FX Activation Research Articles

During warfarin induction, the Fiix‐prothrombin time reflects the anticoagulation level better than the standard prothrombin time

Background The prothrombin time (PT) is equally prolonged by reduction of each of the vitamin K-dependent (VKD) factors (F) II, VII and X. The Fiix-PT is only affected by FII and FX, the main contributors to thrombin generation (TG). Objective To test the hypothesis that variability in warfarin anticoagulation is reduced early during monitoring with the normalized PT-ratio calculated from Fiix-PT (Fiix-International Normalized Ratio [INR]) compared with traditional PT-INR monitoring. Also, that because of its insensitivity to FVII, Fiix-PT more accurately reflects TG when Fiix-INR and PT-INR are discrepant. Methods Samples from Fiix-trial participants monitored with either Fiix-PT or PT were used. VKD coagulation factors and TG were measured in samples from 40 patients during stable anticoagulation and in serial samples obtained from 26 patients during warfarin induction. TG was assessed in relation to selective reduction in single VKD factors. Results During Fiix-warfarin induction full anticoagulation measured as FII or FX activity was achieved at a similar rate to that with PT-warfarin but subsequently stabilized better. Fiix-INR but not PT-INR mirrored total TG during initiation. During induction, FII (R2 = 0.66) and FX (R2 = 0.52) correlated better with TG and with a steeper slope than did FIX (R2 = 0.37) and in particular FVII (R2 = 0.21). In vitro, FII and FX were the main determinants of TG at concentrations observed during VKA anticoagulation, whereas FVII and FIX had little influence. Conclusions Fiix-PT monitoring reduces anticoagulation variability, suggesting that monitoring FVII has a limited role during VKA management. TG is better reflected by Fiix-PT.

Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia.

LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT. Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance (SPR) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells (HUVEC). The dissociation constant (Kd ) for activated platelet binding was in the 1 μm range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25-50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. LR769 was similar to NovoSevenRT in all kinetic assays. Active site titration demonstrated 0.7 mole of active site/mole of protein. The kcat /Km values for activation of FX and FIX with purified recombinant tissue factor and phospholipids were 10.5 s-1 /0.32 μm and 3.3 s-1 /0.44 μm respectively. The apparent second-order rate constant for inactivation by human plasma AT was 5.9 ± 0.4 × 103 m-1 s-1 . The Kd values for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nm and 0.9 nm, respectively, and the Kd for binding to soluble EPCR was 41 nm. Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.

New Hyperactive Factor IX Variants Bioengineered Based upon Biochemical Studies of Factor IX Padua

Recent clinical trial successes of AAV-based gene therapy for hemophilia B utilizing the hyperactive factor (F) IX variant, Padua (Arg338Leu), demonstrate the potential of this strategy to achieve disease ameliorating FIX activity levels at lower vectors doses. However, the underlying molecular mechanisms responsible for the 8-fold increase in specific activity remain unanswered. To address this question, we have undertaken a series of biochemical comparisons between recombinant FIX-Padua and FIX wild-type (WT). We observe FIX-Padua demonstrates increased aPTT-based clotting activity and increased thrombin generation compared to FIX-WT, as both a zymogen and activated (FIXa) enzyme. Furthermore, FIX-Padua and FIX-WT are activated by FXIa at similar rates. Combined, these results suggest that the hyperactivity of FIX-Padua is due to alterations of the activated enzyme and not due to differences in the zymogen or its activation. We therefore investigated the protease ability of FIXa-Padua and FIXa-WT. In the absence of FVIIIa, FIXa-Padua and FIXa-WT display similar rates of cleavage of peptide substrates. Similarly, FIXa-Padua and FIXa-WT are inactivated by the suicide substrate antithrombin at similar rates. However, when combined with FVIIIa within the intrinsic Xase complex, FIXa-Padua, demonstrates a 3-fold increased catalytic rate (kcat) of FX activation compared to FIXa-WT. Thus, the hyperactivity of FIX-Padua appears to be due to enhanced enzymatic activity with its incorporation into the intrinsic Xase during clotting, and not due to differences in activation or inactivation. The additional fold increase in plasma-based clotting activity may be due to a combination of amplification and feedback reactions occurring in plasma-based assays since the relative difference in clotting activity between FIX-Padua and FIX-WT increases with increasing FX concentration. These biochemical studies support the safety of FIX-Padua as a therapeutic since they suggest that FIX-Padua is regulated similarly to FIX-WT. As such, thrombotic complications would only be anticipated at supratherapeutic FIX activity levels and would be similar for FIX-Padua and FIX-WT at a given activity level. The implication of these results is that the active site of FIXa in the intrinsic Xase is improved by the Padua substitution. We also observe that most substitutions in a 20 amino acid screen at position 338 result in FIX variants with at least equal activity compared to WT. FIX-Padua is the most active variant, while surprisingly, FIX-WT is one the least functional that does not cause hemophilia. Since Arg-338 is highly conserved within FIX mammalian orthologs, these results suggest that FIX-WT may have evolved to have limited procoagulant activity; indeed, our biochemical studies suggest that the FIX-WT active site is sub-optimal for FX activation. We, therefore, examined if there were additional positions in FIX where amino acid substitutions would lead to variants with increased clotting activity. To direct our search, we focused on positions within the protease domain that were 1) likely structurally important for interactions with FVIIIa and 2) where hemophilia B causing mutations have not been reported. Using this strategy, we identified 3 additional positions where single amino acid substitutions resulted in FIX variants with 2 - 6 fold increased clotting activity compared to FIX-WT. Moreover, the most active novel substitution can be additively combined with the Padua substitution: recombinant FIX of this two amino-acid substituted variant displayed 15-fold increased specific activity compared to FIX-WT and almost 2-fold increased specific activity compared to FIX-Padua. Given the proven benefit of the enhanced activity of FIX-Padua now being employed in clinical trials, novel hyperactive substitutions of FIX could further enhance the safety and efficacy of these therapeutics. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding; sparK: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Novo Nordisk: Research Funding; Bayer: Consultancy. Arruda:Pfizer: Patents & Royalties, Research Funding.

The Role of Sphingomyelin (SM) in Tissue Factor (TF) Encryption: ATP-Induced Activation of Acid Sphingomyelinase in Macrophages Decrypts Tissue Factor By Hydrolysis of SM in the Outer Leaflet

While tissue factor (TF)-mediated blood coagulation is essential for maintaining hemostasis, the aberrant activation of TF-mediated coagulation is a major determinant of thrombotic occlusions, the precipitating event in acute myocardial infarction, unstable angina, and ischemic stroke. Typically, TF on cell surfaces exists in inactive coagulant status (cryptic TF). Cell injury leads conversion of cryptic TF to coagulant active/prothrombotic TF. Molecular differences between cryptic and procoagulant TF and the mechanisms that are responsible for the conversion from one to the other form are poorly understood and often controversial. A majority of the evidence in the literature suggest that level of anionic phospholipids, such as phosphatidylserine (PS), in the outer leaflet of the plasma membrane plays a critical role in regulating TF procoagulant activity at the cell surface. However, other pathways, such as the thioredoxin system or thiol-disulfide exchange pathways involving protein-disulfide isomerase (PDI), were also shown to contribute to TF activation by inducing structural changes in TF. It is unknown at present whether TF on cell surfaces of naïve cells exists primarily in the cryptic state because of the limited availability of anionic phospholipids at the outer leaflet or phospholipids present in the outer leaflet play an active role in maintaining TF in the cryptic state. In the outer leaflet of mammalian plasma membrane, sphingomyelin (SM) constitutes up to 50% of the total phospholipids present on the cell surface. It is possible that a high SM content in the outer leaflet may be responsible for maintaining TF in its cryptic state at the cell surface in naïve cells, and the hydrolysis of SM on the outer leaflet mediated by factors released in cell injury contributes to TF activation. The present study was carried out to investigate this possibility.First, we tested the potential effect of SM on TF activity in a reconstituted system in which full-length TF was reconstituted into phospholipid vesicles composed of varying molar concentrations of SM with the remainder of the vesicle consisting of phosphatidylcholine (PC). SM, at 35 mol % or higher concentration in the proteoliposome, inhibited TF coagulant activity significantly as measured in factor X activation assay. Ceramide, having a similar sphingosine backbone as of SM, had no inhibitory effect on TF-FVIIa activation of FX. Measurement of FVIIa-TF amidolytic activity showed that SM does not inhibit the amidolytic activity of FVIIa-TF, indicating that SM neither affects FVIIa binding to TF nor TF-FVIIa cleavage of the small substrate peptide. SM also inhibited significantly TF activity of TF reconstituted in PC/PS (94%:6% mol/mol) vesicles. Next, human monocyte-derived macrophages (MDMs) were treated with varying concentrations of bacterial sphingomyelinase (b-SMase) to hydrolyze SM in the outer leaflet. b-SMase treatment increased cell surface TF activity in a dose-dependent manner. SMase treatment also enhanced the release of TF-bearing microparticles (MPs). SMase treatment had no significant effect on cell surface prothrombinase activity or annexin V binding to MDMs, indicating that b-SMase treatment did not increase PS availability at the cell surface under our experimental conditions. Similar to that observed in bone marrow-derived mouse macrophages, ATP (200 µM) stimulation of MDMs increased cell surface TF activity by about 3-fold and triggered the release of TF+ MPs. Immunofluorescence confocal microscopy revealed that ATP stimulation induced in the translocation of acid(a)-SMase from intracellular compartments to the outer leaflet of the plasma membrane. Treatment of MDMs with sphingomyelinase inhibitors, desipramine and imipramine (1 and 5 µM), or silencing a-SMase with siRNA markedly reduced the ATP-induced increased TF activity at the cell surface and TF+ MPs release. Finally, ATP stimulation was shown to increase the hydrolysis of SM in the outer leaflet of MDMs markedly. a-SMase inhibitors or silencing of a-SMase attenuated the ATP-induced SM hydrolysis. In summary, our data indicate that SM plays a critical role in maintaining TF in the cryptic state in resting cells. Activation/translocation of a-SMase to the outer leaflet following the activation of ATP receptor P2X7 leads to hydrolysis of SM and thus relieves the inhibitory effect of SM on TF, leading to TF decryption and the release of TF+ MPs. DisclosuresNo relevant conflicts of interest to declare.

Factor VIIa Interaction with Endothelial Cell Protein C Receptor: Its Role in Hemostatic Effect of rFVIIa in Treating Hemophilia

Recent studies from our laboratory and others established that clotting factor FVII/FVIIa, which binds tissue factor and triggers the coagulation cascade, also binds endothelial cell protein C receptor (EPCR), a receptor that plays a critical role in protein C/activated protein C (APC)-mediated anticoagulation pathway. We postulated that FVIIa binding to EPCR might augment the hemostatic effect of rFVIIa in therapeutic conditions by down-regulating the EPCR-mediated anticoagulation pathway. Our recent studies showing the blockade of endogenous protein C binding to EPCR by administration of EPCR blocking antibodies augmented the hemostatic effect of rFVIIa in a mouse hemophilia model provided an indirect support to this hypothesis. However, studies from other investigators raised a possibility that FVIIa interaction with EPCR may influence the hemostatic effect of rFVIIa in vivo through EPCR-FVIIa directly activating FX or EPCR tethering FVIIa to provide an extended locale of procoagulant reactions on the endothelium. The present study is carried out to investigate the mechanism by which FVIIa interaction with EPCR contributes to the hemostatic effect of FVIIa in hemophilia therapy. The study employed wild-type, transgenic mice expressing no EPCR (EPCR-/-) or overexpressing EPCR (Tie2-EPCR). FVIII mAb (1 mg/kg) were given to mice to induce hemophilia. The saphenous vein incision bleeding model was used to evaluate the hemostatic effect of human rFVIIa. In this model, bleeding is induced by a sharp incision to the saphenous vein and the bleeding from the cut was monitored for 30 min. After each hemostasis incident, the clot was disrupted gently to reinitiate a new bleeding episode and average time to achieve hemostasis (ATH) was calculated.First, we investigated the role of EPCR in hemostasis by comparing the bleeding episodes in unchallenged wild-type, EPCR-/- and Tie2-EPCR mice following the saphenous vein incision. In this model, control wild-type mice had a median ATH of 55 sec (mean 63 ± 7 sec; n=9) and EPCR-/- mice had a median ATH of 67 sec (mean 74 ± 6 sec; n =12). The difference between them is not statistically significant. Surprisingly, the median ATH in EPCR overexpressing Tie2-EPCR mice was significantly lower (a median ATH of 43 sec; mean 46 ± 2 sec; n=10) compared to the ATH in wild-type or EPCR-/- mice. The reason for a shorter ATH observed in EPCR overexpressing mice is unknown at present. Analysis of blood count showed no significant differences among wild-type, EPCR-/- and Tie2-EPCR in their platelet count, RBC, and other blood cell count.Next, we induced acquired hemophilia condition in wild-type, EPCR-/- and Tie2-EPCR by injecting FVIII mAb (1 mg/kg) i.v. 2 h before inducing the bleeding. Administration of FVIII mAb markedly prolonged the bleeding time in wild-type and Tie2-EPCR mice (between 300 to 1800 sec) with a median ATH of 600 sec (mean 730 ± 277 sec) in wild-type mice and 750 sec (mean 939 ± 200 sec) in Tie2-EPCR mice. Interestingly, EPCR-/- mice were protected from the antibody-induced acquired hemophilia. Administration of FVIII mAb prolonged the bleeding time only marginally in this group of mice to a median ATH of 120 sec (mean 163 ± 38 sec). Administration of a low dose of rFVIIa (0.25 mg/kg) fully corrected the mild bleeding defect in EPCR-/- acquired hemophilia mice (median ATH of 67 sec; mean 86 ± 17 sec). Administration of 0.25 mg/kg rFVIIa is corrected the bleeding disorder only partially in wild-type acquired hemophilia mice (median ATH 164 sec; mean 303 ± 94 sec; n=5). Administration of either 1 or 4 mg/kg of rFVIIa was required to correct the bleeding in these mice (1 mg/kg dose: median ATH of 75 sec, mean 74 ± 1 sec; 4 mg/kg dose: median ATH of 37 sec; mean 40 ± 2 sec, n=5). In Tie2-EPCR hemophilia mice, administration of 1 mg/kg rFVIIa was not sufficient to fully achieve the hemostatic effect (median ATH 164 sec; mean 521 ± 237 sec; n=6). Administration of 4 mg/kg rFVIIa was required to restore the hemostasis in these mice (median ATH of 90 sec; mean 94 ± 7 sec, n= 6). In summary, the present data indicate that EPCR levels influence the hemostatic effect of rFVIIa in treating antibody-induced hemophilia. The present study rules out the possibility of direct activation of FX by FVIIa-EPCR complexes or EPCR tethering of FVIIa as probable mechanisms by which EPCR-FVIIa influences the hemostatic effect of rFVIIa. DisclosuresNo relevant conflicts of interest to declare.

Concizumab Promotes Hemostasis Via a TF-FVIIa Dependent Mechanism As Shown in a Rabbit Hemophilia Bleeding Model

Currently, a number of novel hemophilia drugs with different modes of action are being developed for either subcutaneous (sc) and/or less frequent administration as compared to current intravenous (iv) factor replacement therapy. These drugs may provide a major improvement of hemophilia treatment.Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of the factor VIIa/tissue factor/factor Xa (FVIIa/TF/FXa) complex. Abrogation of TFPI function extends FX activation within the initiation phase of the clotting system and may potentially completely bypass the need for factor VIII (FVIII) and factor IX (FIX). Concizumab (previously mAb 2021) is a high-affinity humanized monoclonal antibody against the Kunitz 2 domain of TFPI, intended for sc prophylaxis in hemophilia A and B patients with and without inhibitors, and currently being tested in clinical phase 1/2 trials. Concizumab has been shown to effectively prevent inhibition of FVIIa/TF/FXa and decrease cuticle bleeding in a dose-dependent manner in hemophilic rabbits. Blood loss in hemophilic rabbits was reduced for 7 days after administration of a single high dose of concizumab. It is expected that the inhibition of TFPI is effective as long as the initiation complex FVIIa/TF/FXa is active. In the present study, it was evaluated if concizumab can reduce an ongoing bleeding following administration at different time points after induction of a bleeding in a rabbit hemophilia model.Four separate cuticle bleeding studies in rabbits made hemophilic by iv administration of a monoclonal human FVIII-antibody were performed. In the first study, administration of 2 mg/kg concizumab iv reduced cuticle blood loss to a similar extent as 9 mg/kg recombinant activated factor VII (rFVIIa) iv when dosed 5 minutes after induction of bleeding, with a blood loss of 13 and 11%, respectively, compared to a control group receiving isotype control antibody (n=7-8; p<0.05 for both; Table 1). In a subsequent dose-response study, 0.5, 1, 2 and 4 mg/kg concizumab (iv) dosed 5 minutes after induction of bleeding reduced blood loss to 31, 10, 25 and 7% of control level, respectively (n=7-9; p<0.01 for 1, 2 and 4 mg/kg; Table 1).The ability of concizumab to reduce blood loss when dosed 30 minutes after induction of bleeding was further evaluated. Rabbits received 2 mg/kg concizumab iv and blood loss was 80% of the blood loss in hemophilic control animals (n=8; not significant), whereas 9 mg/kg rFVIIa iv reduced blood loss to 2% of control level (p<0.001; Table 1).Finally, 2 mg/kg concizumab, administered iv to rabbits 15 minutes after induction of bleeding, resulted in a blood loss of 76% compared to hemophilic control animals (n=8; not significant), whereas 2 mg/kg rFVIIa iv reduced blood loss to 5% of control level (p<0.01; Table 1).Neutralization of TFPI will sustain the activity of the TF/FVIIa/FXa initiation complex. As this complex is present only for a short period of time when the blood vessel wall is injured and TF is exposed, concizumab is not expected to be useful for treatment of an ongoing bleeding. Here, we show that concizumab is as potent as rFVIIa in an ongoing bleeding in hemophilic rabbits, when administrated 5 minutes after bleeding induction. In contrast, no effect of concizumab was observed when administered 15 or 30 minutes after induction of bleeding, whereas rFVIIa significantly reduced blood loss when dosed at these time-points. These data support a TF-dependent effect of concizumab and a TF-independent effect of pharmacological doses of rFVIIa. [Display omitted] DisclosuresLauritzen:Novo Nordisk: Employment. Hilden:Novo Nordisk: Employment.

Beyond Antibodies: Development of a Novel Protein Scaffold Based on Human Chaperonin 10.

Human Chaperonin 10 (hCpn10) was utilised as a novel scaffold for presenting peptides of therapeutic and diagnostic significance. Molecular dynamic simulations and protein sizing analyses identified a peptide linker (P1) optimal for the formation of the quarternary hCpn10 heptamer structure. hCpn10 scaffold displaying peptides targeting Factor VIIa (CE76-P1) and CD44 (CP7) were expressed in E. coli. Functional studies of CE76-P1 indicated nanomolar affinity for Factor VIIa (3 nM) similar to the E-76 peptide (6 nM), with undetectable binding to Factor X. CE76-P1 was a potent inhibitor of FX activity (via inhibition of Factor VIIa) and prolonged clot formation 4 times longer than achieved by E-76 peptide as determined by prothrombin time (PT) assays. This improvement in clotting function by CE76-P1, highlights the advantages of a heptamer-based scaffold for improving avidity by multiple peptide presentation. In another example of hCPn10 utility as a scaffold, CP7 bound to native CD44 overexpressed on cancer cells and bound rCD44 with high affinity (KD 9.6 nM). The ability to present various peptides through substitution of the hCpn10 mobile loop demonstrates its utility as a novel protein scaffold.

Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

IntroductionFactor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin.Methods and ResultsPurified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma.ConclusionsOur data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

Coagulative safety of epidural catheters after major upper gastrointestinal surgery: advanced and routine coagulation analysis in 38 patients.

BackgroundThe risk of spinal haematoma in patients receiving epidural catheters is estimated using routine coagulation tests, but guidelines are inconsistent in their recommendations on what to do when results indicate slight hypocoagulation. Postoperative patients are prone to thrombosis, and thromboelastometry has previously shown hypercoagulation in this setting. We aimed to better understand perioperative haemostasis by comparing results from routine and advanced tests, hypothesizing that patients undergoing major upper gastrointestinal surgery would be deficient in vitamin K-dependent coagulation factors because of malnutrition, or hypocoagulative because of accumulation of low molecular weight heparin (LMWH).MethodsThirty-eight patients receiving epidural analgesia for major upper gastrointestinal surgery were included. We took blood at the time of preoperative epidural catheterization and at catheter withdrawal. Prothrombin time-international normalized ratio (PT-INR), activated partial thromboplastin time (aPTT) and platelet count (Plc) were analysed, and also albumin, proteins induced by vitamin K absence (PIVKA-II), rotational thromboelastometry (ROTEM®), multiple electrode aggregometry (Multiplate®) and activities of factors II, VII, IX, X, XI, XII and XIII.ResultsPostoperative coagulation was characterized by thrombocytosis and hyperfibrinogenaemia. Mean PT-INR increased significantly from 1.0 ± 0.1 to 1.2 ± 0.2 and mean aPTT increased significantly from 27 ± 3 to 30 ± 4 s. Activity of vitamin K-dependent factors did not decrease significantly: FIX and FX activity increased. FXII and FXIII decreased significantly. Mean Plc increased from 213 ± 153 × 106/L while all mean ROTEM-MCFs (maximal clot firmnesses) especially FIBTEM-MCF increased significantly to above the reference interval. All mean ROTEM® clotting times were within their reference intervals both before and after surgery. ROTEM® (HEPTEM minus INTEM) results were spread around 0. There were significant correlations between routine tests and the expected coagulation factors, but not any of the viscoelastic parameters or PIVKA-II. Multiplate® area under curve and EXTEM-MCF correlated significantly to Plc as did EXTEM-MCF to fibrinogen, FIX, FX and FXIII; and FIBTEM-MCF to Plc, FII, FXI and FXIII.ConclusionsThe increase in PT-INR may be caused by decreased postoperative FVII while the elevated aPTT may be caused by low FXII. The mild postoperative hypocoagulation indicated by routine tests is not consistent with thromboelastometry. The relevance of ROTEM® and Multiplate® in the context of moderately increased routine tests remains unclear.Trial registration number is not applicable since this is not a clinical trial.Electronic supplementary materialThe online version of this article (doi:10.1186/s13741-016-0053-0) contains supplementary material, which is available to authorized users.

Factor VIII chromogenic assays can be used for potency labeling and postadministration monitoring of N8‐GP

Essentials Chromogenic assays may be less variable than one-stage clot assays for measuring modified factor VIII. Chromogenic assays were evaluated for N8-GP potency labeling and postadministration monitoring. There was no significant difference between chromogenic assay kits for measuring N8-GP potency. Postadministration monitoring of N8-GP was comparable to turoctocog alfa for all kits tested. Background Factor VIII activity ( C) is commonly measured using one-stage activated partial thromboplastin time (aPTT)-based clot assays. Chromogenic assays are, however, an alternative, and potency assessment in Europe is performed using chromogenic assays. One-stage clot assays are in general associated with high variability, and modified FVIII products may add to this variability. FVIII chromogenic assays may be less affected. Objectives To evaluate available chromogenic assay kits for potency labeling of polyethylene glycol-glycoconjugated turoctocog alfa (turoctocog alfa pegol [N8-GP]) and to evaluate selected chromogenic kits for postadministration monitoring of N8-GP using turoctocog alfa (Novoeight(®) ) as comparator. Methods Six FVIII chromogenic assay kits were adapted to the European Pharmacopeia guidelines for potency labeling, including assessment of time to 50% FX activation. Four kits were adapted for postadministration monitoring using an ACL(®) TOP 500 analyzer. Severe hemophilia A plasma was spiked with N8-GP or turoctocog alfa to simulate postadministration samples. The World Health Organization (WHO) 8th International Standard (IS) FVIII concentrate was used as calibrator throughout. In addition, a plasma calibrator was used for postadministration samples. Results When measuring N8-GP potency, no significant difference using a 1% significance level was observed between kits. In simulated postadministration samples, all test kits were highly accurate and precise, except at low concentrations, with no significant difference between C (P > 0.05) measured using the different calibrators. However, values obtained using the WHO 8th IS were closer to labeled values. Conclusions Chromogenic assay kits tested measured consistent C for N8-GP potency and showed comparable results for N8-GP and turoctocog alfa in simulated postadministration samples.

Ringhalexin from Hemachatus haemachatus: A novel inhibitor of extrinsic tenase complex.

Anticoagulant therapy is used for the prevention and treatment of thromboembolic disorders. Blood coagulation is initiated by the interaction of factor VIIa (FVIIa) with membrane-bound tissue factor (TF) to form the extrinsic tenase complex which activates FX to FXa. Thus, it is an important target for the development of novel anticoagulants. Here, we report the isolation and characterization of a novel anticoagulant ringhalexin from the venom of Hemachatus haemachatus (African Ringhals Cobra). Amino acid sequence of the protein indicates that it belongs to the three-finger toxin family and exhibits 94% identity to an uncharacterized Neurotoxin-like protein NTL2 from Naja atra. Ringhalexin inhibited FX activation by extrinsic tenase complex with an IC50 of 123.8 ± 9.54 nM. It is a mixed-type inhibitor with the kinetic constants, Ki and Ki’ of 84.25 ± 3.53 nM and 152.5 ± 11.32 nM, respectively. Ringhalexin also exhibits a weak, irreversible neurotoxicity on chick biventer cervicis muscle preparations. Subsequently, the three-dimensional structure of ringhalexin was determined at 2.95 Å resolution. This study for the first time reports the structure of an anticoagulant three-finger toxin. Thus, ringhalexin is a potent inhibitor of the FX activation by extrinsic tenase complex and a weak, irreversible neurotoxin.

Establishing a rat model for the study of vitamin K deficiency.

The main vitamin K-deficient model, minidose warfarin, is different from the pathological mechanism of vitamin K deficiency, which is a shortage of vitamin K. The objective of this study was to establish a new method of vitamin K-deficient model combining a vitamin K-deficient diet with the intragastrical administration of gentamicin in rats. The clotting was assayed by an automated coagulation analyser. The plasma PIVKA-II was assayed by ELISA. The vitamin K status was detected by an HPLC-fluorescence system. In thediet- and gentamicin-induced vitaminK-deficient 14-day group, the rats had undetected vitamin K1 and vitamin K2 in the liver and a prolonged APTT. In the 21-day group, there was also a prolonged PT and a decrease of the FIX activities. In the 28-day group, the undetected vitamin K1 and vitamin K2, the prolonged PT and APTT, and the decrease of the FII, FVII, FIX, and FX activities prompted the suggestion that there were serious deficiencies of vitamin K and vitamin K-dependent coagulation in rats. It is suggested that thediet- and gentamicin-induced vitamin K-deficient 14-day or 21-day model can be used for studies related to the status of vitamin K. The vitamin K-deficient 28-day model can be appliedto research involving both the status of vitamin K and of vitamin K-dependent coagulation. In conclusion, the combination of a vitamin K-deficient diet with the administration of gentamicin results in a useful model of vitamin K-deficieny.

Rare coagulation disorders: an emerging issue.

Rare coagulation disorders: an emerging issue.

Platelet-Derived Beta Thromboglobulin: A Potential Novel Activator of Coagulation Factor X

BackgroundDuring clotting, factors IXa and VIIa participate in the activation of Factor X, leading to thrombin generation. This process occurs on the surface of activated platelets. Activated platelets release an array of proteins that can modulate thrombin generation. This includes platelet factor 4 (PF4), a chemokine that modulates coagulation by promoting protein C activation and by attenuating the anticoagulant activity of activated protein C. PF4 shares significant homology with another protein released in high concentration by platelets, β-thromboglobulin (βTG). The physiological effects of βTG, including effects on blood coagulation, are poorly characterized. The aim of this study was to assess the effect of βTG on blood coagulation.MethodsThe effect of purified human βTG on coagulation was determined using the prothrombin time (PT), activated partial thromboplastin time (APTT), calibrated automated thrombography (4µM phospholipids ± 1pM tissue factor (TF)) and chromogenic FX and prothrombin activation assays. Mass spectrometry and surface plasmon resonance (SPR) were used to assess the composition of the purified βTG preparation and to measure protein-protein interactions respectively.ResultsMass spectrometry confirmed the absence of TF, FVIIa, FVIIIa, FIXa, FVa, and FXa in the βTG preparation. In normal pooled plasma, βTG dose-dependently increased the rate and extent of TF stimulated thrombin generation. In the absence of βTG, the lagtime was 9±1 min, which was shortened in a dose-dependent manner upon incubation with βTG (100µg/ml; 5±1 min, p<0.05). Incubation with βTG (50µg/ml) also shortened the APTT (35±1 to 25±1 secs, p<0.05) and reduced the lagtime to thrombin generation (26±5 to 7±1 min, p<0.05) and increased the peak thrombin generation (60±30 to 97±23nM, p<0.05) in the absence of an exogenous TF stimulus. Immunodepleted plasmas and inhibitory antibodies were used to determine the underlying mechanism of action. In FVIII-deficient plasma, thrombin generation was not observed in the absence of an exogenous TF stimulus. However, upon incubation with βTG (50µg/ml), thrombin generation was observed and peak thrombin generation increased from 1±1 to 75±12nM IIa (p<0.05). βTG also shortened the APTT in FVIII-deficient plasma from 131±8 to 103±14 secs (50µg/ml, p<0.05). The procoagulant effect of βTG in FVIII-deficient plasma was not inhibited by TF or FVIIa inhibitory antibodies, suggesting that the effect was independent of the intrinsic tenase complex, TF or FVIIa. Interestingly, homologous PF4 did not induce thrombin generation in FVIII-deficient plasma (peak thrombin generation was 75±12nM v 0±0nM IIa upon incubation with 50µg/ml βTG and PF4 respectively). The procoagulant effect of βTG persisted when thrombin generation was independent of FV activation (supplementation of FV-deficient plasma with FVa). In contrast, the effect was not observed when thrombin generation was independent of FX activation (supplementation of FX-deficient plasma with FXa). Collectively, these data raised the possibility that βTG may modulate FX activation. To investigate this hypothesis, chromogenic FX activation was measured. Cleavage of a FXa-specific chromogenic substrate was observed upon incubation of βTG with FX, suggesting a direct effect of βTG upon FX activation. No measurable chromogenic substrate cleavage was observed upon incubation with βTG or FX alone. In contrast, βTG did not induce prothrombin activation, measured using a thrombin-specific chromogenic substrate. Using SPR, βTG was found to bind directly to immobilised FX (KD 1.36±0.7x10-7 M). The kinetics of the interaction between βTG and FX was lower than that between FIXa and FX (KD 4.25±1.90 × 10-9 M).ConclusionIn conclusion, we have identified that βTG is a potential novel platelet-derived activator of coagulation FX. Moreover, βTG is capable of inducing thrombin generation in immunodepleted FVIII and FIX-deficient plasma, a finding that may be of translational relevance to patients with haemophilia. DisclosuresNí Áinle:Actelion Pharma: Research Funding; Leo Pharma: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

Pharmacological Concentrations of Recombinant Factor VIIa Restores Hemostasis in Acquired Hemophilia Primarily Independent of Tissue Factor

High doses of recombinant FVIIa (rFVIIa) are used successfully to treat hemophilia patients with inhibitory antibodies against FVIII and FIX. Despite its widespread and successful use of treating bleeding disorders for many years, the understanding of the mechanism of rFVIIa action in hemophilia therapy is incomplete. A majority of the studies, mostly in vitro studies, suggested that activation of FX by rFVIIa bound to phospholipids exposed on the activated platelets is responsible for the hemostatic effect of rFVIIa in hemophilia patients. However, other in vitro studies and mathematical modeling indicated that the action of rFVIIa at therapeutic doses is dominated by the TF-dependent pathway, partly because of the need to overcome the inhibitory effect of zymogen FVII, with a minor contribution from phospholipid-dependent mechanism. In the present study, we investigated whether TF plays a role in rFVIIa-mediated hemostasis in hemophilia by employing transgenic mice expressing low levels of human TF (Low TF mice) or normal levels of human TF (HTF mice; breeding pairs for these mice were provided by Nigel Mackman). Mice were administered with FVIII mAb to induce acquired hemophilia. The saphenous vein incision bleeding model was used to evaluate the hemostasis. In this model, saphenous vein was exposed and bleeding was induced by a sharp incision of the vein with a fine scissors. The bleeding from the cut was monitored for 30 min while blood was collected onto a filter paper by capillary action. After each hemostasis incident, the clot was disrupted gently to reinitiate a new bleeding episode, and average time to achieve hemostasis (ATH) was calculated.First, we investigated the role of TF in hemostasis by comparing the bleeding episodes in low TF and HTF mice following the saphenous vein incision. In the saphenous vein bleeding model, control wild-type mice had a median ATH of 60 sec (n=6) and hemophilia A mice failed to achieve hemostasis within 30 min experimental time frame (n=6). HTF mice had essentially very similar ATH compared to that of the wild-type mice, i.e., 64 sec (n=5). In contrast, low TF mice had a substantially longer median ATH, 90 sec (n=6), which is statistically significantly longer than that of ATH of wild-type or HTF mice. Administration of TF antibodies (1 mg/kg) to neutralize remaining traces of TF in Low TF mice further prolonged the bleeding time to a median ATH of 309 sec (n=6). These data clearly illustrates the role of TF in hemostasis.Next, we created acquired hemophilic condition in low TF and HTF mice by injecting FVIII mAb (1 mg/kg). Low TF mice also received TF antibodies. Administration of FVIII mAb markedly prolonged the bleeding time in both groups of mice (between 300 to 1800 sec), with a median ATH of 900 sec. Administration of rFVIIa (4 mg/kg) to HTF hemophilic mice reduced the ATH to a median of 51 sec (mean ± SD, 50 ± 5 sec, n=6). The same concentration of rFVIIa also restored hemostasis in hemophilic low TF mice with a median ATH of 66 sec (mean ±SD, 70 ± 18 sec, n=6). This value is not statistically significantly different from that of ATH of wild-type mice. Although rFVIIa restored the hemostasis in both HTF and low TF mice, it appears that rFVIIa is slightly, but statistically significantly, more effective in HTF mice compared to low TF mice in restoring the hemostasis (p = 0.025). However, we did not observe any significant differences between these two groups in the volume of blood loss following rFVIIa administration. In summary, our data strongly support the hypothesis that high concentrations of rFVIIa restore hemostasis in hemophilia primarily in TF-independent manner. More detailed studies are needed to completely eliminate the role of TF in rFVIIa mediated hemostasis. DisclosuresNo relevant conflicts of interest to declare.

The Role of Putative Phosphatidylserine-Interactive Residues of Tissue Factor on Its Coagulant Activity at the Cell Surface

Tissue factor (TF) is the cellular cofactor for the serine protease coagulation factor VIIa (FVIIa). The TF-FVIIa complex formed on the cell surface initiates the coagulation cascade. It is believed that most of the TF molecules on the cell surface of a resting cell exist in an encrypted state with very little procoagulant activity. Encrypted TF must undergo decryption to become fully active. The exact mechanisms by which TF activity on the cell surface is regulated are unknown. Exposure of phosphatidylserine (PS) to the outer leaflet of the cell membrane is thought to play a critical role in TF decryption. Recent studies of molecular dynamics simulation of TF ectodomain in solution and on the surface of anionic phospholipids suggested a direct interaction of PS headgroups with specific residues in TF. At present, the role of the putative lipid interactive residues of TF in TF decryption is unknown. In the present study, we investigated the potential role of TF direct interaction with the cell surface lipids on basal TF activity as well as enhanced TF activity following the decryption using different TF mutants. Plasmids or adenoviral constructs encoding wild-type or mutant TF (mutations in the putative lipid binding region) were used to transduce TF expression in CHO-K1 or monocytic THP-1 cells, respectively. TF protein expression level at the cell surface and FVIIa binding to the cell surface TF were evaluated by radioligand binding studies using 125I-labeled TF mAb or FVIIa, respectively. TF-FVIIa coagulant activity on the cell surface was determined in FX activation assay. Data of these studies showed that all TF mutants were capable of interacting with FVIIa with no apparent defect. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A-exhibited a similar or slightly higher TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A and TFK166A, was reduced substantially to a range of 40% - 70% of that of wild-type TF. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% loss of TF specific activity. Mutation of all nine lipid binding residues together (DLBR) did not further decrease the specific activity of TF anymore than that of mutation of G164 alone. Comparison of the present data with the published data on these mutants revealed that some of the TF residues that are critical for regulating TF activity on liposomes are not crucial for TF activity on the cell surface. To address whether the decreased FXa generation seen with the select TF variants is caused by changes in TF-membrane interaction or by the substrate interaction with TF/FVIIa complex, we performed Michaelis-Menten kinetics of FX activation for two of TF mutants (TFS163A and TFG164A). Results of this study suggested that there were no significant differences in Km values between wild-type TF and TF mutants (wild-type TF, 51 ± 14.6 nM; TFS163A, 68 ± 19.5 nM; TFG164A, 39 ± 18.4 nM, n=4). Interestingly, mutation of the selective residues in the lipid binding region failed to abrogate the PS-dependent TF decryption. The fold-increase in TF activity in cells expressing wild-type TF or TF variants was similar following cell activation with either HgCl2 or calcium ionomycin treatment. Annexin V markedly diminished the increased TF-FVIIa activation of FX in cells expressing wild-type TF as well as cells expressing the TF mutant (DLBR mutant). Overall, our data suggest that the regulation of TF activity at the cell surface milieu may be different from that of PC/PS vesicles and TF region other than earlier identified LBR may be responsible for enhancing TF activity following the PS exposure. DisclosuresNo relevant conflicts of interest to declare.

Binding of rhFVIIa (LR769) to the Endothelial Protein C Receptor

Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in replacement therapies. In such cases, factor replacement becomes ineffective and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new second generation recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB USA was studied. The goal is to provide patients with Hemophilia A and B who develop inhibitors an alternative treatment option. In work presented previously, we demonstrated that LR769 was similar to NovoSevenRT with respect to activation of FX and FIX, tissue factor binding, and inactivation by antithrombin. LR769 bound better to platelets than NovoSevenRT at saturation but had a similar binding constant(1). It is known that FVIIa binds to Endothelial Protein C Receptor (EPCR) with the same affinity as Protein C (2-4). Several studies suggest EPCR is involved in therapeutic mechanism rhFVIIa (5,6,7,8). In addition to its effect on thrombin generation, binding to EPCR may protect against permeability induced by VEGF and LPS (6). In the work presented here, the binding of LR769 to EPCR on the surface of cultured HUVEC cells was determined and compared to NovoSevenRT. A dose-dependent binding was observed at rhFVIIa concentrations expected in patients undergoing treatment with this product. The specificity for EPCR binding was confirmed by competition with Protein C, soluble EPCR, or anti-EPCR which reduced binding to these cells. Addition of soluble tissue factor increased the binding by approximately 20-30%. Overall, the results show that LR769 binds to primary human endothelial cells via EPCR and that tissue factor increases the binding affinity. Binding was found to be higher with LR769 than NovoSevenRT. The significance of the tissue factor effect on rhFVIIa binding with respect to Hemophilic joint arthropathy is discussed. The methods used will be used to further characterize the interactions of rhFVIIa with important components of the vascular system.1) Grandoni, J.A. et al. (2013) ASH 2013 poster2) Gosh et al. (2007) JBC 282 11849-118573) Lopez-Sagaseta et al. (2007) JTH 5 1817-18244) Preston et al. (2006) JBC 281 28850-288575) Pavani, G et al. (2014) Blood 7 1157-656) Sundarham et al. J. Thromb and Hemost (2014)12:690-7007) Monroe DM, et al. (1997) Br J Haematol;99:542-7 DisclosuresGrandoni:LFB USA: Employment.

The Acidic Region of Factor VIII Light Chain Contains the Thrombin-Binding Site(s) Responsible for the Cleavage at Arg1689

Thrombin-catalyzed activation of factor (F)VIII by cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies the activation of FX as a cofactor of FIX. We had already demonstrated that thrombin interacts with FVIII through the residues 392-394 and 484-509 in the A2 domain and the C2 domain, and each association regulates cleavage at Arg740, Arg372, and Arg1689, respectively (Nogami K, JBC 2000, 2005; BJH 2008), and recently reported that the A1 acidic clustered region 340-350 involving the sulfated tyrosine regulate the cleavage of Arg372 (Minami et al. 55th ASH). On the other hand, Fay and colleague suggested that recombinant FVIII lacking the C2 domain retains greater than 50% cofactor activity (JBC 2010), supporting the presence of other thrombin-binding region responsible for cleavage at Arg1689 of the light chain. In this study, we attempted to identify this thrombin-binding site(s). We focused on the acidic residues 1659-1669 and 1675-1685 within the light chain, which had similar sequence to the A1 residues 340-350 in terms of involving the clustered acidic residues and sulfated tyrosine as well as hirugen residues 54-65. We prepared four of synthetic peptides corresponding to the residues 1659-1669 and 1675-1685 with sulfated tyrosine, P(1659-69s) and P(1675-85s), and with non-sulfated tyrosine, P(1659-69) and P(1675-85). The inhibitory effect on the thrombin-catalyzed FVIII activation by each peptide was evaluated in a one-stage clotting assay. Each peptide showed a dose-dependent inhibition on thrombin-catalyzed activation. These inhibitory effects were greater in order of P1675-85s, P1659-69s, P1675-85, P1659-69, and the IC50 were 25, 67, 71 and 225 µM, respectively. The peptides with sulfated tyrosine had approximately 3-fold greater inhibition of the FVIII activation by thrombin than with non-sulfated tyrosine. The IC50 in the presence of mixture of P1675-85s and P1659-69s was 30.4 µM, suggesting that these peptides had no an additive effect. The impacts of P1659-69s and P1675-85s on the thrombin-catalyzed cleavage at Arg1689 were examined by SDS-PAGE/western blotting. These peptides blocked the cleavage at Arg1689 in dose-dependent fashions. In timed-course assay, the presence of P1659-69s and P1675-85s decreased the cleavage rate of Arg1689 by 61.3 % and 81.8 %, respectively compared to its absence. The direct binding of P1659-69s and P1675-85s to thrombin was examined by surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based assay using a Biacore T200TM, thrombin bound to immobilized P1659-69s and P1675-85s directly with high affinity. The Kd values adjusted to 1:1 binding model of global fitting were 203 nM and 94 nM, respectively. EDC cross-linking in fluid-phase assay revealed that formation of EDC cross-linking products between biotinylated P1659-69s or P1675-85s and thrombin were observed in dose-dependent fashions. The products between the biotinylated peptides (800 nM) and thrombin were competitively reduced by the addition of non-biotinylated peptides. Moreover, N-terminal sequence analysis of cross-linking products between both peptides-thrombin indicated that thrombin bound to the residues 1664-1669 and 1683-1684. Taken together, we demonstrated that the A3 residues 1659-1669 (QEEIDYDDTIS) and residues 1675-1685 (EDFDIYDEDEN) contained the thrombin binding-sites responsible for proteolytic cleavage at Arg1689 of the A3 domain. DisclosuresNogami:Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding. Shima:Chugai Pharmaceutical Co., Ltd. and F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Molecular analysis of patients with severe congenital factor X deficiency: First report from north and northwest of Iran.

Background Congenital FX deficiency is an extremely rare autosomal recessive disorder, mostly present in regions with a high rate of consanguinity, with an incidence of approximately 1:2,000,000 in the general population. The aim of this study was to evaluate the clinical manifestations and to provide a molecular analysis of 14 patients with homozygous factor X deficiency. Methods We evaluated the results of clinical and molecular characterization of 14 new cases (10 males, 4 females aged from 4 to 17 years) of FXD (FX activity <1%) in North and Northwest of Iran. FXD was diagnosed by clinical findings and routine laboratory tests. The PCR products of all the eight exons and their intron-exon boundaries were sequenced using DNA sequencer. Results Mean age of patients and mean age at diagnosis were 9 and 2.5 years, respectively. The molecular analysis revealed five different mutations, all of them were previously described. Molecular analysis showed five different homozygous and double heterozygous mutations including Arg-1Thr, Cys81Tyr, Gly78Asp, IVS1+3, and IVS2-3. Discussion The study of a large population of factor X patients from three institutions indicated that FXD was one of the most serious among rare bleeding disorders and that factor X gene mutation may be related to bleeding tendency in patients.

Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

AbstractActivation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI.