The tick‐derived inhibitor Ixolaris prevents tissue factor signaling on tumor cells

Summary Background: Tissue factor (TF) is frequently overexpressed in cancer cells and correlated with more aggressive tumor phenotypes and poor prognosis. In addition to promoting coagulation‐dependent metastasis and cancer‐associated thrombosis, tumor cell‐expressed TF mediates direct cell signaling involving the protease‐activated receptor (PAR) 2. Ixolaris is a tick‐derived inhibitor of the TF–factor (F)VIIa–Xa coagulation initiation complex which blocks primary tumor growth and angiogenesis in glioblastoma and melanoma models. Methods: In this study we address the anti‐tumor effects of Ixolaris in TF–VIIa–PAR2 signaling‐dependent breast cancer models, a xenograft model of highly aggressive human MDA‐MB‐231mfp cells and a syngeneic model of PAR2‐deficient and replete PyMT mouse mammary carcinoma cells. Results: Ixolaris potently inhibited the procoagulant activity of human MDA‐MB‐231mfp or murine PyMT breast cancer cells. Ixolaris blocked signaling by the ternary TF–FVIIa–FXa complex, and, surprisingly, at higher concentrations also the binary TF–FVIIa complex on MDA‐MB‐231 cells. We show that Ixolaris interacts with certain residues in the human VIIa protease domain that are involved in PAR2 cleavage. In contrast to human VIIa, Ixolaris was a poor inhibitor of murine TF–FVIIa signaling and did not attenuate PAR2‐dependent tumor growth in a syngeneic mouse model of breast cancer progression. Conclusion: These data show that Ixolaris inhibits PAR2 cleavage specifically by human TF signaling complexes and suggest that Ixolaris may block tumor growth of human cell models with ectopic FVIIa expression through inhibition of direct TF–FVIIa–PAR2 signaling as well as its anticoagulant activity.     

Plasminogen activator inhibitor‐1 inhibits factor VIIa bound to tissue factor

Summary. Background and objective: A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI‐1). The present study was designed to investigate whether PAI‐1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well‐known procoagulant risk factor. Methods and results: The ability of PAI‐1 to inhibit FVIIa‐TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI‐1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa‐TF (PAI‐1_IC50, 817 and 125 nm , respectively); (ii) FVIIa‐TF‐mediated FX activation (PAI‐1_IC50, 260 and 50 nm , respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI‐1_IC50, 260 and 120 nm , respectively). The association rate constant (k_a) for PAI‐1 inhibition of FVIIa‐TF was determined using a chromogenic assay. K_a for PAI‐1 inhibition of FVIIa bound to relipidated TF is 3.3‐fold higher than that for FVIIa bound to soluble TF (k_a = 0.09 ± 0.01 and 0.027 ± 0.03 μm ^?1 min^?1, respectively). Vitronectin increases k_a for both soluble and relipidated TF by 3.5‐ and 30‐fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 μm ^?1 min^?1). However, only a 3.5‐ to 5.0‐fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI‐1/vitronectin/FVIIa/relipidated TF non‐covalent complex. Conclusions: Our results demonstrate potential anticoagulant activity of PAI‐1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI‐1 and TF are overexpressed.     

Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDA-MB-231 cells

BACKGROUND: Factor VIIa (FVIIa) binding to tissue factor (TF) induces cell signaling via the protease activity of FVIIa and protease-activated receptor 2 (PAR2). OBJECTIVE: We examined how the gene-expression profile induced by FVIIa corresponds to the profiles induced by protease-activated receptor 1 (PAR1) or PAR2 agonists using MDA-MB-231 breast carcinoma cells that constitutively express TF, PAR1 and PAR2. RESULTS AND CONCLUSIONS: Out of 8500 genes, FVIIa stimulation induced differential regulation of 39 genes most of which were not previously recognized as FVIIa regulated. All genes regulated by FVIIa were similarly regulated by a PAR2 agonist peptide confirming FVIIa signaling via PAR2. An appreciable fraction of the PAR2-regulated genes was also regulated by a PAR1 agonist peptide suggesting extensive redundancy between FVIIa/PAR2 signaling and thrombin/PAR1 signaling. The FVIIa regulated genes encode cytokines, chemokines and growth factors, and the gene repertoire induced by FVIIa in MDA-MB-231 cells is consistent with a role for TF-FVIIa signaling in regulation of a wound healing type of response. Interestingly, a number of genes regulated exclusively by FVIIa/PAR2-mediated cell signaling in MDA-MB-231 cells were regulated by thrombin and a PAR1 agonist, but not by FVIIa, in the TF-expressing glioblastoma U373 cell line.     

Mechanisms of factor VIIa‐catalyzed activation of factor VIII

Summary. Background: Factor (F)VIIa, complexed with tissue factor (TF), is a primary trigger of blood coagulation, and has extremely restricted substrate specificity. The complex catalyzes limited proteolysis of FVIII, but these mechanisms are poorly understood. Objectives: In the present study, we investigated the precise mechanisms of FVIIa/TF‐catalyzed FVIII activation. Results: FVIII activity increased ～4‐fold within 30 s in the presence of FVIIa/TF, and then decreased to initial levels within 20 min. FVIIa (0.1 nm ), at concentrations present physiologically in plasma, activated FVIII in the presence of TF, and this activation was more rapid than that induced by thrombin. The heavy chain (HCh) of FVIII was proteolyzed at Arg⁷⁴⁰ and Arg³⁷² more rapidly by FVIIa/TF than by thrombin, consistent with the enhanced activation of FVIII. Cleavage at Arg³³⁶ was evident at ～1 min, whilst little cleavage of the light chain (LCh) was observed. Cleavage of the HCh by FVIIa/TF was governed by the presence of the LCh. FVIII bound to Glu‐Gly‐Arg‐active‐site‐modified FVIIa (K_d, ～0.8 nm ) with a higher affinity for the HCh than for the LCh (K_d, 5.9 and 18.9 nm ). Binding to the A2 domain was particularly evident. Von Willebrand factor (VWF) modestly inhibited FVIIa/TF‐catalyzed FVIII activation, in keeping with the concept that VWF could moderate FVIIa/TF‐mediated reactions. Conclusions: The results demonstrated that this activation mechanism was distinct from those mediated by thrombin, and indicated that FVIIa/TF functions through a ‘priming’ mechanism for the activation of FVIII in the initiation phase of coagulation.     

Subcellular localization of tissue factor and human coronary artery smooth muscle cell migration

Summary. Background: Tissue factor (TF) is the most relevant physiological trigger of thrombosis. Additionally TF is a transmembrane receptor with cell signaling functions. Objectives: The aim of this study was to investigate TF subcellular localization, function and signaling in human coronary artery smooth muscle cell migration. Methods: Coronary arteries and primary cultures of vascular smooth muscle cells (HVSMC) were obtained from human explanted hearts. Wound repair and Boyden chamber assays were used to measure migration in vitro. TF‐pro‐coagulant activity (TF‐PCA) was measured in extracted cellular membranes. Analysis of TF distribution was performed by confocal microscopy. A nucleofector device was used for TF and protease activated receptor 2 (PAR2) silencing. mRNA levels were analyzed by RT‐PCR. Results: In migrating HVSMC TF translocates to the leading edge of the cells showing an intense patch‐like staining in the lamellipodia. In the migrating front TF colocalizes with filamin (FLN) in the polarized lipid rafts. TF‐PCA was increased in migrating cells. Silencing of the TF gene inhibits RSK‐induced FLN‐Ser‐2152 phosphorylation, down‐regulates CDC42, RhoA, and Rac1 protein expression and significantly inhibits cell migration. Silencing PAR2 also inhibits cell migration; however, silencing both TF and PAR2 induces a significantly higher effect on migration. Smooth muscle cells expressing TF have been identified in non‐lipid‐rich human coronary artery atherosclerotic plaques. Conclusions: TF translocates to the cell front in association with cytoskeleton proteins and regulates HVSMC migration by mechanisms dependent and independent of factor (F)VIIa/PAR2. These results extend the functional role of TF to smooth muscle cell trafficking in vessel wall remodeling.     

Regulation of tissue factor coagulant activity on cell surfaces

Summary. Tissue factor (TF) is a transmembrane glycoprotein and an essential component of the factor VIIa‐TF enzymatic complex that triggers activation of the coagulation cascade. Formation of TF‐FVIIa complexes on cell surfaces not only trigger the coagulation cascade but also transduce cell signaling via activation of protease‐activated receptors. Tissue factor is expressed constitutively on cell surfaces of a variety of extravascular cell types, including fibroblasts and pericytes in and surrounding blood vessel walls and epithelial cells, but is generally absent on cells that come into contact with blood directly. However, TF expression could be induced in some blood cells, such as monocytes and endothelial cells, following an injury or pathological stimuli. Tissue factor is essential for hemostasis, but aberrant expression of TF leads to thrombosis. Therefore, a proper regulation of TF activity is critical for the maintenance of hemostatic balance and health in general. TF‐FVIIa coagulant activity at the cell surface is influenced not only by TF protein expression levels but also independently by a variety of mechanisms, including alterations in membrane phospholipid composition and cholesterol content, thiol‐dependent modifications of TF allosteric disulfide bonds, and other post‐translational modifications of TF. In this article, we critically review the key literature on mechanisms by which TF coagulant activity is regulated at the cell surface in the absence of changes in TF protein levels with specific emphasis on recently published data and provide the authors’ perspective on the subject.     

Platelet binding and activity of a factor VIIa variant with enhanced tissue factor independent activity

Summary. Background and objectives: Platelet binding and activity play important roles in the efficacy of factor VIIa (FVIIa) as a bypassing agent for hemophilia treatment. An analog of FVIIa with increased tissue factor (TF)‐independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain. NN1731 has a conformation similar to TF‐bound FVIIa, even in the absence of TF. This results in much greater intrinsic proteolytic activity, but similar activity in the presence of TF. Objectives: We hypothesized that these changes would not alter binding to platelets or phospholipid, a characteristic thought to be localized to the Gla domain. The goal of the current work was to compare platelet binding and activity of NN1731 and wild‐type FVIIa. Methods/Results: FVIIa and NN1731 bound identically to phospholipid vesicles as assessed by both activity assays and electrophoretic quasielastic light scattering techniques. However, NN1731 bound to a greater number of sites on activated platelets than FVIIa, as assessed by flow cytometry. Removal of the Gla domain abolished binding of both FVIIa and NN1731. Inhibition of the active site did not reduce NN1731 binding to the level of FVIIa. When corrected for the amount of protein bound, NN1731 had greater activity than FVIIa on platelet surfaces. Conclusions: While the Gla domain is essential for FVIIa binding to platelets, changes in the protease domain in NN1731 enhanced platelet binding as well as proteolytic activity. Features in addition to lipid composition appear to contribute to binding of rFVIIa and, especially, NN1731 to platelets.     

Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro

To cite this article: Banfi C, Brioschi M, Lento S, Pirillo A, Galli S, Cosentino S, Tremoli E, Mussoni L. Statins prevent tissue factor induction by protease‐activated receptors 1 and 2 in human umbilical vein endothelial cells in vitro. J Thromb Haemost 2011; 9 : 1608–19. Summary. Background: Protease‐activated receptors (PARs) are G‐protein‐coupled receptors that function in hemostasis and thrombosis, as well as in the inflammatory and proliferative responses triggered by tissue injury. We have previously shown that PAR1 or PAR2 occupancy by specific PAR‐agonist peptides (PAR‐APs) induces tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs), where TF regulation by PAR1 (but not by PAR2) requires intact endothelial caveolin‐enriched membrane microdomains in which PAR1 and caveolin‐1 associate. Objectives: The aim of this study was to determine the effects of cholesterol‐lowering agents (statins) and cholesterol‐loading lipoprotein on PAR1‐AP‐mediated and PAR2‐AP‐mediated TF induction in HUVECs. Results: Statins completely prevented TF induction by PAR‐APs in an isoprenoid‐independent manner, induced the delocalization of PAR1 from caveolin‐enriched membrane microdomains without affecting PAR1 mRNA, and decreased PAR2 mRNA and protein levels. Statins also prevented PAR‐AP‐mediated extracellular signal‐related kinase 1/2 activation, which is crucial for TF induction. The redistribution of PAR1 is accompanied by the relocation of the membrane microdomain‐associated G‐protein α, caveolin‐1, and Src, which we previously showed to play a key role in signal transduction and TF induction. Conversely, cholesterol loading potently amplified PAR1‐AP‐induced TF, probably as a result of the increased abundance of PAR1 and the Src and G‐protein α signaling molecules in the caveolin‐1‐enriched fraction, without affecting PAR1 mRNA. Conclusions: As PARs have important functions in hemostasis, cancer, thrombosis, and inflammatory processes, our findings that statins prevent TF induction by PAR‐APs altering the membrane localization of PAR1 and the expression of PAR2 suggest that they may provide health benefits other than reducing atherosclerosis.     

Dynamical view of membrane binding and complex formation of human factor VIIa and tissue factor

Summary. Background: The molecular mechanism of enhancement of the enzymatic activity of factor VIIa by tissue factor (TF) is not fully understood, primarily because of the lack of atomic models for the membrane‐bound form of the TF–FVIIa complex. Objectives: To construct the first membrane‐bound model of the TF–FVIIa complex, and to investigate the dynamics of the complex in solution and on the surface of anionic membranes by using large‐scale molecular dynamics (MD) simulations in full atomic detail. Methods: Membrane‐bound models of the TF–FVIIa complex and the individual factors were constructed and subjected to MD simulations, in order to characterize protein–protein and protein–lipid interactions, and to investigate the dynamics of TF and FVIIa. Results: The MD trajectories reveal that isolated FVIIa undergoes large structural fluctuation, primarily due to the hinge motions between its domains, whereas soluble TF (sTF) is structurally stable. Upon complex formation, sTF restricts the motion of FVIIa significantly. The results also show that, in the membrane‐bound form, sTF directly interacts with the lipid headgroups, even in the absence of FVIIa. Conclusion: The first atomic models of membrane‐bound sTF–FVIIa, FVIIa and sTF are presented, revealing that sTF forms direct contacts with the lipids, both in the isolated form and in complex with FVIIa. The main effect of sTF binding to FVIIa is spatial stabilization of the catalytic site of FVIIa, which ensures optimal interaction with the substrate, FX.     

Characterization of canine coagulation factor VII and its complex formation with tissue factor: canine–human cross‐species compatibility

Summary. Background: Canine models have been good predictors of efficacy of hemophilia treatments, including recombinant human coagulation factor (F)VIIa (hFVIIa). However, canine FVIIa and tissue factor (TF) have remained incompletely characterized. Objective: To explore canine–human cross‐species FVIIa–TF compatibility in order to strengthen the predictive value of canine models in research on FVIIa and TF. Methods: Canine FVIIa (cFVIIa) and canine TF_(1–217) [cTF_(1–217)] were produced by recombinant techniques, and canine–human cross‐species FVIIa–TF interactions were characterized in vitro. Results: Recombinant cFVIIa and soluble cTF_(1–217) were produced and purified to homogeneity. hFVIIa and cFVIIa bound with comparably high affinities to cTF_(1–217) (K_D = 6.0 ± 0.7 nm and K_D = 6.0 ± 0.3 nm , respectively) and to cell surface‐expressed cTF (K_D = 8.4 ± 0.4 nm and K_D = 7.2 ± 1.2 nm , for ¹²⁵I‐labeled hFVIIa and cFVII, respectively). In contrast, cFVIIa bound to human TF (hTF) with decreased affinity, both in solution and on cell surfaces. The decreased binding resulted in reduced activity of cFVIIa in functional assays with hTF_(1–209). In direct comparison, cFVIIa was more active than hFVIIa, both in the absence and the presence of cognate TF. Conclusion: The present finding that hFVIIa binds to cTF essentially as it does to hTF substantiates the hypothesis that human FVIIa–TF biology can be reliably recapitulated in canine models on administration of hFVIIa to dogs.     

Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey

Essentials

• Tissue factor (TF) enhances factor VIIa (FVIIa) activity through structural and dynamic changes.
• We analyzed conservation of TF‐activated FVIIa allosteric networks in extant vertebrate lamprey.
• Lamprey Tf/FVIIa molecular dynamics show conserved Tf‐induced structural/dynamic FVIIa changes.
• Lamprey Tf activation of FVIIa allosteric networks follows molecular pathways similar to human.

Summary

Background

Previous studies have provided insight into the molecular basis of human tissue factor (TF) activation of activated factor VII (FVIIa). TF‐induced allosteric networks of FVIIa activation have been rationalized through analysis of the dynamic changes and residue connectivities in the human soluble TF (sTF)/FVIIa complex structure during molecular dynamics (MD) simulation. Evolutionary conservation of the molecular mechanisms for TF‐induced allosteric FVIIa activation between humans and extant vertebrate jawless fish (lampreys), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest.

Objective

To model the sTf/FVIIa complex from cloned Petromyzon marinus lamprey sequences, and with comparisons to human sTF/FVlla investigate conservation of allosteric mechanisms of FVIIa activity enhancement by soluble TF using MD simulations.

Methods

Full‐length cDNAs of lamprey tf and f7 were cloned and characterized. Comparative models of lamprey sTf/FVIIa complex and free FVIIa were determined based on constructed human sTF/FVIIa complex and free FVIIa models, used in full‐atomic MD simulations, and characterized using dynamic network analysis approaches.

Results

Allosteric paths of correlated motion from Tf contact points in lamprey sTf/FVIIa to the FVIIa active site were determined and quantified, and were found to encompass residue–residue interactions along significantly similar paths compared with human.

Conclusions

Despite low conservation of residues between lamprey and human proteins, 30% TF and 39% FVII, the structural and protein dynamic effects of TF activation of FVIIa appear conserved and, moreover, present in extant vertebrate proteins from 500 million years ago when TF/FVIIa‐initiated extrinsic pathway blood coagulation emerged.

     

Neutrophil activation by the tissue factor/Factor VIIa/PAR2 axis mediates fetal death in a mouse model of antiphospholipid syndrome

Women with antiphospholipid syndrome (APS), a condition characterized by the presence of antiphospholipid antibodies (aPL), often suffer pregnancy-related complications, including miscarriage. We have previously shown that C5a induction of tissue factor (TF) expression in neutrophils contributes to respiratory burst, trophoblast injury, and pregnancy loss in mice treated with aPL. Here we analyzed how TF contributes to neutrophil activation and trophoblast injury in this model. Neutrophils from aPL-treated mice expressed protease-activated receptor 2 (PAR2), and stimulation of this receptor led to neutrophil activation, trophoblast injury, and fetal death. An antibody specific for human TF that has little impact on coagulation, but potently inhibits TF/Factor VIIa (FVIIa) signaling through PAR2, inhibited aPL-induced neutrophil activation in mice that expressed human TF. Genetic deletion of the TF cytoplasmic domain, which allows interaction between TF and PAR2, reduced aPL-induced neutrophil activation in aPL-treated mice. Par2^–/– mice treated with aPL exhibited reduced neutrophil activation and normal pregnancies, which indicates that PAR2 plays an important role in the pathogenesis of aPL-induced fetal injury. We also demonstrated that simvastatin and pravastatin decreased TF and PAR2 expression on neutrophils and prevented pregnancy loss. Our results suggest that TF/FVIIa/PAR2 signaling mediates neutrophil activation and fetal death in APS and that statins may be a good treatment for women with aPL-induced pregnancy complications.     

Tissue factor at the crossroad of coagulation and cell signaling

Summary

The tissue factor (TF) pathway plays a central role in hemostasis and thrombo‐inflammatory diseases. Although structure‐function relationships of the TF initiation complex are elucidated, new facets of the dynamic regulation of TF's activities in cells continue to emerge. Cellular pathways that render TF non‐coagulant participate in signaling of distinct TF complexes with associated proteases through the protease‐activated receptor (PAR) family of G protein‐coupled receptors. Additional co‐receptors, including the endothelial protein C receptor (EPCR) and integrins, confer signaling specificity by directing subcellular localization and trafficking. We here review how TF is switched between its role in coagulation and cell signaling through thiol‐disulfide exchange reactions in the context of physiologically relevant lipid microdomains. Inflammatory mediators, including reactive oxygen species, activators of the inflammasome, and the complement cascade play pivotal roles in TF procoagulant activation on monocytes, macrophages and endothelial cells. We furthermore discuss how TF, intracellular ligands, co‐receptors and associated proteases are integrated in PAR‐dependent cell signaling pathways controlling innate immunity, cancer and metabolic inflammation. Knowledge of the precise interactions of TF in coagulation and cell signaling is important for understanding effects of new anticoagulants beyond thrombosis and identification of new applications of these drugs for potential additional therapeutic benefits.

     

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.     

The many faces of tissue factor

Summary.  Tissue factor (TF) is a member of the cytokine receptor superfamily and binds FVII/VIIa. The TF:FVIIa complex has both procoagulant and signaling activities. It functions in many biological processes, including hemostasis, thrombosis, inflammation, angiogenesis and tumor growth. Importantly, TF is essential for hemostasis. However, increased TF expression within atherosclerotic plaques and elevated levels of circulating TF-positive micro particles promote thrombosis. TF increases inflammation by enhancing intravascular fibrin deposition, by increasing the formation of pro-inflammatory fragments of fibrin and by generating coagulation proteases, including FVIIa, FXa and thrombin, that activate protease-activated receptors (PARs). In endotoxemia and sepsis, TF-dependent thrombin generation and activation of PAR1 on dendritic cells enhance inflammation. Finally, the TF:FVIIa complex contributes to tumor growth by activating PAR2.     

Zn2+‐containing protein S inhibits extrinsic factor X‐activating complex independently of tissue factor pathway inhibitor

Summary. Background: Protein S (PS) has direct anticoagulant activity, independently of activated protein C (APC). The mechanisms underlying this activity remain unclear, because PS preparations differ in activity, giving rise to conflicting results. Some purification procedures result in loss of intramolecular Zn²⁺, which is essential for inhibition of prothrombinase. Objective: To investigate the inhibition of extrinsic factor (F)Xase by Zn²⁺‐containing PS. Methods: Purified component extrinsic FXase assays were used to determine FXa generation in the presence and absence of PS and/or tissue factor pathway inhibitor (TFPI). Binding assays, immunoblots and thrombin generation assays in plasma supported the FXase data. Results: Zn²⁺‐containing PS potently inhibited extrinsic FXase in the presence of saturating phospholipids, independently of TFPI, whereas inhibition of extrinsic FXase by Zn²⁺‐deficient PS required TFPI. Immunoblots for FXa and functional assays showed that Zn²⁺‐containing PS inhibited primarily the quantity of FXa formed by tissue factor (TF)–FVIIa, rather than FXa amidolytic activity. Zn²⁺‐containing PS, but not Zn²⁺‐deficient PS, bound to TF with high affinity (K_{d app} = 41 nm ) and targeted TF function. Binding of PS to FVIIa was negligible, whereas PS showed appreciable binding to FX. Increasing FX concentrations 10‐fold reduced PS inhibition five‐fold, suggesting that PS inhibition of FXase is FX‐dependent. PS also exhibited TFPI‐independent and APC‐independent anticoagulant activity during TF‐initiated thrombin generation in plasma. Conclusions: PS that retains native Zn²⁺ also retains anticoagulant functions independently of APC and TFPI. Inhibition of extrinsic FXase by PS at saturating levels of phospholipids depends on PS retention of intramolecular Zn²⁺, interaction with FX, and, particularly, interaction with TF.