalyze the inhibition of factor Xa (or thrombin) by added AT. Although approximately 30% of ATH molecules contain two AT-binding sites on their heparin chains, the secondary site does not solely account for the increased activity of ATH. We studied the possibility that all pentasaccharide AT-binding sequences in ATH may catalyze factor Xa inhibition. Chromatography of ATH on Sepharose-AT resulted in >80% binding of the load. Similar chromatographies of non-covalent AT + H mixtures lead to a lack of binding for AT and fractionation of H into unbound (separate from AT) or bound material. Gradient elution of ATH from Sepharose-AT gave 2 peaks, a peak containing higher affinity material that had greater anti-factor Xa catalytic activity (708 units/mg heparin) compared with the peak containing lower affinity material (112 units/mg). Sepharose-AT chromatography of the ATH component with short heparin chains (相似文献   

Inhibition of fibrin-bound thrombin by a covalent antithrombin-heparin complex

Numerous studies have shown that fibrin-bound thrombin (IIa) is protected from inhibition by antithrombin (AT) + heparin (H) due to the formation of a ternary fibrin.IIa.H complex. We investigated factors affecting the inhibition of fibrin.IIa by a covalent complex of AT and H (ATH). The rate of IIa reaction with ATH was decreased 2-3-fold by fibrin monomer as compared to 57-fold for AT + heparin with high AT affinity. Furthermore, although the reaction of AT + H with a IIa mutant with decreased H binding (RA-IIa) was inhibited 2-3-fold in the presence of fibrin, reaction rates of ATH + RA-IIa were not reduced by fibrin. The relative difference in the effect of fibrin on the ATH reaction with RA-IIa compared to that for reactions of AT + H with RA-IIa is consistent with the fact that, in the absence of fibrin, the rate of the ATH reaction with RA-IIa relative to IIa was much less reduced (8-fold) compared to the corresponding reactions of AT + H (decreased 306 fold). Similarly, the addition of excess H in the absence of fibrin gave only a small decrease in rate of ATH + IIa reaction. However, in the presence of fibrin, the addition of 40-fold excess H decreased the rate of ATH inhibition of IIa by 1 order of magnitude. Experiments with ATH containing low molecular weight heparin chains with low AT affinity showed that IIa inhibition requires ATH with long chains that activate the AT moiety. Finally, electrophoresis of fibrin +/- ((125)I-)IIa +/- ((125)I-)ATH on native and denaturing gels showed that ATH forms ATH-IIa complexes that remain bound to fibrin through the ATH component. Thus, ATH is a potent inhibitor of fibrin-bound IIa, likely due to the formation of fibrin.ATH-IIa as opposed to fibrin.IIa.H ternary complexes.     

Analysis of inhibition rate enhancement by covalent linkage of antithrombin to heparin as a potential predictor of reaction mechanism

Antithrombin (AT) inhibition of coagulation enzymes is catalyzed by unfractionated heparin (UFH) and other heparinoids. Reaction proceeds either via conformational activation of the inhibitor or template-mediated binding of both inhibitor and protease. We investigated if the relative inhibition rates of AT + UFH and covalent AT-heparin conjugate (ATH) with coagulation factors might be indicative of the mechanism involved. Rates were determined by discontinuous assay and mechanisms were probed by a variety of binding studies with UFH or ATH heparin chains. Rates were increased more than 2-fold with ATH over AT + UFH in reactions with thrombin, factor (F) VIIa + tissue factor + Ca2+ + lipid, FIXa and FXIa, but not with FXa or FXIIa. In comparison, UFH or ATH heparin binding (evidence of a template mechanism) was only observed with thrombin, tissue factor, FIXa and FXIa. Thus, inhibition rate enhancement by conjugation of AT with heparin were predictive of inhibitor.enzyme template bridging by heparin. Rationales behind this novel concept are discussed.     

Isolation of a pure octadecasaccharide with antithrombin activity from an ultra-low-molecular-weight heparin

Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP [U.S. Pharmacopeia] heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.     

High affinity interaction between a synthetic, highly negatively charged pentasaccharide and alpha- or beta-antithrombin is predominantly due to nonionic interactions

Idraparinux is a synthetic O-sulfated, O-methylated pentasaccharide that binds tightly to antithrombin (AT) and thereby specifically and efficiently induces the inactivation of the procoagulant protease, factor Xa. In this study, the affinity and kinetics of the interaction of this high-affinity pentasaccharide with alpha- and beta-AT were compared with those of a synthetic pentasaccharide comprising the natural AT-binding sequence of heparin. Dissociation equilibrium constants, Kd, for the interactions of Idraparinux with alpha- and beta-AT were approximately 0.4 and 0.1 nM, respectively, corresponding to an over 100-fold enhancement in affinity compared with that of the normal pentasaccharide. This large enhancement was due to a approximately 400-fold tighter conformationally activated complex formed in the second binding step, whereas the encounter complex established in the first step was approximately 4-fold weaker. The high-affinity and normal pentasaccharides both made a total of four ionic interactions with AT, although the high-affinity saccharide only established one ionic interaction in the first binding step and was compensated by three in the second step, whereas the normal pentasaccharide established two ionic interactions in each step. In contrast, the affinities of the nonionic interactions (Kd approximately 450 and 90 nM for the binding to alpha- and beta-AT, respectively) were considerably higher than those for the normal pentasaccharide and the highest of all AT-saccharide interactions reported so far. The nonionic contribution to the total free energy of the high-affinity pentasaccharide binding to AT thus amounted to approximately 70%. These findings show that nonionic interactions can play a predominant role in the binding of highly charged saccharide ligands to proteins and can be successfully exploited in the design of such biologically active ligands.     

Recombinant Factor IX Fc Fusion Protein Maintains Full Procoagulant Properties and Exhibits Prolonged Efficacy in Hemophilia B Mice

Introduction

Hemophilia B is an inherited X chromosome–linked disorder characterized by impaired blood clotting owing to the absence of functional coagulation factor IX. Due to the relatively short half-life of factor IX, patients with hemophilia B require frequent factor IX infusions to maintain prophylaxis. We have developed a recombinant factor IX (rFIX) fused to the Fc region of IgG (rFIXFc) with an extended half-life in animals and humans.

Materials and Methods

Procoagulant properties of rFIXFc and rFIX (BENEFIX^®) were compared to determine the effect of the Fc region on rFIXFc hemostatic function. Specifically, we assessed rFIXFc activation, intermolecular interactions within the Xase complex, inactivation by antithrombin III (AT) and thrombin generation potential compared with rFIX. We also assessed the acute and prophylactic efficacy profiles of rFIXFc and rFIX in vivo in hemophilia B mouse bleeding models.

Results and Conclusions

The activation by factor XIa or factor VIIa/tissue factor, inhibition by AT, interaction profiles with phospholipids, affinities for factor VIIIa within the context of the Xase complex, and thrombin generation profiles were similar for rFIXFc and rFIX. Xase complexes formed with either molecule exhibited similar kinetic profiles for factor Xa generation. In acute efficacy models, mice infused with rFIXFc or rFIX were equally protected from bleeding. However, in prophylactic efficacy models, protection from bleeding was maintained approximately three times longer in rFIXFc-dosed mice than in those given rFIX; this prolonged efficacy correlates with the previously observed half-life extension. We conclude that rFIXFc retains critical FIX procoagulant attributes and that the extension in rFIXFc half-life translates into prolonged efficacy in hemophilia B mice.     

Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin

Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS)     

The new oral anticoagulants

In patients with nonvalvular atrial fibrillation oral anticoagulation with the vitamin K antagonists acenocoumarol, phenprocoumon and warfarin reduces the risk of stroke by more than 60%, whereas single or double antiplatelet therapy is much less effective and sometimes associated with a similar bleeding risk as vitamin K antagonists. Besides bleeding, INR monitoring and high interindividual variability remain the largest drawbacks of vitamin K antagonists. In the last decade oral agents have been developed that directly block the activity of thrombin (factor IIa), as well as drugs that directly inhibit activated factor X (Xa), which is the first protein in the final common pathway to the activation of thrombin. These agents have huge advantages in that they do not need monitoring and have a fast onset and offset of action. This survey addresses the role of classical and modern anticoagulation in stroke prevention in atrial fibrillation. (Neth Heart J 2010;18:314-8.)     

Role of P2 glycine in determining the specificity of antithrombin reaction with coagulation proteases

Structural data suggests that bulky hydrophobic residues at the S2-S4 sub-sites of factor Xa (fXa) restrict the preference of this pocket for small and non-polar residues like Gly at the P2 position of substrates and inhibitors. However, kinetic studies monitoring the cleavage specificity of 10-residue peptides by fXa have identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser as the most preferred P3-P3′ residues for recognition by fXa. To determine whether this mechanism of specificity is also true for fXa reaction with antithrombin (AT), we prepared two AT mutants having either a Phe at the P2 or Gln-Phe-Arg-Ser-Leu-Ser at the P3-P3′ positions of the reactive center loop. Inhibition kinetic studies indicated that the reactivity of P2-Phe with fXa was significantly (∼5-fold) impaired, however, the P3-P3′ mutant exhibited 1.5-fold improved reactivity with the protease, suggesting cooperative effects between P3-P3′ residues influence the P2 specificity of AT. Substitution of Tyr-99 of fXa with a Gly dramatically impaired the reactivity of fXa with wild-type AT, but improved its reactivity with the serpin mutants in the absence, but not in the presence of pentasaccharide. AT with a P2-Phe inhibited thrombin with >150-fold impaired reactivity, however, the defect was restored by either pentasaccharide or by replacing Leu-99 of thrombin with a Gly. The P3-P3′ mutant rapidly inhibited factors VIIa and XIa independent of pentasaccharide. These results indicate that P2-Gly plays a key role in determining the S2 sub-site specificity and target protease selectivity of AT in circulation.     

Identification of anti-thrombin antibodies in the antiphospholipid syndrome that interfere with the inactivation of thrombin by antithrombin.

The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in vitro blood coagulation tests, and are comprised mainly of Ab against beta(2) glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient-derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti-thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients.     

Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin-heparin complex

Factor-Xa assembly into the prothrombinase complex decreases its availability for inhibition by antithrombin + unfractionated heparin (AT + UFH). We have developed a novel covalent antithrombin-heparin complex (ATH), with enhanced anticoagulant actions compared with AT + UFH. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase. Discontinuous inhibition assays were performed to determine final k(2) values for inhibition of Xa. Fluorescent microscopy was conducted to evaluate inhibitor-prothrombinase interactions. The k(2) for inhibition of prothrombinase versus free Xa by AT + UFH was lower, whereas for ATH were much higher. Relative to intact prothrombinase, rates for Xa inhibition by AT + UFH in complexes devoid of prothrombin/vesicles/factor-Va were higher. For ATH, exclusion of prothrombin decreased k(2), removal of vesicles increased k(2) and exclusion of factor-Va gave no effect. While UFH may displace Xa from prothrombinase, Xa is detained within prothrombinase during ATH reactions. We confirm prothrombinase hinders inhibitory action of AT + UFH, whereas ATH is less affected with prothrombin being a key component in the complex responsible for the opposing effects. Overall, the results suggest that covalent linkage between AT-heparin assists access and neutralization of complexed Xa, with concomitant inhibition of prothrombinase function compared with conventional non-conjugated heparin.     

Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.     

The new oral anticoagulants in atrial fibrillation: an update

In patients with nonvalvular atrial fibrillation, oral anticoagulation with the vitamin K antagonists acenocoumarol, phenprocoumon and warfarin reduces the risk of stroke by more than 60 %, whereas single or double antiplatelet therapy is much less effective and sometimes associated with a similar bleeding risk as vitamin K antagonists. Besides bleeding, and intracranial haemorrhage in particular, INR monitoring remains the largest drawback of vitamin K antagonists. In the last decade oral agents have been developed that directly block the activity of thrombin (factor IIa), as well as drugs that directly inhibit activated factor X (Xa), which is the first compound in the final common pathway to the activation of thrombin. These agents have been approved for stroke prevention in atrial fibrillation and are now reimbursed under a national guideline for their safe use. They have advantages in that they do not need monitoring and have a fast onset and offset of action, but lack an established specific antidote. This survey addresses the role of modern anticoagulation for stroke prevention in atrial fibrillation.     

Protective Effects of Non-Anticoagulant Activated Protein C Variant (D36A/L38D/A39V) in a Murine Model of Ischaemic Stroke

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t_½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t_½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.     

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.     

Thrombin-activable factor X re-establishes an intrinsic amplification in tenase-deficient plasmas

Classical hemophilia results from a defect of the intrinsic tenase complex, the main factor X (FX) activator. Binding of factor VIIa to tissue factor triggers coagulation, but little amplification of thrombin production occurs. Handling of hemophilia by injection of the deficient or missing (thus foreign) factor often causes immunological complications. Several strategies have been designed to bypass intrinsic tenase complex, but none induce true auto-amplification of thrombin production. In an attempt to re-establish a cyclic amplification of prothrombin activation in the absence of tenase, we prepared a chimera of FX having fibrinopeptide A for the activation domain (FX(FpA)). We reasoned that cascade initiation would produce traces of thrombin that would activate FX(FpA) (contrary to its normal homologue). Given that the activation domain of FX is released upon activation, thrombin cleavage would produce authentic FXa that would produce more thrombin, which in turn would activate more chimeras. FX(FpA) was indeed activable by thrombin, albeit at a relatively low rate (5 x 10(3) M(-1) s(-1)). Nevertheless, FX(FpA) allowed in vitro amplification of thrombin production, and 100 nM efficiently corrected thrombin generation in tenase-deficient plasmas. A decisive advantage of FX(FpA) could be that the artificial cascade is self-regulating: FX(FpA) had little influence on the clotting time of normal plasma, yet corrected that of tenase deficiency. Another advantage could be the half-life of FX(FpA) in blood; FX has a half-life of about 30 h (less than 3 h for FVIIa). It is also reasonable to expect little or no immunogenicity, because FX and fibrinopeptide A both circulate normally in the blood of hemophiliacs.     

P1 variant antithrombins Glasgow (393 Arg to His) and Pescara (393 Arg to Pro) have increased heparin affinity and are resistant to catalytic cleavage by elastase Implications for the heparin activation mechanism

The heparin affinity of normal and two P1 variants of antithrombin-III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin-Sepharose. At pH 7.4 normal AT eluted art [Na⁺] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na⁺] 0.86 mol/l and AT Glasgow at [Na⁺] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg-393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.     

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Linkage of protease-inhibitor-heparin interactions in the reaction with thrombin

Heparin (H) was previously shown to accelerate the inactivation of alpha-thrombin (T) by antithrombin III (AT) primarily by promoting the initial binding of thrombin and AT in a ternary T.AT.H complex intermediate without significantly influencing the subsequent product formation step (Olson, S. T., and Shore, J. D. (1982) J. Biol. Chem. 257, 14891-14895). In the present study, the protein-heparin interactions which contribute to the assembly of the ternary complex intermediate and their linkage were quantitated by equilibrium binding and stopped-flow kinetic studies at pH 7.4, I 0.3, 25 degrees C, using p-aminobenzamidine (P) as a fluorescence probe. Equilibrium binding studies of the AT.H and T.H binary complex interactions monitored by the 40% enhancement in AT fluorescence or the 16-18% quenching of thrombin-bound p-aminobenzamidine fluorescence, respectively, indicated a 100-fold greater affinity of AT for heparin (KAT,H 0.23 microM) as compared to thrombin for heparin (KT,H 35-42 microM). Consistent with this large difference in affinities, rapid kinetic studies indicated that assembly of the ternary complex occurred predominantly as a bimolecular association between the AT.H binary complex and free thrombin. Thus, under pseudo-first order conditions ([AT]o, [H]o much greater than [T]o much less than [P]o), the observed thrombin inactivation rate constant (kobs) exhibited a saturable dependence on [AT]o or [H]o when [H]o much less than KT,H, reflecting a KAT,H (0.25 microM) similar to that directly determined by equilibrium binding. Moreover, competitive inhibition of the reaction by T.H binary complexes was indicated from the hyperbolic decrease in kobs produced by heparin with either high or low affinity for AT or active-site blocked thrombin at concentrations comparable to KT,H. This behavior was consistent with values of KT,H (27-51 microM) similar to those determined directly from equilibrium binding measurements. Comparison of the affinities of the binary protein-heparin interactions with the affinity of thrombin for AT.H complex in the ternary complex measured previously, indicated that the affinity of either protein for heparin was enhanced about 10-fold by the prior binding of the other protein to heparin. This linkage of the protein-heparin interactions implies that the ternary complex will be assembled at thrombin, AT, and heparin concentrations considerably lower than those predicted from previous reaction models which fail to account for this linkage.