Extrinsic Tenase Research Articles

Structural Findings and Molecular Modeling Approach of a TFPI-Like Inhibitor

Specific blood coagulation inhibitors from hematophagous organisms, with different structures and novel mechanism of action, have been described and they represent promising agents for the treatment of a variety of human diseases related to coagulation and cancer. In our lab, the salivary glands transcriptome of the adult Amblyomma cajennense tick was previously characterized by expressed sequence tags (EST). A transcript that codes for a tissue factor pathway inhibitor (TFPI)-like protein with unique structure was found, and the recombinant form of this protein was named Amblyomin-X. This protein was able to inhibit the factor Xa amidolytic activity and the activation of factor X by the extrinsic tenase complex (FVIIa/TF). Herein, it was performed functional and structural evaluation of Amblyomin-X. The CD assay and molecular dynamics simulations revealed that Amblyomin-X is structurally stable and the naturally unfolded regions as well as the presence of three disulfide bridges in its Kunitz-type domain seem to sustain its inhibitory activity. Regarding the electrostatic potential mapping on the Kunitz-type region, the pattern of charged residues was not quite the same in comparison to human TFPI-1 and TFPI-2, pointing out there might be distinct functional and structural features, which are going to be experimentally exploited.

Influence of temperature on spatial fibrin clot formation process in thrombodynamics

In this study we have investigated the process of spatial fibrin clot formation in non-steered platelet-free plasma at the temperatures from 20°C to 43°C using thrombodynamics - the novel in vitro hemostasis assay, which imitates the process of hemostatic clot growth in vivo. During data processing the following parameters were calculated: initial (V i ) and stationary (V st ) rates of clot growth which characterize initiation and propagation phases of clotting process, and clot size on the 30 th minute. The temperature dependence of extrinsic and intrinsic tenase activities, which determine values of the initial and stationary clot growth rates, respectively, have been also measured. It was established that the temperature lowering from 37°C to 24°C extends mainly on the initiation phase of clot growth, while the stationary rate of clot growth changes insignificantly. Meanwhile none of the thrombodynamics parameters shows the dramatic change of plasma coagulation system condition at the temperature of 24°C (acute hypothermia). Using the thrombodynamics assay an assumption, that the temperature lowering does not change the state of plasma hemostasis system significantly has been confirmed.

Identification and characterisation of novel inhibitors on extrinsic tenase complex from Bungarus fasciatus (banded krait) venom.

Snake venoms are excellent sources of pharmacologically active proteins and peptides, and hence are potential sources of leads for drug developments. It has been previously established that krait (Bungarus genus) venoms contain mainly neurotoxins. A screening for anticoagulants showed that Bungarus fasciatus venom exhibits potent anticoagulant effect in standard clotting assays. Through sequential fractionation of the venom by size exclusion and high performance liquid chromatographies, coupled with functional screening for anticoagulant activities, we have isolated and purified two anticoagulant proteins, termed BF-AC1 (Bungarus fasciatusanticoagulant 1) and BF-AC2. They have potent inhibitory activities (IC50 of 10 nM) on the extrinsic tenase complex. Structurally, these proteins each has two subunits covalently held together by disulfide bond(s). The N-terminal sequences of the individual subunits of BF-AC1 and BF-AC2 showed that the larger subunit is homologous to phospholipase A2, while the smaller subunit is homologous to Kunitz type serine proteinase inhibitor. Functionally, in addition to their anticoagulant activity, these proteins showed presynaptic neurotoxic effects in both in vivo and ex vivo experiments. Thus, BF-AC1 and BF-AC2 are structurally and functionally similar to β-bungarotoxins, a class of neurotoxins. The enzymatic activity of phospholipase A2 subunit plays a significant role in the anticoagulant activities. This is the first report on the anticoagulant activity ofβ-bungarotoxins and these results expand on the existing catalogue of haemostatically active snake venom proteins.

The Effect Of Physiological Metal Ions On The Procoagulant Enzyme-Cofactor Complexes Of The Extrinsic Pathway Of Coagulation

IntroductionThe rapid coagulation response to vascular injury is mediated by the formation of three enzyme cofactor complexes (extrinsic tenase, intrinsic tenase and prothrombinase) on membrane surfaces. A common structural feature of these proteases is their GLA domains, each of which requires the binding of divalent metal ions at multiple sites to achieve the conformation necessary for optimal membrane and cofactor binding. Both Ca2+ and Mg2+ ions have been reported to bind to GLA domain sites. However almost all studies kinetically characterizing these complexes have been done in the presence of Ca2+ (2-5 mM) as the sole metal ion, despite the relatively equivalent availability in plasma of both free Ca2+ (∼1.1 mM) and Mg2+ (∼0.6 mM) (Ca2+/Mg2+). A recent study has systematically examined the effects of various Ca2+ concentrations with and without Mg2+ on the membrane binding of activated protein C (APC) and FVIIa and enzymatic activity of APC and the extrinsic tenase complex which were enhanced in Ca2+/Mg2+ relative to Ca2+ alone (Vadivel, K., et al, 2013 JMB). In the current study we compare the effects of plasma levels of Ca2+ and Mg2+ versus Ca2+alone on the catalytic performances of the extrinsic tenase, intrinsic tenase and prothrombinase complexes individually and collectively. MethodsAll experiments were conducted in Hepes buffered saline pH 7.4 containing 0.1% PEG and either 2 mM Ca2+ or 1.1 mM Ca2+/0.6 mM Mg2+ (Ca2+/Mg2+). In closed system experiments, enzyme-cofactor complexes were assembled on phospholipid vesicles composed of a 3:1 ratio of synthetic phosphatidylcholine and phosphatidylserine (PCPS), and zymogen activation monitored via sampling into assay mixtures containing the appropriate chromogenic substrate. In open system experiments complexes were preassembled on PCPS coated capillaries, the zymogen delivered in the flowing phase and the extent of zymogen activation monitored in the effluent as described previously (Haynes, LM., et al, 2011 Biophys J). The combined interaction of the procoagulant enzyme cofactor complexes under both metal ion conditions was studied in a synthetic coagulation proteome monitoring thrombin (IIa) generation as previously described (van't Veer, C., and Mann, KG, 1997 JBC). ResultsExtrinsic tenase The extrinsic tenase complex had an approximately two-fold higher rate of FXa generation in the presence of Ca2+/Mg2+ (1.78 ±0.05 pM/s) versus Ca2+ alone (0.88 ± 0.02 pM/s) (N=3, p<0.001). Experiments were also conducted in the absence or presence of tissue factor pathway inhibitor (TFPI); in the presence of TFPI and Ca2+/Mg2+ ions the extrinsic tenase complex was three times as catalytically active compared to TFPI and Ca2+ only (N=3, p<0.05). Direct FXa inhibition by TFPI was not significantly different between the Ca2+ and Ca2+/Mg2+ containing buffers. Intrinsic tenase In closed system experiments the intrinsic tenase complex showed impaired FXa generation in the presence of Ca2+/Mg2+ (29.3 ± 3.4 pM/s) compared to Ca2+ alone (51.7 ± 3.0 pM/s) (N=3, p<0.001). Thrombin activation of FVIII was not statistically different between both buffers, however the presence of Ca2+/Mg2+ resulted in a more rapid loss of cofactor activity over Ca2+ alone. Prothrombinase Under flow, IIa generation measured from prothrombinase was not statistically different between both buffers when measured at five different shear rates (100-1000s-1, N≥2). Plasma proteome In the synthetic coagulation proteome (N=4) the presence of Ca2+/Mg2+ led to an average 31% increase in maximum IIa levels compared to Ca2+alone and an average decrease of 1 minute to reach maximum levels. ConclusionsIn summary, the presence of plasma levels of Ca2+ and Mg2+, compared to Ca2+ alone, enhances the initiation phase of the extrinsic pathway of coagulation by enhancing the rate of FXa generation from the extrinsic tenase complex while also impairing the TFPI inhibition of the extrinsic tenase complex. These procoagulant effects are potentially abrogated by the suppressive effects of Ca2+/Mg2+ on the intrinsic tenase complex, potentially caused by the increased rate of spontaneous inactivation of FVIIIa. These results highlight the importance of complementing the assessments of individual enzyme complex systems with studies of the larger complex systems in which they function to identify the net physiologic impact of metal ions that target various components in the coagulation cascade. Disclosures:Mann:Haematologic Technologies, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Mechanism Of Peak Protraction In Thrombin Generation After Administration Of Direct Factor Xa Inhibitors

IntroductionThrombin generation testing is becoming an increasingly important tool to measure the variables of the clotting system. Under normal conditions the curve is bell-shaped, yet after addition of direct, reversible factor Xa (FXa) inhibitors, the shape of the curve changes. Direct FXa inhibitors cause a strong inhibition (in vitro and ex vivo) on the peak, whereas the ETP is affected to a smaller extent, due to a protraction of the thrombin generation process. This leads to a long plateau or a ‘double peak'. AimTo investigate the mechanism behind the protraction of the thrombin generation curve. Materials & MethodsThrombin generation (TG) was determined by using Calibrated Automated Thrombinography (CAT) in platelet poor plasma (PPP), platelet rich plasma and whole blood. The effect of direct FXa inhibitors was investigated with both a parenteral (otamixaban) and an oral agent (rivaroxaban). The two inhibitors were tested at different concentrations, including the plasma concentrations that would be found after therapeutic dosing (otamixaban: 100, 250 and 400 nM and rivaroxaban: 200, 400 and 800 nM). Thrombin generation was performed in control plasma, factor VII (FVII), FVIII, FIX, FXI and TFPI (tissue factor pathway inhibitor) deficient plasmas, activated with tissue factor (TF), FIXa or kaolin. ResultsThrombin generation curves in PPP in the presence of direct FXa inhibitors display a protracted shape with a strong inhibition of the peak. When the intrinsic pathway is bypassed (e.g. in FVIII or FIX deficient plasma), the first part of the peak is no longer present. When the extrinsic pathway is evaded (e.g. by activating with FIXa), the second part of the peak disappears. This leads us to believe that when a direct FXa inhibitor is present, the first part of thrombin generation is due to the intrinsic tenase complex and the second part can be attributed to the extrinsic tenase (prothrombinase). When TFPI deficient plasma is activated with TF, the curve returns to its normal bell-shape. So, TFPI is an important contributor to the second peak. When TG was activated with kaolin, we could not distinguish an effect. In platelet rich plasma and whole blood, a stronger effect on peak height than on ETP is found, however the distinct shape that can be discerned in platelet poor plasma is not seen. ConclusionsAddition of direct FXa inhibitors to PPP results in a protracted TG curve, which could also be described as a double peak. The first peak can be attributed to the actions of the intrinsic pathway (FVIII and FIX), the second part of the peak is due to mechanisms in the extrinsic pathway. The protraction of the peak can also be restored by activating TFPI deficient plasma with TF, so this also contributes to the effect of the extrinsic pathway. The inhibition of the negative feedback on the TF-FVIIa complex by the TFPI-FXa complex will prevent the shutting off of the extrinsic pathway. Disclosures:No relevant conflicts of interest to declare.

Phosphatidylserine index as a marker of the procoagulant phenotype of acute myelogenous leukemia cells

Patients with acute myelogenous leukemia (AML) are at risk for thrombotic complications. Risk to develop thrombosis is closely tied to leukemia subtype, and studies have shown an association between leukocytosis and thrombosis in AML M3. We evaluated the relative roles of cell count and the surface expression of tissue factor (TF) and phosphatidylserine (PS) in the procoagulant phenotype of AML cell lines. The TF-positive AML M3 cell lines, NB4 and HL60, and AML M2 cell line, AML14, exhibited both extrinsic tenase and prothrombinase activity in a purified system and promoted experimental thrombus formation. In contrast, the TF-negative AML cell line, HEL, exhibited only prothrombinase activity and did not affect the rate of occlusive thrombus formation. In plasma, NB4, HL60 and AML14 shortened clotting times in a cell-count, PS- and TF-dependent manner. Exposure of cultured NB4, HL60, and AML14 cells to the chemotherapeutic agent daunorubicin increased their extrinsic tenase activity and PS expression. Clot initiation time inversely correlated with logarithm of PS index, defined as the product of multiplying leukocyte count with cell surface PS exposure. We propose that leukemia cell PS index may serve as a biomarker for procoagulant activity.

Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex

Hemophilia is caused by deficiencies in coagulation factor VIII or IX, resulting in direct blockade of the intrinsic tenase complex and indirect blockade of the extrinsic tenase complex which is rapidly inhibited upon binding of factor Xa to tissue factor pathway inhibitor. We evaluated the ability of Gla-domainless factor Xa, a truncated form of factor Xa devoid of procoagulant properties, to bind to tissue factor pathway inhibitor and to alleviate the physiological inhibition of the extrinsic tenase. Using a thrombin generation assay triggered by a low concentration of tissue factor, we evaluated the ability of Gla-domainless factor Xa to restore blood coagulation in plasma from hemophilia A and B patients without and with inhibitors. We then compared its efficacy to generate thrombin to depletion of antithrombin or tissue factor pathway inhibitor by specific antibodies. Finally, we compared the kinetics of neutralization of factor Xa and Gla-domainless factor Xa by antithrombin and tissue factor pathway inhibitor. Gla-domainless factor Xa was able to restore thrombin generation in plasma samples from hemophiliacs. This effect was observed for plasma from hemophilia A patients without or with inhibitors and for plasma from hemophilia B patients. Gla-domainless factor Xa had a lower affinity than factor Xa for tissue factor pathway inhibitor whereas the affinities of both proteins for antithrombin were similar. Finally, despite a short half-life in plasma, the effect of Gla-domainless factor Xa on thrombin generation was sustained for at least 1 hour. As Gla-domainless factor Xa was able to restore thrombin generation in plasma from hemophilia patients, our results suggest that it may be an effective alternative to current treatments for hemophilia with or without an inhibitor.

Membrane Binding Events in the Initiation and Propagation Phases of Tissue Factor-initiated Zymogen Activation under Flow

This study investigates the dynamics of zymogen activation when both extrinsic tenase and prothrombinase are assembled on an appropriate membrane. Although the activation of prothrombin by surface-localized prothrombinase is clearly mediated by flow-induced dilutional effects, we find that when factor X is activated in isolation by surface-localized extrinsic tenase, it exhibits characteristics of diffusion-mediated activation in which diffusion of substrate to the catalytically active region is rate-limiting. When prothrombin and factor X are activated coincident with each other, competition for available membrane binding sites masks the diffusion-limiting effects of factor X activation. To verify the role of membrane binding in the activation of factor X by extrinsic tenase under flow conditions, we demonstrate that bovine lactadherin competes for both factor X and Xa binding sites, limiting factor X activation and forcing the release of bound factor Xa from the membrane at a venous shear rate (100 s(-1)). Finally, we present steady-state models of prothrombin and factor X activation under flow showing that zymogen and enzyme membrane binding events further regulate the coagulation process in an open system representative of the vasculature geometry.

Peptides Inhibiting Tissue Factor Pathway Inhibitor Improve Hemostasis in Mice

Abstract 24Tissue factor pathway inhibitor (TFPI) is an activated (a) factor X (FXa)-dependent inhibitor of the extrinsic factor X (FX) activation complex and efficiently regulates the extrinsic pathway of coagulation. We are developing peptide inhibitors of TFPI with a view to improving hemostasis in hemophilia. The goal is to inhibit the interaction of TFPI with the factor Xa (FXa)-tissue factor (TF)-factor VIIa (FVIIa) complex, and thereby enhance thrombin formation to the extent that a stable clot is formed. Successful development of these peptides could allow treatment of hemophilia via a non-intravenous route of administration.By screening mRNA display libraries, we identified a de novo peptide which binds to and efficiently inhibit TFPI. The peptide was optimized by iterative amino acid substitution resulting in affinity-improved peptide with a well-characterized structure-activity relation. The affinity of the peptide was analyzed by Biacore and ELISA experiments. One of our optimized peptides bound to immobilized TFPI with an affinity below 1 nM. Kunitz domain 1 of TFPI was identified as the interaction site by NMR studies.We characterized the inhibitory activity of this TFPI binding peptide using in vitro model assay systems including inhibition of FXa and of the extrinsic tenase (TF-FVIIa-PL-Ca2+ complex). Inhibition of cell-associated TFPI was verified in a TF- and TFPI-dependent cell-based assay using TNFalpha-stimulated human umbilical vein endothelial cells (HUVECs) as a source of TF and TFPI. Inhibition of plasma TFPI was probed by thrombin generation experiments using FVIII-deficient patient plasma and ROTEM experiments using FVIII-inhibited whole blood. Furthermore, a cross-reactivity of the peptide to murine TFPI was demonstrated in thrombin generation experiments and in murine FXa inhibition model assays.Conjugation of a 40-kDa polyethyleneglycol (PEG) to the peptide base structure resulted in a peptide with significantly reduced renal clearance. The in vivo terminal half-life of this 40kD-PEG modified peptide was 21.5h and 19.8h after 1 mg/kg i.v and s.c. administration, respectively, with 73% bioavailability after s.c. administration.In a tail-tip bleeding model, administration of an anti-murine-TFPI antibody and 40kD-PEG-peptide in combination with sub-therapeutical doses of recombinant coagulation factors (10 IU/kg of rFVIII in FVIII ko mice and rFIX in FIX ko mice) led to a marked reduction of blood loss compared with buffer-treated or rFVIII/rFIX-treated mice.In addition, we established a murine nail-cut model using C57Bl6 wild-type mice for testing in vivo efficacy in a normal FVIII background as well. The TFPI sensitivity of the nail-cut model was verified by an anti-murine TFPI antibody. 40kD-PEG-peptide significantly reduced blood loss after i.v. and s.c. administration in all treatment groups compared with vehicle-treated animals. Furthermore, the i.v. administration of the peptide was well tolerated in all animals across all treatment groups without any signs of acute toxicity.In conclusion, we identified a low-molecular-weight peptide which efficiently inhibit all forms of naturally occurring TFPI proteins, including plasma TFPI and TFPI in vivo. Our results demonstrate that targeting TFPI efficiently improves hemostasis in hemophilia and provide an in vivo proof of concept for a non i.v. route of administration. Thus, the TFPI inhibitory peptides could be useful to prevent bleeding in hemophilia patients. Moreover, this new approach would probably allow treatment of inhibitor patients. Disclosures:Dockal:Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Polakowski:3B Pharmaceuticals GmbH: Employment. Frank:3B Pharmaceuticals GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Reinecke:3B Pharmaceuticals GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Lung Cancer Chemotherapy Agents Increase Procoagulant Activity In Vitro and In Vivo

Abstract 35 Introduction:Cancer patients have an elevated risk for thrombosis, with venous thromboembolism (VTE) being the second leading cause of death in cancer patients. The risk of VTE is further increased in patients undergoing standard chemotherapy. However, the mechanism(s) by which chemotherapy increases the risk for thrombosis in lung cancer patients is not well understood. Previous studies have shown that treatment of endothelial cells with chemotherapy agents (such as cisplatin, gemcitabine, and paclitaxel) promotes coagulation through an increase in tissue factor (TF) procoagulant activity. Objective:The purpose of this study was to determine in vitro and in vivo procoagulant effects of anti-lung cancer therapy agents cisplatin, carboplatin, paclitaxel, gemcitabine and bevacizumab. We studied the effects of these agents individually as well as in combinations (cisplatin/gemcitabine and carboplatin/paclitaxel) on cell surface TF activity, TF antigen levels, and phosphatidylserine (PS) exposure levels. We also examined the effects of lung cancer chemotherapy agents on thrombin generation in healthy mice. Methods:Human umbilical vein endothelial cells (HUVECs), blood monocytes, and A549 cells were treated for 24 hours with clinically relevant concentrations of chemotherapy agents alone as well as in combination. Cell surface TF activity was measured by factor Xa (FXa) generation in the presence or absence of factor VIIa and CaCl2 on drug-treated cells. To demonstrate that FXa generation was TF-dependent, Xa generation assays were performed on chemotherapy-treated cells incubated with a TF-inhibitory antibody. Cell surface TF antigen and PS levels were measured by flow cytometry. Healthy Balb/C mice received intraperitoneal injections of lung cancer chemotherapy agents alone or in combination. Blood was collected 24 hours post-chemotherapy by carotid artery cannulation. Plasma levels of thrombin-antithrombin (TAT) complex were measured by ELISA. Results:Treatment of all cell lines with single agent and combination chemotherapy significantly increased FXa generation on HUVEC, A459 cells and monocytes. To confirm that chemotherapy-induced FXa generation was TF dependent, FXa generation was repeated in the presence or absence of factor VIIa, an essential component of the extrinsic tenase complex. We also performed the FXa generation assay in the presence or absence of a TF inhibiting antibody HTF-1. Absence of factor VIIa as well as pre-incubation of cells with HTF-1 resulted in ablation of FXa generation, confirming that generation of FXa is due to cell surface TF activity. Through flow cytometry methods we determined that cell surface TF antigen levels remain unchanged on chemotherapy treated HUVEC and A459 cells. In contrast to HUVEC and A549 cells, monocytes treated with high-dose cisplatin alone or in combination with gemcitabine, expressed a significant increase in TF antigen levels. Measurement of cell surface PS exposure levels revealed a significant increase in PS levels only on HUVEC treated with high-dose cisplatin and gemcitabine combinations. However, inhibition of PS with Annexin V on HUVEC treated with high-dose cisplatin/gemcitabine combination did not decrease FXa generation. Furthermore, inhibition of protein disulfide isomerase (PDI) on A549 cells treated with cisplatin/gemcitabine combination but not carboplatin/paclitaxel combination resulted in moderately diminished levels of FXa. Our in vivo results demonstrated that treatment of healthy mice with bevacizumab, paclitaxel and carboplatin moderately increased plasma TAT levels in healthy mice. Conclusions:Our studies are the first to explore the effects of lung cancer chemotherapy agents on monocyte and A549 cell TF activity levels, as well as the first to investigate the mechanisms by which lung cancer agents may promote TF decryption on these cell lines. We are also the first to report in vivo increases in thrombin generation in response to treatment of healthy mice with anti-lung cancer agents. These studies reveal potential mechanisms by which lung cancer chemotherapy induce a hypercoagulable state. Disclosures:No relevant conflicts of interest to declare.

Chemotherapy, but Not Bevacizumab, Increases Tissue Factor Activity Levels In Endothelial Cells, Blood Monocytes, and Non-Small Cell Lung Cancer Cells

AbstractAbstract 5117 Introduction:Venous Thromboembolism (VTE) is a complication commonly seen in patients with lung cancer. The risk of VTE is further increased in patients undergoing standard chemotherapy as well as anti-angiogenesis therapies such as bevacizumab. However, the molecular mechanisms between lung cancer, anti-cancer agents, and thrombosis remain poorly understood. In vitro studies suggest that treatment of endothelial cells with particular lung cancer chemotherapy drugs such as cisplatin, gemcitabine and paclitaxel promote coagulation through an increase in tissue factor (TF) activity. However, the effect of these drugs on non-small cell lung cancer cells and monocytes remains largely unknown. It is also unknown if blockage of VEGF function by bevacizumab affects TF activity in endothelial cells and A549 lung cancer cells, both of which express VEGFR-1 and VEGFR-2 receptors. Objective:The purpose of this study was to determine the effects of standard lung cancer chemotherapy agents cisplatin, carboplatin, paclitaxel and gemcitabine on TF activity levels on vascular endothelial cells, blood monocytes, and the non-small cell lung cancer cell line A549. We studied the effects of these chemotherapy agents individually as well as in established combination chemotherapy (cisplatin/gemcitabine and carboplatin/paclitaxel). We also studied the effects of the anti-angiogenesis agent bevacizumab on TF activity levels in endothelial cells and A549 cells. Methods:Human umbilical vein endothelial cells (HUVECs), blood monocytes, and A549 cells were exposed for 24 hours to clinically relevant concentrations of chemotherapy agents or bevacizumab. At the end of treatment time, cell surface TF activity was measured by the generation of factor Xa in the presence of factor VIIa and CaCl2 on drug-treated cells. Cell viability was examined by Trypan Blue exclusion assays. Factor Xa generation was adjusted to exclude non-viable cells (at most, 15% of the cells were non-viable after 24-hour exposure to the anti-cancer agents). Results:Treatment of all cell lines with single chemotherapy agents increased cell surface TF activity. The combinations of cisplatin/gemcitabine and carboplatin/paclitaxel on HUVECs resulted in synergistic and additive effects on TF activity levels, respectively. The increase in TF activity levels were observed in the absence of cell death. In contrast, treatment of HUVECs and A549ccells with bevacizumab did not affect TF activity levels on HUVECs and A549 cells. To confirm that chemotherapy-induced factor Xa generation was TF dependent, we repeated the same assay in the absence of factor VIIa, an essential component of the extrinsic tenase complex. Absence of factor VIIa resulted in no cell surface generation of factor Xa confirming that the generation of factor Xa is due to cell surface TF activity. Conclusions:Our studies are the first to explore the effects of chemotherapy agents on TF activity levels in A549 cells and monocytes, as well as the effects of bevacizumab on TF activity levels in endothelial cells and A549 cells. Our studies suggest that, unlike chemotherapy agents, bevacizumab does not induce a procoagulant phenotype on endothelial cells and A549 cells via the upregulation of TF activity. Disclosures:No relevant conflicts of interest to declare.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model.

The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Elevated Procoagulant Activity Due to Phosphatidylserine Exposure on Red Blood Cells during Storage.

Serious complications and mortality after cardiac surgery are increased when transfused red cells are stored for more than 2 weeks (Koch, N Engl J Med. 2008). However, the mechanism remains unclear and the relationship of procoagulant activity to PS exposure on banked RBCs has not been clarified, although prior studies suggest that banked cells went to apoptosis. Procoagulant activity of RBCs was measured in a modified prothrombin time in which RBCs replaced thromboplastin. The presence of PS in neutrophils and mononuclear cells from healthy donors and banked RBCs was investigated by flow-cytomety and confocal microscopy. The assembly of extrinsic tenase, intrinsic tenase, and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Lactadherin, a glycoprotein, was utilized as a probe to track PS exposure and as an agent to block exposed PS. Coagulant activity increased approx 5-fold after cells were banked for 14 days and progressively increases. The percent of phosphatidylserine (PS)-positive cells increased from 7.4% on day 14 to 30% on day 42. RBCs with exposed PS were nearly absent in samples at 7 days but readily evident at 14 days on confocal microscopy. Banked RBCs exhibited patched, a rim, and diffuse-pattern that stained with lactadherin but neutrophils and MNC did not, indicating more PS exposure on banked RBCs from 7 days than the other two cell types. These data reveal a positive correlation between PCA and PS exposure on stored RBCs. Inhibition of PCA by lactadherin further confirms that PS supports the PCA in stored RBCs. Thus, we propose the possible role of PCA due to increased PS exposure on aged RBCs as a potential pathogenetic factor leading to organ injury, aggravated infections and finally higher mortality.

Contributions of Phosphatidylserine Exposure on Leukemic Cells to Unregulated Procoagulant Activity in Acute Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) causes coagulation, which can worsen following initiation of chemotherapy and is improved or corrected by the differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As2O3). The relationship of procoagulant activity to phosphatidylserine (PS) exposure on leukemic cells has not been clarified, although prior studies indicate that APL cells expose tissue factor (TF). Procoagulant activity of leukemic cells was measured in a modified prothrombin time in which leukemic cells replaced thromboplastin. The presence of phosphatidylserine (PS) in neutrophils and mononuclear cells (MNC) from healthy donors and APL cells from patients was investigated by flow-cytometry and confocal microscopy. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Lactadherin was utilized as a probe to track PS exposure and as an agent to block exposed PS. Isolated APL cells exhibited patches that stained with lactadherin, but neutrophils and MNC did not, indicating more PS exposure on APL cells than the other two cell types. Exclusion of propidium iodide by the leukemic cells indicated that PS exposure occurs in the absence of frank apoptosis. Coagulant activity increased approx 20-fold after cells were exposed to 0.1 μM daunorubicin for 24 hr with 70% of apoptosis and decreased by 85% after 24 hr treatment with 1 μM ATRA or As2O3 with partial differentiation. Inhibition of procoagulant activity by ATRA and As2O3 corresponded to decreased PS and decrease activity for all three enzyme complexes. Lactadherin, which blocks PS binding sites, inhibited Xase and prothrombin conversion by their respective APL-assembled activating complexes. APL cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant PS and TF exposure at the outer leaflet of cell membrane. In contrast, less PS on neutrophils and MNC do not support the coagulation.

Effect of Plasmatic Procoagulant Factors in Thrombin Generation Parameters in a Platelet-Poor Plasma Model System.

The evolution of the thrombin generation (TG) concept and the recent technical advances in automated TG measurement have made its clinical utility practical but the clinical relevance of all parameters thrombin generation provided still remain unclear. In the current study we investigated effects of various procoagulant plasmatic factors in the TG parameters, such as rate, peak, lag time and area under the curve (AUC), in a platelet poor plasma model system using the calibrated automated thrombography (CAT). The plasma model system was made by mixing well-characterized commercial normal pooled human plasma with plasmas that are deficient in various procoagulant factors (factor VIII, IX, II, X, V and VII) to reach factor levels between <1 % to 100% normal. Initially 5 pM of tissue factor and 4 μM of phospholipid were used and all TG parameters (lag time, peak, rate, and AUC) were compared with various levels of all procoagulant factors. Our result showed that the AUC is proportional to concentrations of factor II and X but not sensitive to factor VIII, IX and VII levels under such experimental conditions. Factor VII affects only the lag time and factor X affects both the lag time and the rate of thrombin generation. Factor II and X affect the peak as well. Factor VIII and IX affect both the rate and peak but not the AUC. Factor V plays very little role on affecting these parameters. Furthermore different concentrations of tissue factor (0.5 to 5 pM) were used to explore the sensitivity of TG for screening hemophiliac factor deficiencies. When ≤ 2pM of tissue factor and 4 μM of phospholipid were used the AUC showed three fold difference for factor VIII deficient plasma versus the normal plasma pool as compared with the less than 20% of difference in AUC (SD=15% for normal population) under the 5 pM concentration of tissue factor. Using the AUC and the rate of TG factor VIII and IX levels were well differentiated under such further diluted tissue factor concentrations (≤ 2 pM). Our study indicated that, under the standard CAT assay condition, 1) the lag time is mainly an indicator of extrinsic tenase reaction (factor VII, X); 2) the peak and rate reflect the intrinsic tenase reaction (factor X, IX, and VIII); and 3) the AUC and peak are largely affected by the prothrombinase factors (II, X) indicating the total free thrombin available for its overall plasmatic procoagulant activity. When proper concentration of triggering agent is used (≤ 2pM of tissue factor and 4 μM of phospholipid) TG becomes sensitive to all plasmatic procoagulant factors and CAT can be used for screening factor deficiencies in hemophiliac patients. A combination of these TG parameters can be used in an in-vitro screening assay to indicate the overall hemostasis balance in plasma under physiological conditions and an individual factor deficiency in related to hemostasis unbalance. Such further diluted triggering agent needs to be optimized for the CAT assay and a standardized large scale clinical retrospective study is needed to prove the utility of these parameters.

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells.

That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes. C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.

Hemextin AB Complex – A Snake Venom Anticoagulant Protein Complex That Inhibits Factor VIIa Activity

Snake venom is a veritable gold mine of bioactive molecules, capable of binding to a wide variety of pharmacological targets, including the blood coagulation cascade. Here, we report the isolation and characterization of two synergistically acting anticoagulant three-finger proteins, hemextin A and hemextin B, from the venom of Hemachatus haemachatus (African Ringhals cobra). Hemextin A but not hemextin B exhibits mild anticoagulant activity. However, hemextin B interacts with hemextin A and forms a complex (hemextin AB complex), and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Using a ‘a dissection approach’, we found that hemextins A and AB complex prolong clotting by inhibiting extrinsic tenase activity. Further studies showed that hemextin AB complex potently inhibits the proteolytic activity of factor VIIa (FVIIa) and its complexes. Kinetic studies showed that hemextin AB complex is a non-competitive inhibitor of FVIIa-soluble tissue factor proteolytic activity with a K_i of 25 nM. Hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require either tissue factor or factor Xa scaffold to mediate its inhibitory activity. Molecular interactions of hemextin AB complex with FVIIa/tissue factor-FVIIa may provide a new paradigm in the search for anticoagulants inhibiting the initiation of blood coagulation.

Spatial Propagation and Localization of Blood Coagulation Are Regulated by Intrinsic and Protein C Pathways, Respectively

Blood coagulation in vivo is a spatially nonuniform, multistage process: coagulation factors from plasma bind to tissue factor (TF)-expressing cells, become activated, dissociate, and diffuse into plasma to form enzymatic complexes on the membranes of activated platelets. We studied spatial regulation of coagulation using two approaches: 1), an in vitro experimental model of clot formation in a thin layer of plasma activated by a monolayer of TF-expressing cells; and 2), a computer simulation model. Clotting in factor VIII- and factor XI-deficient plasmas was initiated normally, but further clot elongation was impaired in factor VIII- and, at later stages, in factor XI-deficient plasma. The data indicated that clot elongation was regulated by factor Xa formation by intrinsic tenase, whereas factor IXa was formed by extrinsic tenase on activating cells and diffused into plasma, thus sustaining clot growth. Far from the activating cells, additional factor IXa was produced by factor XIa. Exogenously added TF had no effect on the clot growth rate, suggesting that plasma TF does not contribute significantly to the clot propagation process in a reaction-diffusion system without flow. Addition of thrombomodulin at 3–100 nM caused dose-dependent termination of clot elongation with a final clot size of 2–0.2 mm. These results identify roles of specific coagulation pathways at different stages of spatial clot formation (initiation, elongation, and termination) and provide a possible basis for their therapeutic targeting.

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) in the blood with freshly exposed tissue factor (TF) to form the TF.FVIIa complex. However, unwanted clot formation can lead to death and debilitation due to vascular occlusion, and hence, anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences and CD spectra of the native proteins indicate that these proteins belong to the three-finger toxin family. Hemextin A (but not hemextin B) exhibits mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Complex formation was supported by size-exclusion chromatography. Using a "dissection approach," we determined that hemextin A and the hemextin AB complex prolong clotting by inhibiting TF.FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF.FVIIa complex. Furthermore, we demonstrated their specificity of inhibition by studying their effects on 12 serine proteases; the hemextin AB complex potently inhibited the amidolytic activity of FVIIa in the presence and absence of soluble TF. Kinetic studies showed that the hemextin AB complex is a noncompetitive inhibitor of soluble TF.FVIIa amidolytic activity, with a Ki of 50 nm. Isothermal titration calorimetric studies showed that the hemextin AB complex binds directly to FVIIa with a binding constant of 1.62 x 10(5) m(-1). The hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require a scaffold to mediate its inhibitory activity. Molecular interactions of the hemextin AB complex with FVIIa/TF.FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation.

Structure–function relationships and mechanism of anticoagulant phospholipase A 2 enzymes from snake venoms

Phospholipase A 2 (PLA 2) enzymes from snake venom are toxic and induce a wide spectrum of pharmacological effects, despite similarity in primary, secondary and tertiary structures and common catalytic properties. Thus, the structure–function relationships and the mechanism of this group of small proteins are subtle, complex and intriguing challenges. This review, taking the PLA 2 enzymes from spitting cobra ( Naja nigricollis) venom as examples, describes the mechanism of anticoagulant effects. The strongly anticoagulant CM-IV inhibits both the extrinsic tenase and prothrombinase complexes, whereas the weakly anticoagulant PLA 2 enzymes (CM-I and CM-II) inhibit only the extrinsic tenase complex. CM-IV binds to factor Xa and interferes in its interaction with factor Va and the formation of prothrombinase complex. In contrast, CM-I and CM-II do not affect the formation of prothrombinase complex. In addition, CM-IV inhibits the extrinsic tenase complex by a combination of enzymatic and nonenzymatic mechanisms, while CM-I and CM-II inhibit by only enzymatic mechanism. These functional differences explain the disparity in the anticoagulant potency of N. nigricollis PLA 2 enzymes. Similarly, human secretory enzyme binds to factor Xa and inhibits the prothrombinase complex. We predicted the anticoagulant region of PLA 2 enzymes using a systematic and direct comparison of amino acid sequences. This region between 54 and 77 residues is basic in the strongly anticoagulant PLA 2 enzymes and neutral or negatively charged in weakly and non-anticoagulant enzymes. The prediction is validated independently by us and others using both site directed mutagenesis and synthetic peptides. Thus, strongly anticoagulant CM-IV binds to factor Xa (its target protein) through the specific anticoagulant site on its surface. In contrast, weakly anticoagulant enzymes, which lack the anticoagulant region fail to bind specifically to the target protein, factor Xa in the coagulation cascade. Thus, these studies strongly support the target model which suggests that protein–protein interaction rather than protein–phospholipid interaction determines the pharmacological specificity of PLA 2 enzymes.