Inhibition of intrinsic Xase by protein S: a novel regulatory role of protein S independent of activated protein C.

OBJECTIVE: Protein S is a vitamin K-dependent plasma protein that functions in the feedback regulation of thrombin generation. Our goal was to determine how protein S regulates the intrinsic pathway of blood coagulation. METHODS AND RESULTS: We used plasma, including platelet-rich plasma, and in vitro methods to determine how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: (1) activated partial thromboplastin time assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time; (2) a modified activated partial thromboplastin time assay with factor IX (fIX)-deficient plasma confirmed that protein S affects fIX-initiated clotting; (3) a fIXa/factor VIIIa (fVIIIa)-mediated thrombin generation assay with either platelet-rich plasma or factor-deficient plasma, initiated with a limiting amount of tissue factor, was regulated by protein S; (4) in the presence of phosphatidylserine vesicles, protein S inhibited fIXa in the absence and presence of fVIIIa; and (5) protein S altered only the K(M) for factor X activation by fIXa in the absence of fVIIIa and both k(cat) and K(M) in the presence of fVIIIa. CONCLUSIONS: From our findings, it can be concluded that protein S inhibits fIXa in the presence or absence of fVIIIa in an activated protein C-independent way.

Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.

Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary.

Highly purified fucosylated chondroitin sulfate oligomers with selective intrinsic factor Xase complex inhibition.

Fucosylated chondroitin sulfate (FCS) oligosaccharides of specific molecular weight have shown potent anticoagulant activities with selectivity towards intrinsic factor Xase complex. However, the preparation of FCS oligosaccharides by traditional methods requires multiple purification steps consuming large amounts of time and significant resources. The current study focuses on developing a method for the rapid preparation of FCS oligomers from sea cucumber Pearsonothuria graeffei having 6-18 saccharide residues. The key steps controlling molecular weight (Mw) and purity of these FCS oligomers were evaluated. Structural analysis showed the resulting FCS oligomers were primarily l-Fuc3,4diS-α1,3-d-GlcA-ß1,3-(d-GalNAc4,6diS-ß1,4-[l-Fuc3,4diS-α1,3-]d-GlcA-ß1,3-)nd-anTal-ol4,6diS (n = 1˜5) accompanied by partial de-fucosylation and/or de-sulfation. In vitro and in vivo experiments demonstrate that these FCS oligomers selectively inhibit intrinsic factor Xase complex and exhibit remarkable antithrombotic activity without hemorrhagic and hypotension side effects. This method is suitable for large-scale preparation of FCS oligosaccharides as clinical anticoagulants.

Synthesis and anticoagulation studies of "short-armed" fucosylated chondroitin sulfate glycoclusters.

Fucosylated chondroitin sulfate (FuCS) is a structurally complex glycosaminoglycan found in sea cucumbers with a wide spectrum of biological activities, among which anticoagulant activity is particularly attractive for the development of alternative anticoagulant drugs with decreased adverse effects and risks of bleeding. Previous studies show that FuCS glycomimetics bearing several trisaccharide epitopes displayed promising anticoagulant activity and did not change the mode of action of FuCS. To simplify synthetic difficulty of high valent glycoclusters and obtain candidate compounds with relatively low molecular weights, here we report the synthesis of two FuCS glycoclusters with low valence and more compact structures. Anticoagulation studies showed that these simplified "short-armed" glycoclusters demonstrated comparable potency with "long-armed" high valent glycoclusters, offering a concise approach for the development of novel anticoagulant agents.

Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex.

A method for measuring activated factor VIII in plasma.

A method is described which enables a quantitative measurement of the concentration of activated factor VIII (VIIIa) in plasma. Based on the ability of factor VIIIa to accelerate the activation of factor X by factor IXa, phospholipid and calcium ions, the course of factor X activation in time is measured using a chromogenic substrate. Free factor Xa is able to activate nonactivated factor VIII present in a plasma sample, which increases the factor X activation velocity, and thus disturbs the measurement of factor VIIIa. Furthermore, factor Xa was found to be inactivated by serine protease inhibitors from the plasma sample. By adding surplus chromogenic substrate these reactions of factor Xa are inhibited and at the same time the rate of substrate conversion is a measure of the amount of factor Xa present. Factor X activation and amidolysis of chromogenic substrate then take place simultaneously. It is shown that under proper conditions the factor X activation velocity is linearly proportional to the factor VIIIa concentration. This causes the optical density to increase as a parabolic function of time. The concentration of factor VIIIa can be obtained from the quadratic coefficient of the equation describing the parabola. The method is specific for factor VIIIa in that the extrinsic factor X activator is shown to have no influence on the measurement of factor VIIIa in thromboplastin activated plasma. We conclude that a sensitive and reliable method for assessing factor VIIIa concentrations in plasma has been developed on the basis of simultaneous inhibition and measurement of factor Xa by a high concentration of chromogenic substrate.

Synergistic cofactor function of factor V and protein S to activated protein C in the inactivation of the factor VIIIa - factor IXa complex -- species specific interactions of components of the protein C anticoagulant system.

Human factor V has been shown not only to be a precursor to procoagulant factor Va but also to express anticoagulant properties. Thus, factor V was recently found to potentiate the effect of protein S as cofactor to activated protein C (APC) in the inactivation of the factor VIIIa-factor IXa complex. The purpose of this study was to determine whether the APC-cofactor function of factor V was also expressed in the bovine protein C system and to elucidate the molecular background for the species specificity of APC. For this purpose, the effects of protein S and factor V on APC-mediated inactivation of factor VIIIa were studied using purified APC, protein S and factor V of human and bovine origin. The factor VIIIa investigated here was part of a Xase complex (i.e. factor IXa, factor VIIIa, phospholipid and calcium) and the APC-mediated inhibition of factor VIIIa was monitored by the ability of the Xase complex to activate factor X. Synergistic APC-cofactor function of factor V and protein S was demonstrated in the bovine system. The effect of bovine APC was potentiated by bovine protein S but not by human protein S, whereas both human or bovine protein S stimulated the function of human APC. Factor V did not express species specificity in its APC-cofactor activity even though bovine factor V was more potent than its human counterpart. Recombinant human/bovine protein S chimeras were used to demonstrate that the thrombin sensitive region and first epidermal growth factor-like module of protein S determine the species specificity of the APC-protein S interaction. In conclusion, both human and bovine factor V were found to express APC-cofactor activity which depends on the presence of protein S. The species specificity of APC was shown to be caused by the interaction between APC and protein S.

C4b-binding protein inhibits the factor V-dependent but not the factor V-independent cofactor activity of protein S in the activated protein C-mediated inactivation of factor VIIIa.

Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equimolar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.

Mechanisms of human neutrophil elastase-catalysed inactivation of factor VIII(a).

Mechanisms of inflammation and coagulation are linked through various pathways. Human neutrophil elastase (HNE), can bind to activated platelets, might be localised on platelet membranes that provide negatively-charged phospholipid essential for the optimum function of tenase complex. In this study, we examined the effect of HNE on factor (F)VIII. FVIII activity was rapidly diminished in the presence of HNE and was undetectable within 10 minutes. The inactivation rate was ~8-fold greater than that of activated protein C (APC). This time-dependent inactivation was moderately affected by von Willebrand factor. HNE proteolysed the heavy chain (HCh) of FVIII into two terminal products, A11-358 and A2375-708, by limited proteolysis at Val358, Val374, and Val708. Cleavage at Val708 was much slower than that at Val358 in the >90-kDa A1-A2-B compared to the 90-kDa A1-A2. The 80-kDa light chain (LCh) was proteolysed to 75-kDa product by cleavage at Val1670. HNE-catalysed FVIIIa inactivation was markedly slower than that of native FVIII (by ~25-fold), due to delayed cleavage at Val708 in FVIIIa. The inactivation rate mediated by HNE was ~8-fold lower than that by APC. Cleavages at Val358 and Val708 were regulated by the presence of LCh and HCh, respectively. In conclusion, HNE-catalysed FVIII inactivation was associated with the limited-proteolysis that led to A11-358, A2375-708, and A3-C1-C21671-2332, and subsequently to critical cleavage at Val708. HNE-related FVIII(a) reaction might play a role in inactivation of HNE-induced coagulation process, and appeared to depend on the amounts of inactivated FVIII and active FVIIIa which is predominantly resistant to HNE inactivation.

Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation.

The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/Phe2200 phospholipid binding beta-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 beta-sandwich. MAbs from groups A, AB, and B inhibit the binding of fVIIIa to phospholipid membranes. Group BC was the most common group and displayed the highest specific fVIII inhibitor activities. MAbs in this group are type II inhibitors that inhibit the activation of fVIII by either thrombin or factor Xa and poorly inhibit the binding of fVIII to phospholipid membranes or von Willebrand factor (VWF). Group BC MAbs are epitopically and mechanistically distinct from the extensively studied group C MAb, ESH8. These results reveal the structural and functional complexity of the anti-C2 domain antibody response and indicate that interference with fVIII activation is a major attribute of the inhibitor landscape.

Elevated alpha1-antitrypsin is a risk factor for arterial ischemic stroke in childhood.

Alpha1-antitrypsin (alpha1-AT) is a physiological inhibitor of activated protein C (APC) and therefore decreased APC activity. APC itself causes an anticoagulant effect by inactivating factors Va and VIIIa. The present case-control study was performed to evaluate the role of the elevated alpha1-AT concentration in pediatric patients with ischemic stroke (IS). alpha1-AT concentrations were measured along with established prothrombotic risk factors 6-12 months after the acute thrombotic onset in 81 Caucasian children with IS aged 1 month to 18 years. The cutoff values defined as age-dependent 90th percentiles were obtained from 229 healthy controls. Median (range) values of alpha1-AT were significantly higher in patients compared with control subjects [122.0 mg/dl (61.4-224.0) vs. 114.0 mg/dl (66.8-156.0); p = 0.016]. In addition, 14 of the 81 patients (17.3%) compared with 10 of the 162 controls (6.2%) had alpha1-AT concentrations above the 90th age-dependent percentiles (p = 0.012). Multivariate analysis performed in a 1:2 matched case-control setting adjusted for the presence of established prothrombotic risk factors showed a significantly increased odds ratio (OR) and 95% confidence interval (CI) for patients with elevated alpha1-AT >90th percentiles and IS (OR/CI: 4.0/1.64-9.92; p = 0.0024). Data shown here give evidence that total alpha1-AT concentrations above the 90th age-dependent percentiles independently increase the risk of IS 4.0-fold in Caucasian children.

New anticoagulant therapy.

The development of new anticoagulants is expanding the list of drugs that can be used to prevent and treat venous and arterial thrombosis. New parenteral anticoagulants have been developed to overcome the limitations of heparin and low-molecular-weight heparin, whereas novel orally active anticoagulants have been designed to provide more streamlined therapy than vitamin K antagonists. This review identifies the molecular targets of new anticoagulants, describes the results of clinical trials, and provides clinical perspective on the opportunities for new anticoagulants.

Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity.

Human factor VIII and factor VIIIa were proteolytically inactivated by activated protein C. Cleavages occurred within the heavy chain (contiguous A1-A2-B domains) of factor VIII and in the heavy chain-derived A1 and A2 subunits of factor VIIIa, whereas no proteolysis was observed in the light chain or light chain-derived A3-C1-C2 subunit. Reactivity to an anti-A2 domain monoclonal antibody and NH2-terminal sequence analysis of three terminal digest fragments from factor VIII allowed ordering of fragments and identification of cleavage sites. Fragment A1 was derived from the NH2 terminus and resulted from cleavage at Arg336-Met337. The A2 domain was bisected following cleavage at Arg562-Gly563 and yielded fragments designated A2N and A2C. A third cleavage site is proposed at the A2-B junction (Arg740-Ser741) since fragment A2C was of equivalent size when derived either from factor VIII or factor VIIIa. The site at Arg562 was preferentially cleaved first in factor VIII(alpha) compared with the site at Arg336, and it was this initial cleavage that most closely correlated with the loss of cofactor activity. Factor VIIIa was inactivated 5-fold faster than factor VIII, possibly as a result of increased protease utilization of the site at Arg562 when the A2 subunit is not contiguous with the A1 domain. When initial cleavage occurred at Arg336, it appeared to preclude subsequent cleavage at Arg562, possibly by promoting dissociation of the A2 domain (subunit) from the A1/light chain dimer. This conclusion was supported by the failure of protease treated A1/A3-C1-C2 dimer to bind A2 subunit and gel filtration analysis that showed dissociation of the A2 domain-derived fragments, A2N and A2C, from the A1 fragment/light chain dimer. These results suggest a mechanism for activated protein C-catalyzed inactivation of factor VIII(alpha) involving both covalent alteration and fragment dissociation.

Identification of a sequence of human activated protein C (residues 390-404) essential for its anticoagulant activity.

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify the regions on the surface that mediate anticoagulant activity, 26 synthetic peptides were prepared representing 90% of the human protein C heavy chain primary structure and tested for their ability to inhibit APC anticoagulant activity. Peptide-(390-404) specifically inhibited APC activity in activated partial thromboplastin time and Xa-1-stage coagulation assays in normal, in protein S-depleted and Factor VIII-deficient plasma with 50% inhibition at 5 microM peptide. Polyclonal antibodies raised against this peptide and immunoaffinity-purified on a protein C-Sepharose column inhibited APC anticoagulant activity in activated partial thromboplastin time and Xa-1-stage assays in normal, protein S-depleted, and Factor VIII-deficient plasma with half-maximal inhibition at 30 nM anti-(390-404) antibody. Neither the peptide-(390-404) nor the anti-(390-404) antibodies inhibited APC amidolytic activity or the reaction of APC with recombinant [Arg358] alpha 1-antitrypsin. Furthermore, in a purified system, peptide-(390-404) inhibited APC-catalyzed inactivation of Factor Va in the presence as well as in the absence of phospholipids with 50% inhibition at 4 microM peptide. These data suggest that the region containing residues 390-404 in APC is essential for anticoagulant activity and is available to interact with antibodies or with other proteins such as the macromolecular substrates Factors Va or VIIIa.

Targeting tissue factor as an antithrombotic strategy.

It is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of cell-surface protein, such as tissue factor (TF). TF is exposed to the flowing blood as a consequence of vascular injury induced, for instance, by PTCA, or by spontaneous rupture of an atherosclerotic plaque. Expression of TF may also be induced in monocytes and endothelial cells in several conditions such as sepsis and cancer, causing a more generalized activation of clotting. In addition to its essential role in hemostasis, TF may be also implicated in several pathophysiological processes, such as intracellular signaling, cell proliferation, and inflammation. For all these reasons, TF has been the subject of intense research focus. Many experimental studies have demonstrated that inhibition of TF:factor VIIa procoagulant activity is a powerful inhibitor of in vivo thrombosis and that this approach usually results in a less-pronounced bleeding tendency compared with other "more classical" antithrombotic interventions. Alternative approaches may be represented by antibodies directed against TF, by transfection of the arterial wall with natural inhibitors of the TF:factor VIIa complex, such as the TF pathway inhibitor, or with catalytic RNA (ribozyme), which could inhibit the expression of the TF protein by the disruption of cellular TF mRNA. All these approaches seem particularly attractive because they may result in complete inhibition of local thrombosis without incurring potentially harmful systemic effects. Further studies are warranted to determine the efficacy and safety of such approaches in patients.

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa.

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

The role of the tissue factor-thrombin pathway in cardiac ischemia-reperfusion injury.

This article focuses on the role of the tissue factor (TF)-thrombin pathway in cardiac ischemia-reperfusion (I/R) injury. We and others have used rabbit models of cardiac I/R injury to show that anti-TF therapy prevents the transient decrease in regional myocardial blood flow, reduces platelet and fibrin(ogen) accumulation, and reduces infarct size. At present, the mechanism by which TF-initiated coagulation contributes to myocardial injury is not established. Inhibition of TF may decrease intravascular fibrin deposition and thrombosis. However, immunohistochemical studies demonstrated that fibrin deposition was predominantly within the myocardium and depletion of fibrinogen did not reduce infarct size. In contrast, inhibition of thrombin reduced infarct size to a similar extent as anti-TF therapy. We propose that the TF-thrombin pathway may contribute to myocardial injury by an additional mechanism that is not dependent on fibrin deposition but involves activation of protease activated receptor 1 (PAR-1) on vascular endothelial cells and cardiac myocytes. Anti-TF therapy would inhibit both thrombin-dependent fibrin deposition and thrombin-dependent PAR-1 signaling.

Heparin inhibits the intrinsic tenase complex by interacting with an exosite on factor IXa.

The specific molecular target for direct heparin inhibition of factor X activation by intrinsic tenase (factor IXa-factor VIIIa) was investigated. Comparison of size-fractionated oligosaccharides demonstrated that an octasaccharide was sufficient to inhibit intrinsic tenase. Substitution of soluble dihexanoic phosphatidylserine (C6PS) for phospholipid (PL) vesicles demonstrated that inhibition by low-molecular weight heparin (LMWH) was independent of factor IXa-factor VIIIa membrane assembly. LMWH also inhibited factor X activation by the factor IXa-PL complex via a distinct mechanism that required longer oligosaccharides and was independent of substrate concentrations. The apparent affinity of LMWH for the factor IXa-PL complex was higher in the absence of factor VIIIa, suggesting that the cofactor adversely affected the interaction of heparin with factor IXa-phospholipid. LMWH did not interact directly with the active site, as it failed to inhibit chromogenic substrate cleavage by the factor IXa-PL complex. LMWH induced a modest decrease in factor IXa-factor VIIIa affinity [K(D(app))] on PL vesicles that did not account for the inhibition. In contrast, LMWH caused a substantial reduction in factor IXa-factor VIIIa affinity in the presence of C6PS that fully accounted for the inhibition. Factor IXa bound LMWH with significantly higher affinity than factor X by competition solution affinity analysis, and the K(D(app)) for the factor IXa-LMWH complex agreed with the K(I) for inhibition of the factor IXa-PL complex by LMWH. Thus, LMWH binds to an exosite on factor IXa that antagonizes cofactor activity without disrupting factor IXa-factor VIIIa assembly on the PL surface. This exosite may contribute to the clinical efficacy of heparin and represents a novel target for antithrombotic therapy.

Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism.

Phosphorothioate oligonucleotides (PS ODNs) prolong the activated partial thromboplastin time in human plasma by inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity. This inhibition was characterized using ISIS 2302, a 20-mer antisense PS ODN. ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of factor X activation by the intrinsic tenase complex. The decrease in V(max(app)) was analyzed by examining complex assembly, cofactor stability, and protease catalysis. ISIS 2302 did not inhibit factor X activation by the factor IXa-phospholipid complex, or significantly affect factor VIII-phospholipid affinity. Inhibitory concentrations of ISIS 2302 modestly decreased the affinity of factor IXa-factor VIIIa binding in the presence of phospholipid (K(D) = 11.5 vs 4.8 nM). This effect was insufficient to explain the reduction in V(max(app)). ISIS 2302 did not affect the in vitro half-life of factor VIIIa, suggesting it did not destabilize cofactor activity. In the presence of 30% ethylene glycol, the level of factor X activation by the factor IXa-phospholipid complex increased 3-fold, and the level of chromogenic substrate cleavage by factor IXa increased more than 50-fold. ISIS 2302 demonstrated partial inhibition of factor X activation by the factor IXa-phospholipid complex, and chromogenic substrate cleavage by factor IXa, only in the presence of ethylene glycol. Like the intact enzyme complex, ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of chromogenic substrate cleavage by factor IXa (K(I) = 88 nM). Equilibrium binding studies with fluorescein-labeled ISIS 2302 demonstrated a similar affinity (K(D) = 92 nM) for the PS ODN-factor IX interaction. These results suggest that PS ODNs bind to an exosite on factor IXa, modulating catalytic activity of the intrinsic tenase complex.