Coagulation factors VII, IX and X are effective antibacterial proteins against drug-resistant Gram-negative bacteria.

Infections caused by drug-resistant "superbugs" pose an urgent public health threat due to the lack of effective drugs; however, certain mammalian proteins with intrinsic antibacterial activity might be underappreciated. Here, we reveal an antibacterial property against Gram-negative bacteria for factors VII, IX and X, three proteins with well-established roles in initiation of the coagulation cascade. These factors exert antibacterial function via their light chains (LCs). Unlike many antibacterial agents that target cell metabolism or the cytoplasmic membrane, the LCs act by hydrolyzing the major components of bacterial outer membrane, lipopolysaccharides, which are crucial for the survival of Gram-negative bacteria. The LC of factor VII exhibits in vitro efficacy towards all Gram-negative bacteria tested, including extensively drug-resistant (XDR) pathogens, at nanomolar concentrations. It is also highly effective in combating XDR Pseudomonas aeruginosa and Acinetobacter baumannii infections in vivo. Through decoding a unique mechanism whereby factors VII, IX and X behave as antimicrobial proteins, this study advances our understanding of the coagulation system in host defense, and suggests that these factors may participate in the pathogenesis of coagulation disorder-related diseases such as sepsis via their dual functions in blood coagulation and resistance to infection. Furthermore, this study may offer new strategies for combating Gram-negative "superbugs".

Prohemostatic Activity of Factor X in Combination With Activated Factor VII in Dilutional Coagulopathy.

BACKGROUND: Recombinant activated factor VII (rFVIIa) concentrate reduces allogeneic blood transfusions, but it may increase thromboembolic complications in complex cardiac surgery. The mixture of activated factor VII (FVIIa) and factor X (FX) (FVIIa/FX) (FVIIa:FX = 1:10) is a novel bypassing agent for hemophilia patients. We hypothesized that the combination of FX and FVIIa could improve thrombin generation (TG) in acquired multifactorial coagulation defects such as seen in cardiac surgery and conducted in vitro evaluation of FVIIa/FX in parallel with other coagulation factor concentrates using in vitro and in vivo diluted plasma samples. METHODS: Plasma samples were collected from 9 healthy volunteers and 12 cardiac surgical patients. We measured TG (Thrombinoscope) using in vitro 50% dilution plasma and in vivo dilution plasma after cardiopulmonary bypass, in parallel with thromboelastometry (ROTEM) and standard coagulation assays. In vitro additions of FVIIa/FX (0.35, 0.7, and 1.4 µg/mL, based on the FVIIa level), rFVIIa (1.4, 2.8, and 6.4 µg/mL), prothrombin complex concentrate (0.3 international unit), and 20% plasma replacement were evaluated. RESULTS: In diluted plasma, the addition of either FVIIa/FX or rFVIIa shortened the lag time and increased the peak TG, but the effect in lag time of FVIIa/FX at 0.35 µg/mL was more extensive than rFVIIa at 6.4 µg/mL. Prothrombin complex concentrate increased peak TG by increasing the prothrombin level but failed to shorten the lag time. No improvement in any of the TG variables was observed after 20% volume replacement with plasma. The addition of factor concentrates normalized prothrombin time/international normalized ratio but not with plasma replacement. In cardiac patients, similar patterns were observed on TG in post-cardiopulmonary bypass samples. FVIIa/FX shortened clotting time (CT) in a concentration-dependent manner on CT on thromboelastometry. Plasma replacement did not improve CT, but a combination of plasma and FVIIa/FX (0.35 µg/mL) more effectively shortened CT than FVIIa/FX alone. CONCLUSIONS: The combination of FVIIa and FX improved TG more efficiently than rFVIIa alone or plasma in dilutional coagulopathy models. The required FVIIa dose in FVIIa/FX was considerably lower than those reported during bypassing therapy in hemophilia patients (1.4-2.8 µg/mL). The combination of plasma could restore coagulation more efficiently compared to FVIIa/FX alone. Lesser FVIIa requirement to exert procoagulant activity may be favorable in terms of reducing systemic thromboembolic complications.

Independent anti-angiogenic capacities of coagulation factors X and Xa.

Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.

Differential effects of murine and human factor X on adenovirus transduction via cell-surface heparan sulfate.

Serum coagulation factor X (FX) is proposed to play a major role in adenovirus tropism, promoting transduction by bridging the virus to cell-surface heparan sulfate proteoglycans (HSPGs). Both murine FX and human FX increased transduction by Ad.CMVfLuc, an adenovirus vector, in murine hepatocyte-like cells and human hepatocarcinoma cells. In contrast, only hFX increased transduction of several non-hepatic cancer cell lines and Chinese hamster ovary (CHO) cells. Not only was mFX unable to promote transduction in these cells, it competitively blocked hFX-enhanced transduction. Competition and HSPG digestion experiments suggested mFX- and hFX-enhanced transduction in hepatocyte-derived cells, and hFX-enhanced transduction in epithelial cancer cells were dependent on HSPGs. Ad·hFX-mediated transduction of CHO mutants unable to produce HSPGs was also curtailed. Hepatocyte-derived cells expressed substantially more HSPGs than the cancer cell lines. Dose-response curves and heparin-Sepharose binding suggested Ad·hFX has greater affinity for HSPGs than does Ad·mFX. In coagulation factor-depleted mice hFX also had enhanced ability, compared with mFX, to reconstitute hepatic adenovirus transduction. The results suggest that differences in Ad·hFX and Ad·mFX affinity to HSPGs may result in differences in their ability to enhance adenovirus transduction of many cells. These findings may have implications for murine models of adenovirus vector targeting.

The roles of cell surface attachment molecules and coagulation Factor X in adenovirus 5-mediated gene transfer in pancreatic cancer cells.

Transduction of 11 pancreatic cancer cell lines with a replication-deficient adenovirus 5 expressing enhanced green fluorescent protein (Ad5EGFP) was analyzed and variable EGFP levels were observed, ranging from <1% to ∼40% of cells transduced, depending on the cell line. Efficient Ad5EGFP transduction was associated mainly with higher levels of cell surface Coxsackie and adenovirus receptor (CAR) but not with expression of α(v)ß(3) and α(v)ß(5) integrins and was fiber dependent. Reduction of CAR by RNA interference resulted in a corresponding decrease in Ad5EGFP transduction. Pre-treatment of Ad5EGFP with blood coagulation Factor X increased virus entry even in the presence of low CAR levels generated by RNA interference, suggesting a potential alternative route of Ad5 entry into pancreatic cancer cells. Immunohistochemistry carried out on 188 pancreatic ductal adenocarcinomas and 68 matched controls showed that CAR was absent in 102 (54%) of adenocarcinomas, whereas moderate and strong staining was observed in 58 (31%) and 28 (15%) cases, respectively. Weak or absent CAR immunolabeling correlated with poor histological differentiation of pancreatic cancer. In normal tissue, strong immunolabeling was detected in islet cells and in the majority of inter- and intralobular pancreatic ducts.

Protein S down-regulates factor Xase activity independent of activated protein C: specific binding of factor VIII(a) to protein S inhibits interactions with factor IXa.

Protein S functions as an activated protein C (APC)-independent anticoagulant in the inhibition of intrinsic factor X activation, although the precise mechanisms remain to be fully investigated. In the present study, protein S diminished factor VIIIa/factor IXa-dependent factor X activation, independent of APC, in a functional Xa generation assay. The presence of protein S resulted in an c. 17-fold increase in K(m) for factor IXa with factor VIIIa in the factor Xase complex, but an c. twofold decrease in K(m) for factor X. Surface plasmon resonance-based assays showed that factor VIII, particularly the A2 and A3 domains, bound to immobilized protein S (K(d); c. 10 nmol/l). Competition binding assays using Glu-Gly-Arg-active-site modified factor IXa showed that factor IXa inhibited the reaction between protein S and both the A2 and A3 domains. Furthermore, Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the cleavage rate of factor VIIIa at Arg(336) by factor IXa was c. 1.8-fold lower in the presence of protein S than in its absence. These data indicate that protein S not only down-regulates factor VIIIa activity as a cofactor of APC, but also directly impairs the assembly of the factor Xase complex, independent of APC, in a competitive interaction between factor IXa and factor VIIIa.

Factor VIIa stimulates endothelin-1 synthesis in TNF-primed endothelial cells by activation of protease-activated receptor 2.

The mechanisms linking prothrombotic changes to endothelial dysfunction and accelerated atheroma formation have yet to be fully defined. Expression of TF (tissue factor) on the endothelium is potentially an initiating event as binding and activation of FVII (factor VII) can result in thrombosis. Although PAR2 (protease-activated receptor-2) is expressed on vascular endothelium, its precise physiological significance and mechanism of activation have yet to be defined. In the present study, we investigated whether PAR2 can be activated by FVIIa (activated FVII) and induce ET-1 (endothelin-1) synthesis. In bovine aortic endothelial cells pretreated with TNF (tumour necrosis factor-alpha) to increase TF expression, FVIIa stimulated ET-1 synthesis via activation of PAR2. Although FX (factor X) alone was inactive, this response was enhanced by using FVII and FX in combination. Inhibition of the proteolytic activity of FVIIa abolished the response. The PAR2 agonist peptide SLIGKV also enhanced ET-1 release on TNF-pretreated cells. The response to FVIIa was inhibited by a PAR2 antagonist peptide FSLLRY. Inhibition of the p38 MAPK (mitogen-activated protein kinase) reduced PAR2 expression and the ET-1 response. In summary, FVIIa can stimulate ET-1 synthesis in endothelial cells by activating PAR2, demonstrating a potential link between thrombotic processes and endothelial cell dysfunction.

Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts.

UNLABELLED: Coagulation factors (F)VIIa, FXa and thrombin are implicated in cellular responses in vascular, mesenchymal and inflammatory cells. Fibroblasts are the most abundant cells in connective tissue, and damage to blood vessels places coagulation factors in contact with these and other cell types. OBJECTIVES: To investigate cellular responses of primary dermal fibroblasts to FVIIa, FXa and thrombin by following changes in expression of candidate proteins: monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF), and to determine the expression of receptors implicated in signaling by these coagulation factors. METHODS: Steady-state mRNA levels were quantified by RNase protection assay, and protein secretion by ELISA. PAR gene expression was assessed by ribonuclease protection assay and conventional and quantitative reverse-transcription-polymerase chain reaction. RESULTS: FVIIa did not induce the candidate genes. In contrast, FXa and thrombin induced MCP-1 mRNA and protein secretion strongly, IL-8 moderately, and IL-6 weakly. Neither FXa nor thrombin induced VEGF mRNA or protein secretion, although FXa induced VEGF protein secretion in lung fibroblasts. Comparison of the presence of candidate receptors in the two fibroblast subtypes demonstrated higher levels of PAR-1 and PAR-3 in lung fibroblasts relative to their dermal counterparts and the additional expression of PAR-2. CONCLUSIONS: FXa and thrombin induce expression of MCP-1, IL-8 and IL-6, and distribution and expression of PARs on dermal fibroblasts is reduced relative to their lung counterparts. Tissue origin may influence the cellular response of fibroblasts to coagulation proteases.

Recombinant factor VIIa enhances deposition of platelets with congenital or acquired alpha IIb beta 3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor-independent thrombin generation.

A novel approach to treat bleeding episodes in patients with Glanzmann thrombasthenia (GT) and perhaps also in patients receiving alpha IIb beta 3 inhibitors is the administration of recombinant factor VIIa (rFVIIa). The mechanism of action of rFVIIa in these patients is, however, still unclear. We studied the effect of rFVIIa-mediated thrombin formation on adhesion of alpha IIb beta 3-deficient platelets under flow conditions. Adhesion of alpha IIb beta 3-deficient platelets to the extracellular matrix (ECM) of stimulated human umbilical vein endothelial cells or to collagen type III was studied using a model system with washed platelets and red cells. When alpha IIb beta 3-deficient platelets were perfused over the surface at arterial shear rate for 5 minutes, a low surface coverage was observed (GT platelets, mean +/- SEM, 37.5% +/- 5.0%; normal platelets preincubated with an RGD-containing peptide, 7.4% +/- 2.1%). When rFVIIa, together with factors X and II, was added to the perfusate, platelet deposition significantly increased (GT platelets, mean +/- SEM, 67.0% +/- 4.3%; normal platelets preincubated with an RGD-containing peptide, 48.2% +/- 2.9%). The same effect was observed when normal platelets were pretreated with the commercially available anti-alpha IIb beta 3 drugs abciximab, eptifibatide, or tirofiban. It was shown that tissue factor-independent thrombin generation (presumably induced by binding of rFVIIa to adhered platelets) was responsible for the increase in platelet deposition. In conclusion, defective adhesion of alpha IIb beta 3-deficient platelets to ECM can be restored by tissue factor-independent rFVIIa-mediated thrombin formation. The enhanced generation of platelet procoagulant surface facilitates fibrin formation, so that lack of platelet aggregate formation might be compensated for.

Platelet aggregation through prothrombinase activation induced by non-aggregant doses of platelet agonists.

Activation of the prothrombinase complex, which catalyzes the formation of thrombin from prothrombin, is crucial for the (patho)physiological processes of hemostasis and thrombosis. We here report that washed platelets supplemented with prothrombin can be irreversibly aggregated with otherwise non-aggregant doses of adenosine diphosphate (10 micromol/l), thrombin (0.06 U/ml), or collagen (1 microg/ml). Prothrombinase-catalyzed prothrombin to thrombin conversion most probably supports this aggregation response, since inhibitors of thrombin (hirudin or heparin) and an inhibitor of activated factor X (DX-9065a) impair the response. A certain degree of agonist-induced platelet activation seems to be required for this prothrombin-supported aggregation response, since prothrombin alone does not induce aggregation, and blockade of glycoprotein Ia/IIa with a specific antibody inhibits the platelet aggregation response to collagen and prothrombin. These results may suggest that activation of the prothrombinase complex could be a common step of the platelet response to distinct agonists, which may be achieved at low levels of platelet stimulation.

Tissue factor is the only activator of coagulation in cultured human lung cancer cells.

It is a long-known principle that tumour cells tend to exploit the host's physiologic systems in order to get support in terms of, for example, nutrition, growth or metastasis. One of these physiologic systems is the blood coagulation cascade, which has been found activated in many tumour patients. The mechanisms of the activation of coagulation have been assessed in numerous animal and in vitro experiments, and the results appeared to point to several distinct activators. The present study used a large panel of different cultivated human lung cancer cell lines and experimental systems involving normal plasma, plasmas deficient of factors V, VII or X, purified coagulation factors II and X, recombinant tissue factor (TF), and specific inhibitory antibodies against factor VII and TF. The results provide strong evidence that there is no activator of coagulation besides TF in the wide array of lung cancer cells examined. However, this work reveals a striking variability of TF content among the cell lines. This might explain ambiguous results of clinical trials of anticoagulation as an adjunct to antineoplastic therapy in lung cancer. By sensitive diagnostic tools like the plasma thrombin-antithrombin complex levels it might be possible to select patients with activated coagulation, who might benefit from anticoagulation.

Macrophages synthesize factor X and secrete factor X/Xa-containing prothrombinase activity into the surrounding medium.

Activation of the coagulation cascade, mediated by various monocyte/macrophage procoagulants, is an important component in the pathology of inflammatory disease. The type of procoagulant expressed may vary between different monocyte/macrophage subtypes and may differ depending on how the cells are treated. In the present study we show that both murine peritoneal macrophages and human adherent synovial cells from rheumatoid arthritis lesions express prothrombinase activity that was inhibited by anti-Factor X antibodies. Northern blot analysis showed that Factor X was transcribed by the murine peritoneal cells and Western blot analysis showed the presence of Factor X antigen. Further experiments showed that the prothrombinase activity was secreted by the cells into the medium in a detergent-sensitive form, suggesting that the prothrombinase is released on small lipid-containing vesicles.

Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa.

Tissue factor (TF) assembled with activated factor VII (FVIIa) initiates the coagulation cascade. We recently showed that TF was essential for FVIIa-induced vascular endothelial growth factor (VEGF) production by human fibroblasts. We investigated whether this production resulted from TF activation by its binding to FVIIa or from the production of clotting factors activated downstream. Incubation of fibroblasts with a plasma-derived FVIIa concentrate induced the generation of activated factor X (FXa) and thrombin and the secretion of VEGF, which was inhibited by hirudin and FXa inhibitors. By contrast, the addition of recombinant FVIIa to fibroblasts did not induce VEGF secretion unless factor X was present. Moreover, thrombin and FXa induced VEGF secretion and VEGF mRNA accumulation, which were blocked by hirudin and FXa inhibitors, respectively. The effect of thrombin was mediated by its specific receptor, protease-activated receptor-1; in contrast, the effect of FXa did not appear to involve effector cell protease receptor-1, because it was not affected by an anti-effector cell protease receptor-1 antibody. An increase in intracellular calcium with the calcium ionophore A23187 or intracellular calcium chelation by BAPTA-AM had no effect on either basal or FXa-induced VEGF secretion, suggesting that the calcium signaling pathway was not sufficient to induce VEGF secretion. Finally, FVIIa, by itself, had no effect on mitogen-activated protein (MAP) kinase activation, contrary to thrombin and FXa, which activate the p44/p42 MAP kinase pathway, as shown by the blocking effect of PD 98059 and by Western blotting of activated MAP kinases. These findings indicate that FVIIa protease induction of VEGF expression is mediated by thrombin and FXa generated in response to FVIIa binding to TF-expressing fibroblasts; they also exclude a direct signaling involving MAP kinase activation via the intracellular domain of TF when expressed by these cells.

Thrombin activates factor XI on activated platelets in the absence of factor XII.

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.

Cloning and recombinant expression of mouse coagulation factor X.

Engineering of recombinant coagulation factor X variants, which can be activated by tumor-associated proteinases may lead to the development of new therapeutic molecules. However, the evaluation of such variants requires an appropriate animal model. Therefore, we isolated the complete coding sequence of mouse coagulation factor X from mouse liver cDNA by polymerase chain reaction. The deduced amino acid sequence codes for a prepro protein of 481 amino acids homologous to factor X sequences from various species. Recombinant mouse factor X was expressed in human embryonic kidney cells and secreted into cell culture supernatant as zymogen, which could be converted to catalytically active factor Xa by Russell's viper venom. Purified recombinant mouse factor X restored coagulation in human factor X deficient plasma, demonstrating that mouse factor X is able to functionally interact with the human blood coagulation system. Recombinant mouse factor X opens the possibility to analyze therapeutically useful variants in the mouse system.

Effector protease receptor 1 mediates the mitogenic activity of factor Xa for vascular smooth muscle cells in vitro and in vivo.

The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.

The effect of heparin on the regulation of factor VIIa-tissue factor activity by tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) inhibits factor VIIa-tissue factor and factor Xa through its first and second Kunitz-type domains, respectively. Previous studies have shown that heparin binds to TFPI at two sites and enhances the inhibitory activity of TFPI towards factor Xa. We have studied the effect of heparin of the TFPI-mediated inhibition of factor VIIa-relipidated tissue factor amiodolytic activity and factor VIIa-tissue factor proteolytic activity towards factor IX and factor X on a human bladder carcinoma cell line, J82. The inhibitory activity of full-length TFPI on factor VIIa-tissue factor amidolytic and proteolytic activities was greatly enhanced by heparin, whereas the inhibitory activity of a truncated form of TFPI lacking the third Kunitz-type domain and carboxy-terminal tail (TFPI1-161) was not affected by heparin. Optimal inhibition of factor VIIa-tissue factor by TFPI was observed at 0.1 U/ml heparin. Heparin enhanced the inhibitory activity of TFPI towards factor Xa even in the presence of factor VIIa-tissue factor complexes in solution phase. In addition, heparin augmented the ability of TFPI to inhibit factor VIIa-tissue factor amidolytic activity in the presence of active-site mutated factor X (S376A factor X). Our collective results suggest that heparin may play a significant role in augmenting the physiological regulation of factor VIIa-tissue factor activity by TFPI.

The inhibition of human factor VIIa-tissue factor by antithrombin III-heparin is enhanced by factor X on a human bladder carcinoma cell line.

Previous studies have shown that antithrombin III-heparin effectively inhibited the factor VIIa-tissue factor complex. Herein, we show that the neutralization of factor VIIa in complex with the cell surface tissue factor by antithrombin III-heparin was markedly enhanced by plasma levels of factor X. Active site-mutated factor X (S376A factor X) and factor Xa previously inactivated with dansyl-Glu-Gly-Arg-chloromethyl ketone were as effective as plasma-derived factor X in this reaction, indicating that the active site serine residue of factor Xa was not involved in this mechanism. Furthermore, Gla-domainless factor X had no effect in this system, emphasizing the importance of the factor X Gladomain in this reaction. Antibody experiments revealed that this effect was not due to trace levels of a tissue factor pathway inhibitor contaminating either the factor X or antithrombin III preparations. The presence of heparin in this system was essential, as deletion of heparin resulted in a factor VIIa-tissue factor neutralization rate essentially identical to that observed for antithrombin III alone. Plasma levels of factor IX also accelerated the inhibition of factor VIIa-tissue factor by antithrombin III-heparin, although its effect was not as pronounced as that of factor X. Other vitamin K-dependent plasma proteins including protein S, protein C and prothrombin failed to augment the inhibition of factor VIIa-tissue factor by antithrombin III-heparin. Factor X did not enhance the neutralization rate of factor VIIa-tissue factor by antithrombin III-heparin when a carboxyl-terminal truncated tissue factor construct (TF1-219) was used, even in the presence of mixed phospholipids. Our collective finding suggest that antithrombin III and factor X bind to heparin at distinct sites on the heparin molecule resulting in a transient ternary complex of antithrombin III-heparin-factor X that represents the anticoagulant species. Factor X conceivably guides complex to a phosphatidylserine-rich site on the cell surface in close proximity to the factor VIIa-tissue factor complex and facilitates rapid neutralization of factor VIIa. Our findings also suggest that the effect of heparin on the regulation of the extrinsic pathway of blood coagulation may be more profound than previously recognized.

Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity.

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.