Factor Xa Generation Research Articles

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.

Depletion of Systemic Concentrations of Coagulation Factors in Blood From Patients with Atherosclerotic Vascular Disease

Abstract 1231 Introduction.Thrombosis complicating the rupture of atherosclerotic plaques leads to arterial occlusion. Tissue factor (Tf), a membrane-bound glycoprotein, is expressed to a greater extent in atherosclerotic plaques and is likely a key mediator of micro and macro thrombosis. Atherosclerotic vascular disease is a widespread process that can lead to stroke, myocardial infarction and peripheral artery occlusion. Because thrombosis consumes coagulation factors, the goal of the present study was to determine whether the extent of atherosclerosis correlated with the concentration or activity of coagulation factors in blood and to relate, using novel computational approaches, the thrombin generation potential of the arterial blood to the extent of atherosclerosis. Methods. Patients scheduled for diagnostic cardiac catheterization (n=57) for clinical indications were enrolled. The average age was 65 (range 35–92) with a predominance of males (71%). Most patients were treated with aspirin (54/57 – 95%) and a small group had been treated with both aspirin plus clopidogrel (7/57 – 12%). After review of their angiograms, patients were divided into 3 groups based on their maximal extent of stenosis, 1) no coronary artery disease (no CAD, n=10), 2) coronary arterial stenosis <50% (CAD<50%, n=9), and 3) coronary arterial stenosis >50% (CAD>50%, n=38). For patients with CAD>50%, we evaluated those not treated with clopidogrel (n=31) separately from the group taking clopidogrel (n=7). Blood was taken from catheters placed in the femoral artery before administration of anticoagulants. Thrombin and factor Xa generation was assessed computationally based on measurements of plasma coagulation factors (II, V, VII, IX, X) and inhibitors (antithrombin and free tissue factor pathway inhibitor, TFPI) in all patients. Results. Plasma composition was not significantly different between individuals with no CAD versus CAD. In individuals with CAD, the concentrations of fX and prothrombin were significantly less in blood from patients with more than 50% stenosis. This difference was apparent in both those taking clopidogrel and those not taking clopidogrel. Thrombin and fXa generation after a 5 pM Tf initiator was significantly increased in all individuals compared to a physiologic control. Individuals with >50% CAD had significantly lower total thrombin then individuals with CAD<50% (83±16μM.s vs. 96 ±20μM.s, respectively). Kinetic analysis suggested that depletion of fX and prothrombin would decrease the amount of thrombin generated in patients with CAD>50%. To determine whether selected factors influenced the generation of fXa and thrombin, linear regression analysis was performed. The concentrations in blood of TFPI and fVIII were strongly associated (R2=0.5) with the time to the maximum rate of thrombin generation and maximum concentration of fXa generated. In simulations performed with increasing Tf concentrations (12.5 pM and 25 pM) TFPI and fVIII continued to correlate with thrombin and fXa generation. Also, at increasing Tf concentrations, fXa generation had greater (11 fold range) power to resolve each individual’s thrombin generation profile (4 fold range). Conclusions. In summary, a reduced concentration of prothrombin and fX was observed in patients with more severe CAD, which could reflect greater consumption of these coagulation factors, reduced production or some combination. The computational analyses indicated important roles for TFPI and fVIII in the thrombin generation potential of individuals with CAD. The kinetics of fXa and thrombin generation suggests that the lower concentrations of fX and prothrombin may have functional implications consistent with clinical observations of patients with severe CAD having a narrower therapeutic window when treated with vitamin K antagonists. Disclosures:No relevant conflicts of interest to declare.

Thrombin Generation Inhibition by Newer Anti Xa and Anti IIa Drugs in Prothrombin Complex Concentrates: Relevance to Endogenous Modulation of Thrombogenesis

Abstract 1196Several anti-Xa agents (apixaban, otamixaban, rivaroxaban and edoxaban) and anti-IIa agents (dabigatran and melagatran) have been developed for clinical use. These agents are strong inhibitors of factor Xa and IIa and their potency is usually expressed in terms of the inhibitory parameters of these enzymes. These synthetic agents can also modulate thrombin and factor Xa generation in complex systems such as the platelet rich and platelet poor plasma and whole blood. The order of potency differs depending upon the type of matrix used due to cellular and protein binding and interaction with generated proteases and protein fragments. In order to compare these agents for their relative thrombin generation inhibitory profile, prothrombin complex based assay systems are developed utilizing 5 units/mL of non activated prothrombin complex such as Konyne® and Preconativ. Thrombin generation is triggered by tissue factor and monitored using a thrombin specific florometric substrate based assay (Technoclone, Vienna, Austria). All agents were tested in a concentration range of 0–1000 ng/mL. The relative inhibition of thrombin generation was calculated in terms of IC50. The factor Xa inhibitors were found to be relatively potent inhibitors of thrombin generation exhibiting an IC50 value of 25–96 ng/mL with the following order of potency- Rivaroxaban>Otamixaban>Apixaban. The anti IIa agents exhibited potency in the range of 125–225 ng/mL. Melagatran was more potent than dabigatran. In the plasma based anticoagulant assays such as PT, aPTT, heptest, the anti IIa agents were more potent than factor Xa drugs. Assay based differences were noted in all agents which did not follow the same patterns as thrombin generation inhibition in the prothrombin complex systems. These observations suggest that although considered to be single target, the newer anticoagulant drugs produce variable inhibitory effects on the protease network in complex systems such as plasma and prothrombin complexes. These effects may be related to the relative safety and efficacy profile of these agents and should be included in the preclinical profiling of these agents. Disclosures:No relevant conflicts of interest to declare.

Low Concentrations of rhFVIIa or FEIBA Significantly and Rapidly Reverse the Anticoagulant Effects of Supratherapeutic Edoxaban

Abstract 1252 Introduction:Edoxaban is an oral, direct factor Xa (FXa) inhibitor currently in phase 3 clinical development for stroke prevention in nonvalvular atrial fibrillation (AF) and the treatment and secondary prevention of venous thromboembolism (VTE). Guidance for the emergency management of serious bleeding and strategies for reversing the anticoagulant effects edoxaban are needed. Both recombinant human FVIIa (rhFVIIa; NovoSeven®) and a human plasma fraction containing nonactivated forms of FII, FIX, and FX/proteins C and S and activated FVII (FEIBA; factor eight inhibitor bypassing activity) have been identified as potential reversal agents for edoxaban. The minimum effective doses and the time course required for either rhFVIIa or FEIBA to reverse supratherapeutic concentrations of edoxaban in an anticoagulated patient have yet to be established. This ex vivo study was conducted to assess the effects and establish the time course of various concentrations of rhFVIIa and FEIBA on the reversal of edoxaban’s anticoagulant effects. Methods:Edoxaban at supratherapeutic concentrations of 500 and 1000 ng/mL (therapeutic level is ∼250 ng/mL for Cmax of a 60-mg dose) was added to fresh blood drawn from healthy subjects; control samples were diluent alone. After 60 min of incubation, rhFVIIa, FEIBA, or control was added to the samples. rhFVIIa was added to produce final concentrations of 0.8 and 1.8 μg/mL, which correspond to known maximal observed human plasma concentrations at doses of 40 and 90 μg/kg. FEIBA was added to produce concentrations of 0.75 and 1.4 U/mL, which correspond to human plasma concentrations at the therapeutic doses of FEIBA of 50 to 100 U/kg. In order to determine reversal of anticoagulant effects over time, coagulation assays (PT, aPTT, anti-Xa, intrinsic FXa activity, thrombin generation assay [TGA], D-dimer, and FVIIa activity) were measured at times 0, 0.25, 0.50, 1, 2, and 4 h after incubation with rhFVIIa, FEIBA, or control. Results:In edoxaban alone and control samples, PT, aPTT, and anti-Xa results reflected previously established anticoagulant activity. Both 500 and 1000 ng/mL of edoxaban completely inhibited intrinsic FXa and thrombin generation as measured by TGA (peak). Measures of FVIIa activity indicated that the concentrations of rhFVIIa utilized were consistent with previously reported responses. With the exception of thrombin levels, reversal of edoxaban anticoagulant activity by both rhFVIIa and FEIBA was observed for PT, aPTT, and anti-Xa beginning at 0.25 h and was maintained across the experimental period. At 1000 ng/mL edoxaban, the observed decrease at 0.25 h post-reversal of anticoagulant activity was 82% for anti-Xa, 72% for PT, and 58% for aPTT. Compared with baseline, the maximum reversal of intrinsic FXa activity for edoxaban 500 ng/mL was ∼30% and for 1000 ng/mL was 15% across all concentrations of rhFVIIa and FEIBA. The TGA assay indicated both rhFVIIa and FEIBA reversed ∼45% and 20% of the effect of edoxaban at 500 ng/mL and 1000 ng/mL, respectively, 4 h after adding the reversal agents. With the exception of 1 baseline sample, levels of D-dimer did not show significant changes with the addition of edoxaban or with its subsequent reversal by either rhFVIIa or FEIBA. Conclusion:Low therapeutic concentrations of rhFVIIa and FEIBA showed significant and rapid reversal of supratherapeutic concentrations of the anticoagulant activity induced by edoxaban based on PT, aPTT, and anti-Xa activity. Disclosures:Halim:Daiichi Sankyo Pharma Development: Employment. Li:Daiichi Sankyo Pharma Development: Employment. Stein:Daiichi Sankyo: Consultancy, Research Funding; AACC: Consultancy, Honoraria, Research Funding; Abbott: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; FDA: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; ISIS: Consultancy, Honoraria, Research Funding; Merck & Co: Consultancy, Honoraria, Research Funding; National Lipid Association: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Reliant: Consultancy, Honoraria, Research Funding; Regeneron: Consultancy, Honoraria, Research Funding; SanofiAventis: Consultancy, Honoraria, Research Funding; Schering-Plough: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Wyeth: Consultancy, Honoraria, Research Funding. Mendell:Daiichi Sankyo Pharma Development: Employment.

Increased Heparanase Level and Procoagulant Activity in Orthopedic Surgery Patients Receiving Prophylactic Dose of Enoxaparin,

Abstract 3333 Background:Orthopedic hip and knee surgeries are followed by a hypercoagulable state. Heparanase is implicated in inflammation, coagulation activation and angiogenesis. Recently, heparanase was shown to directly interact with tissue factor (TF) and to enhance generation of factor Xa (Nadir et al, Haematologica, 2010). In addition, an assay evaluating heparanase procoagulant activity has been lately developed (Nadir et al, Thromb Res, 2011). In the present study, heparanase level and procoagulant activity in patients undergoing orthopedic surgery were assessed. Methods: The study group included 50 orthopedic patients. Thirty one patients underwent hip operation and 19 had knee surgery. Fifteen individuals suffered from traumatic hip fractures and 35 had osteoarthrosis of hip or knee joints. All patients received a prophylactic dose of enoxaparin starting 6–8 hours post operation and lasting for 5 weeks. Plasma samples were drawn preoperatively and at 1 hour, 1 week and 1 month post surgery. Samples were tested for heparanase levels by ELISA and TF/heparanase complex activity, TF activity, heparanase procoagulant activity, factor Xa and thrombin levels were estimated using chromogenic substrates. Results: Heparanase levels were significantly higher 1 hour and 1 week postoperatively compared to preoperative levels (p<0.05, p<0.005, respectively). The most dramatic changes were observed in heparanase procoagulant activity, reaching a 2-fold increase 1 week postoperatively and a 1.7-fold increase 1 month after surgery (p<0.0001 and p<0.0001, respectively). Levels of factor Xa and thrombin did not significantly change. Conclusions: Heparanase is found to be involved in coagulation activation of orthopedic surgery patients, with heparanase procoagulant activity being highest 1 week postoperatively and still remaining high 1 month after operation. Consideration of extending prophylactic anticoagulant therapy or evaluation of heparanase procoagulant activity may potentially prevent late thrombotic events in this patient population. Disclosures:No relevant conflicts of interest to declare.

A Novel FVIIa Variant with Increased Potency and Duration of Effect Compared to Wildtype FVIIa. A Study in a Dog Model of Hemophilia A

Abstract 2252Hemophilia patients are treated by replacement therapy, receiving the plasma-derived or recombinant clotting factor in which they are deficient. A significant number of hemophilia patients, however, develop inhibitory antibodies against the factor they receive, becoming refractory to replacement therapy. In these inhibitor patients, hemostasis can be achieved using bypass agents, activated plasma-derived prothombin complex concentrates (aPCC) or recombinant activated Factor VIIa (FVIIa). However, these treatments do not match the efficacy seen in traditional replacement therapy. To overcome this limitation, 813, a modified recombinant human Factor VIIa with enhanced biological properties was developed using a rational protein design approach. 813 was selected from a series of FVIIa variants and is characterized by increased catalytic activity and prolonged duration of effect in vivo. Compared to recombinant wild-type FVIIa, 813 has 7-fold increased catalytic activity, measured by the rate of Factor Xa generation in vitro, both in the presence and absence of tissue factor (TF).813 was studied in Factor VIII (FVIII)-deficient dogs with NovoSeven RT (wt-FVIIa) as comparator. The FVIII-deficient dogs exhibit a severe hemophilia A phenotype, with less than 1% normal coagulant activity. Dogs were dosed intravenously with 813 or wt-FVIIa and blood and plasma were collected at various time points post dosing. Plasma FVIIa levels were measured using a FVIIa-specific clotting assay. Starting from initial peak plasma levels, a time-dependent decrease in FVIIa plasma concentration was observed for wt-FVIIa and variant. Compared to wt-FVIIa, 813 exhibited a smaller volume of distribution (35–48 mL/kg vs. 108–134 ml/kg), a lower rate of clearance (10–18 mL/hr/kg vs. 45 mL/kg/hr) and consequently an increased dose adjusted exposure. Hemophilia A dogs have a prolonged activated partial thromboplastin time (aPTT); treatment with wt-FVIIa or 813 led to a dose-dependent decrease in aPTT. At equivalent doses the peak effect of 813 on decreasing aPTT was approximately two-fold compared to wt-FVIIa. Similar dose-dependent effects were observed when studying whole blood clotting profiles by thromboelastography (TEG). Treatment effects were seen for all TEG parameters monitored (time to clotting onset, rate and strength of clot formation). However, compared to wt-FVIIa, 813 given at equivalent doses caused significantly stronger effects on all TEG parameters; acute peak effects on TEG parameters measured for 813 at 10 ug/kg, were similar to those seen with 50 ug/kg wt-FVIIa. This difference became even more pronounced when monitoring treatment effect over time. All dogs used in this study were monitored throughout the study for possible treatment-related changes; both 813 and wt-FVIIa were well tolerated. No overt adverse events, no significant changes in plasma clinical chemistry or cellular blood counts were observed.In conclusion, 813 is a novel FVIIa protein with increased potency, prolonged duration of effect and a preclinical safety profile comparable to wt-FVIIa. 813 is currently in early clinical development for acute and prophylactic treatment of hemophilia patients with inhibitors. Disclosures:Pittman:Pfizer: Employment. Weston:Pfizer: Employment. Shields:Pfizer: Employment. Parng:Pfizer: Employment. Arkin:Pfizer: Employment. Madison:Catalyst Biosciences: Employment. Nichols:Pfizer: Research Funding. Fruebis:Pfizer: Employment.

Glycosylation of tissue factor is not essential for its transport or functions

Glycosylation plays an important role in protein function. The importance of glycosylation for tissue factor (TF) function is unclear. The aim of the present study is to investigate the importance of TF glycosylation in transport to the cell surface and its coagulant and signaling functions. Endothelial cells and peripheral blood mononuclear cells (PBMC) were treated with tunicamycin to inhibit N-linked glycosylation. Site-specific mutagenesis of one or more potential N-linked glycosylation sites in TF was used to generate TF mutants lacking glycans. TF expression at the cell surface was determined in binding assays using (125)I-FVIIa or (125)I-TF mAb and confocal microscopy. TF coagulant activity was measured by factor (F) Xa generation assay, and TF signaling function was assessed by measuring cleavage of protease activated receptor 2 (PAR2) and activation of p44/42 MAPK. Tunicamycin treatment reduced TF activity at the endothelial cell surface; however, this reduction was found to be the result of decreased TF protein production in tunicamycin-treated cells. Tunicamycin treatment had no significant effect on TF activity or antigen levels in PBMC. No significant differences were observed in TF protein expression and procoagulant activity among cells transfected to express either wild-type TF or TF mutants. A fully non-glycosylated TF is shown to bind FVIIa and interact with FX with the same efficiency as that of wild-type TF. Non-glycosylated TF is also capable of supporting FVIIa cleavage of PAR2 and PAR2-dependent p44/42 MAPK activation. Glycosylation is not essential for TF transport and coagulant or signaling functions.

Increasing Hydrophobicity or Disulfide Bridging at the Factor VIII A1 and C2 Domain Interface Enhances Procofactor Stability

Factor VIII (FVIII) consists of a heavy (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. FVIII x-ray structures show close contacts between A1 and C2 domains. To explore the role of this region in FVIII(a) stability, we generated a variant containing a disulfide bond between A1 and C2 domains by mutating Arg-121 and Leu-2302 to Cys (R121C/L2302C) and a second variant with a bulkier hydrophobic group (A108I) to better occupy a cavity between A1 and C2 domains. Disulfide bonding in the R121C/L2302C variant was >90% efficient as judged by Western blots. Binding affinity between the A108I A1 and A3C1C2 subunits was increased ∼3.7-fold in the variant as compared with WT as judged by changes in fluorescence of acrylodan-labeled A1 subunits. FVIII thermal and chemical stability were monitored following rates of loss of FVIII activity at 57 °C or in guanidinium by factor Xa generation assays. The rate of decay of FVIIIa activity was monitored at 23 °C following activation by thrombin. Both R121C/L2302C and A108I variants showed up to ∼4-fold increases in thermal stability but minimal improvements in chemical stability. The purified A1 subunit of A108I reconstituted with the A3C1C2 subunit showed an ∼4.6-fold increase in thermal stability, whereas reconstitution of the variant A1 with a truncated A3C1 subunit showed similar stability values as compared with WT A1. Together, these results suggest that altering contacts at this A1-C2 junction by covalent modification or increasing hydrophobicity increases inter-chain affinity and functionally enhances FVIII stability.

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.

Atorvastatin neutralises the thrombin-induced tissue factor expresion in endothelial cells via geranylgeranyl pyrophosphate

Statins may have beneficial effects in atherogenesis given their antithrombotic properties involving non-lipid mechanisms that modify endothelial function of tissue factor induction by thrombin. In this study, we investigate the effect of atorvastatin on tissue factor (TF) activity in thrombin-stimulated endothelial cells and its regulation through mevalonate or its derivatives. First subculture of human umbilical endothelial cells was used for this study. Cells were treated with thrombin and atorvastatin for different time intervals and dosage. Tissue factor activity was measured as Factor Xa generation induced by Tissue Factor-Factor VIIa complex on confluent cells. Our results show that atorvastatin prevents the thrombin-induced up-regulation of tissue factor activity in a concentration-dependent manner. Mevalonate and geranylgeranyl pyrophosphate reversed this inhibitory effect of atorvastatin on tissue factor activity, while the presence of farnesyl pyrophosphate did not prevent the atorvastatin effect on thrombin-induced tissue factor activity. Rho-kinase inhibitor did not affect the thrombin stimulation of tissue factor activity. High amount of hydrophobic isoprenoid groups decreases the thrombin-induced TF activity and may promote endothelial cell anti-thrombotic action. Rho kinase pathways do not have a major role in the thrombin-mediated TF activity. The inhibitory effect of atorvastatin on thrombin-induced TF activity was partially reversed by MVA and GGPP but not FPP.

Oncogenic epidermal growth factor receptor up-regulates multiple elements of the tissue factor signaling pathway in human glioma cells

Cancer cells frequently overexpress tissue factor (TF) and become procoagulant. This conversion may be driven by genetic transformation, including through the expression of the oncogenic epidermal growth factor receptor (EGFR) and its mutant, EGFRvIII, present in glioblastoma multiforme (GBM). Here we show that the EGFRvIII-dependent GBM cell transformation is associated with the onset of the simultaneous overexpression of TF, protease-activated receptors 1 and 2 (PAR1 and PAR2), and ectopic synthesis of factor VII (FVII). Efficient generation of factor Xa by these cells still requires exogenous FVIIa. However, as a result of EGFRvIII-dependent transformation, GBM cells become hypersensitive to TF/PAR-mediated signaling and produce ample angiogenic factors (vascular endothelial growth factor and interleukin-8) on exposure to FVIIa and PAR1- or PAR2-activating peptides. Thus, oncogenes may cause complex changes in the ability of GBM cancer cells to interact with the coagulation system, thereby exacerbating its influence on angiogenesis and disease progression.

Tobacco Smoke Induces the Generation of Procoagulant Microvesicles From Human Monocytes/Macrophages

To investigate whether exposure of human monocytes/macrophages to tobacco smoke induces their release of membrane microvesicles (MVs) that carry tissue factor (TF) released from cells, whether smoke-induced MVs are procoagulant, and what cellular processes might be responsible for their production. We found that exposure of human THP-1 monocytes and primary human monocyte-derived macrophages to 3.75% tobacco smoke extract (TSE) significantly increased their total and TF-positive MV generation. More importantly, MVs released from TSE-treated human monocytes/macrophages exhibited 3 to 4 times the procoagulant activity of control MVs, as assessed by TF-dependent generation of factor Xa. Exposure to TSE increased TF mRNA and protein expression and cell surface TF display by both THP-1 monocytes and primary human monocyte-derived macrophages. In addition, TSE exposure caused activation of C-Jun-N-terminal kinase (JNK), p38, extracellular signal regulated kinase (ERK) mitogen-activated protein kinases (MAPK), and apoptosis (a major mechanism for MV generation). Treatment of THP-1 cells with inhibitors of ERK, MAP kinase kinase (MEK), Ras, or caspase 3, but not p38 or JNK, significantly blunted TSE-induced apoptosis and MV generation. Surprisingly, neither ERK nor caspase 3 inhibition altered the induction of cell surface TF display by TSE, indicating an effect solely on MV release. Inhibition of ERK or caspase 3 essentially abolished TSE-induced generation of procoagulant MVs from THP-1 monocytes. Tobacco smoke exposure of human monocytes/macrophages induces cell surface TF display, apoptosis, and ERK- and caspase 3-dependent generation of biologically active procoagulant MVs. These processes may be novel contributors to the pathological hypercoagulability of active and secondhand smokers.

Enhanced cell-associated plasminogen activator pathway but not coagulation pathway activity contributes to motility in metastatic breast cancer cells.

Activation of tumor cell-associated coagulation and plasminogen activator pathways occurs in malignant disease processes, including breast cancer, and may promote metastatic activity. To compare the coagulation and plasminogen activator pathways of normal and metastatic cells, we examined two cell lines from the MCF-10 family of breast cells: near-normal immortalized MCF-10A cells, and metastatic MCF-10CA1 cells. MCF-10CA1 cell motility was significantly increased as compared with that of MCF-10A cells. The two cell types supported similar rates of factor Xa generation, plasma thrombin generation, and fibrin formation. MCF-10A cells produced a stable fibrin network, whereas MCF-10CA1 cells lysed the surrounding fibrin network within 24 h of network formation. Importantly, fibrin located proximal to (within 10 microm) the MCF-10CA1 cell surface lysed substantially faster than fibrin located 100 microm from the surface. MCF-10CA1 cells supported significantly increased plasmin generation rates as compared with MCF-10A cells, providing a mechanism for the increased fibrinolytic activity of these cells towards the fibrin network. Metastatic MCF-10CA1 cells had increased expression (mRNA and protein) levels of urokinase plasminogen activator (u-PA) and decreased levels of plasminogen activator inhibitor-1 as compared with MCF-10A cells. Blocking u-PA activity with the active site-directed protease inhibitor amiloride substantially decreased MCF-10CA1 cell motility. Phosphorylated Akt levels were elevated in MCF-10CA1 cells, which partially explains the increased u-PA expression. These results suggest that the tumor-associated plasminogen activator pathway, not the coagulation pathway, is a key distinguishing feature between metastatic MCF10-CA1 cells and normal MCF-10A cells.

Protease Generation by Chondroitin Sulfate, Semisynthetic OSCS Preparations and Contaminants Isolated From Heparin.

Abstract 3133Poster Board III-70The main contaminant in heparins recalled by the FDA in 2008 was reported to be oversulfated chondroitin sulfate (OSCS) (Kishimoto et al 2008). The adverse reactions were reportedly linked to contact activation and the generation of kallikrein. Because of the molecular and biologic heterogeneity of the contaminants isolated from heparin and LMWHs and the potential presence of other carryover contaminants it was hypothesized that heparin contaminants may also activate other proteases such as plasmin, C1 esterase factor Xa (FXa), and thrombin by direct or indirect mechanisms. To investigate the protease activation profile of heparin, porcine chondroitin sulfate (PCS) and OSCS preparations of porcine (POSCS), bovine (BOSCS) shark (SOCS) and squid (SqOSCS) origin along with contaminants isolated from five different recalled batches of heparin were profiled for the generation of kallikrein, plasmin, C1 esterase, FXa, and thrombin. Normal human pooled plasma (NHP) was supplemented with each of the different agents over a concentration range of 100ug/ml to 0.1ug/ml. Specific substrates for kallikrein (Chromozyme PK), plasmin (Chromozyme PL), C1 esterase (S2302), FXa (Chromozyme Xa), and thrombin (Chromozyme Th) were used to measure the generation of specific enzymes. The PCS produced relatively weaker generation of kallikrein, however, the contaminants isolated from the recalled batches of heparin produced a much stronger generation of this enzyme (absorbance 0.45-0.70). The different OSCS preparations produced a lower generation of kallikrein (absorbance .34-.52). While heparin and PCS produced significant activation of C1 esterase, the contaminants produced an even stronger activation of C1 esterase (abs .33-.44). The OSCS preparations from different sources produce relatively weaker activation which was comparable to the starting material. The C1 esterase activation profile was not proportional to the kallikrein activation. The PCS, UFH, and contaminants along with the different OSCS preparations produced varying degrees of plasmin generation. UFH and PCS also produced significant generation of plasmin which was higher than their C1 esterase and prekallikrein generation. With the exception of PCS and UFH, all of the agents produced varying degrees of Xa and thrombin generation. The generation of Xa was higher by all of the OSCS preparations in comparison to the IIa generation. Interestingly each of the OSCS preparations and different contaminants exhibited their own specific protease generation profile. There was no correlation between the kallikrein formation and the generation of other enzymes. These data highly suggest that the molecular composition of OSCS's and the contaminants play a major role in the mediation of the pathophysiologic responses observed with the use of contaminated heparin. The presence of other glycosaminoglycans may influence the protease generation profile of contaminants present in recalled heparins. Moreover, the relative generation of these proteases may also be influenced by the pathologic predisposition of patients where the generation of these proteases may influence the overall pathophysiologic response. DisclosuresNo relevant conflicts of interest to declare.

Enhanced Cell-Associated Fibrinolytic Pathway but Not Coagulation Pathway Activity Contributes to Motility in Metastatic Breast Cancer Cells.

Abstract 3170Poster Board III-105Activation of tumor cell-associated coagulation and fibrinolytic pathways occurs in many different malignant and metastatic disease processes, including breast cancer. It has been proposed that coagulation and fibrinolytic pathway activation highjack host hemostatic mechanisms and facilitate the metastatic process. However, depending on tumor cell gene expression, tumor microenvironment, and tissue of origin, it is not fully understood whether the coagulation pathway or fibrinolytic pathway, alone or together in concert, contributes to the metastatic phenotype. To characterize and compare the coagulation and fibrinolytic pathways of normal and metastatic cells, we utilized the MCF-10 family of breast cell lines. The MCF-10 family of breast cell lines was originally derived from a woman with benign fibrocystic breast disease, which led to the near normal immortalized MCF-10A cells. After transformation with T24-Ha-ras, more aggressive cell lines were derived, ultimately leading to the metastatic MCF-10CA1 cell line. Using an in vitro modified-Boyden chamber model, MCF-10CA1 cells were significantly more motile than MCF-10A cells both at baseline using BSA as the chemoattractant, and when using EGF as the chemoattractant. Thus, as predicted by properties previously reported in the literature, the metastatic MCF-10CA1 cells were substantially more motile than the near normal parental cell line, MCF-10A. Both cell types supported similar rates of factor Xa generation, plasma thrombin generation, and fibrin formation. Using laser scanning confocal microscopy, we observed that MCF-10A cells produced a stable fibrin network, whereas MCF-10CA1 cells lysed the surrounding fibrin network within 24 hours of network formation. Importantly, fibrin located proximal to (within 10 microns of) the MCF-10CA1 cell surface lysed significantly faster than fibrin located 100 microns from the surface. These results suggest that the tumor-associated fibrinolytic pathway is a key distinguishing feature between the metastatic MCF10-CA1 cells and normal MCF-10A cells. A cell-surface plasminogen activation assay showed that MCF-10CA1 cells supported substantially increased plasmin generation rates compared to MCF-10A cells, providing a mechanism for the increased fibrinolytic activity of these cells towards the fibrin network. Metastatic MCF-10CA1 cells expressed significantly increased levels of urokinase (uPA) and decreased levels of plasminogen activator inhibitor-1 (PAI-1) compared to the MCF-10A cells. Blocking uPA activity with an active-site directed-inhibitor (amiloride) decreased MCF-10CA1 cell motility to essentially the same level as MCF-10A cells. Inhibiting the phosphatidylinositol 3-kinase/Akt signaling axis of MCF-10CA1 cells with LY294002 similarly decreased cell surface plasminogen activation activity and cell motility. Collectively, these results suggest that the tumor-associated fibrinolytic pathway is a key distinguishing feature between the metastatic MCF10-CA1 cells and normal MCF-10A cells. Our results support continued investigation of urokinase inhibition, either by directly blocking uPA activity or by down-regulating uPA expression, as an attractive adjunctive therapeutic target to reduce metastatic breast cancer. DisclosuresNo relevant conflicts of interest to declare.

Interactions of Factor VIII with Tissue Factor Contributes to the Acceleration of Factor Xa Generation in the Initiation Phase of Blood Coagulation.

Abstract 3177Poster Board III-116Activated factor (F)VII complex with tissue factor (FVIIa/TF) initiates the blood coagulation by generating FXa as extrinsic Xase complex (ex-Xase). Although FVIIa/TF also activates FIX, FIXa little functions without its cofactor, FVIIIa. A tiny amount of thrombin generated by FXa activates FV and FVIII, followed by forming of intrinsic Xase complex (in-Xase) and prothrombinase complex, respectively. These formations result in ‘thrombin burst' and successful hemostasis. Although thrombin is thought to be a unique potent activator of FVIII in vivo, FXa and FVIIa/TF also activate FVIII in vitro. We have recently reported the detailed mechanism by which FVIIa/TF activated FVIII more rapidly in early timed-phase than thrombin (Blood Abst.1036, 2008). In this study, we further developed to examine whether TF affected FVIII(a) function. (1) FVIIa/TF rapidly increased FVIII activity by 4.7-fold of initial in the presence of Ca2+ and phospholipid (PL), following by inactivation, in one-stage clotting assay. However, since even in the presence of TF alone, FVIII activity elevated by 1.8-fold of initial, actual increase of FVIII activity by FVIIa/TF was 2.6-fold. A possibility that TF might bind to FVIIa contained in FVIII-deficient plasmas used, was negligible, since FVIIa-inhibitor used blocked an ex-Xase effect >95%. In the presence of FVIIa-inhibitor, residue FVIII activity with TF was ∼50%, thus TF alone affected FVIII cofactor activity independently of FVIIa. (2) Using SDS-PAGE, the addition of TF accelerated FVIII cleavage by FVIIa, whilst decelerated that by thrombin and FXa. (3) Surface plasmon resonance-based assays showed that FVIII(a) directly bound to TF with high affinity (Kd; ∼3 nM). (4) The effect of FVIIa/TF on in-Xase was evaluated in FXa generation assay. 0.1 nM FVIIa/TF, 1 nM FVIII, 90 nM FIX and 20 μM PL were reacted with 150 nM FX at various combinations. FVIIa/TF and FVIIa/TF/FVIII/FIX generated FXa with 3.9 and 10.4 nM/min, respectively. When FVIIa-inhibitor was added prior to addition of FX, FXa generated by FVIIa/TF and FVIIa/TF/FVIII/FIX were 5% and 46% (0.2 and 4.8 nM/min) of those without FVIIa-inhibitor, respectively. The latter was considered as FXa generated by in-Xase. Therefore, FXa derived from in-Xase was ∼40% of total FXa in this condition. (5) FVIIIa/FIXa (1 nM/2 nM)-dependent FXa generation in the presence of TF was evaluated. FXa generation in the presence of TF (0.02 and 0.3 nM) increased by ∼2 and ∼6-folds, respectively, of that in its absence. Furthermore, the functional affinity of FVIIIa for in-Xase complex in the presence of TF (0.1 nM), showed an ∼1.5-fold greater than that in its absence (Km; 4.9 ± 0.4 and 7.1 ± 0.9 nM, respectively). In conclusion, FVIIa/TF can generate FVIIIa in early timed-phase in vitro as well as FXa and FIXa, and possess potential of forming in-Xase. In addition, TF directly binds to FVIII(a), and functions in-Xase complex more efficiently by enhancing the affinity of FVIIIa for in-Xase. Although TF-dependent these reactions may be terminated rapidly via anticoagulant systems such as tissue factor pathway inhibitor, our data suggest that interactions of FVIII with TF might contribute to the acceleration of FXa generation in the initiation phase of blood coagulation. DisclosuresOkuda:Sysmex Corporation: Employment.

Abstract 4939: Hypercholesterolemia Increases Microparticle Tissue Factor and Activates Coagulation in LDL Receptor Deficient Mice

Objective: Tissue factor (TF) is the initiator of the extrinsic pathway of coagulation and contributes to thrombosis. Previous studies demonstrated that the risk of arterial thrombosis is elevated in patients with increased plasma cholesterol concentrations. In addition, increased levels of circulating TF, bound to microparticles (MPs), may induce thrombosis. Therefore, we tested the hypothesis that hypercholesterolemia increases plasma levels of MP TF and activation of coagulation in low density lipoprotein receptor deficient (LDLr −/− ) mice. Methods and Results: Male LDLr −/− mice were fed a high fat diet (HFD) consisting of 21% saturated fat, and 0.15% cholesterol for 0, 1, 2, 4, and 12 weeks (n=3–7 each time point). Mice were then euthanized, blood was collected from the inferior vena cava (into 3.8% sodium citrate), and plasma prepared. Mice had a sustained elevation in plasma cholesterol concentrations after 1 week of feeding HFD, which were unchanged in all subsequent feeding intervals (945±35 mg/dL). MP TF was analyzed by detection of Factor Xa generation in the presence or absence of an inhibitory rat anti mouse TF antibody. MP TF activity increased in a time-dependent manner at 1 (75%), 2 (112%), 4 (650%), and 12 (1140%) weeks (all times versus 0 week control; P&lt;0.001). Importantly, a similar time-dependent increase in plasma thrombin-antithrombin (TAT) activity was observed at 2 (362%), 4 (1203%), and 12 (927%) weeks (all times versus 0 week control; P&lt;0.001). Moreover, the levels of MP TF activity and TAT were linearly correlated (R=0.70, P&lt;0.001). The inflammatory cytokine IL-6 was also increased in a time-dependent manner from 0 weeks (22.7±10.1 pg/ml) to 12 weeks (112.3±20.2 pg/ml; P&lt;0.023). Conclusion: These data indicate that hypercholesterolemia results in increased MP TF activity and subsequently activates coagulation. Further studies are required to determine if TF positive MPs are required for hypercholesterolemia -induced coagulation.

Identification of Residues in the 558-Loop of Factor VIIIa A2 Subunit That Interact with Factor IXa

Factor VIIIa is comprised of A1, A2, and A3C1C2 subunits. Several lines of evidence have identified the A2 558-loop as interacting with factor IXa. The contributions of individual residues within this region to inter-protein affinity and cofactor activity were assessed following alanine scanning mutagenesis of residues 555-571 that border or are contained within the loop. Variants were expressed as isolated A2 domains in Sf9 cells using a baculovirus construct and purified to >90%. Two reconstitution assays were employed to determine affinity and activity parameters. The first assay reconstituted factor Xase using varying concentrations of A2 mutant and fixed levels of A1/A3C1C2 dimer purified from wild type (WT), baby hamster kidney cell-expressed factor VIII, factor IXa, and phospholipid vesicles to determine the inter-molecular K(d) for A2. The second assay determined the K(d) for A2 in factor VIIIa by reconstituting various A2 and fixed levels of A1/A3C1C2. Parameter values were determined by factor Xa generation assays. WT A2 expressed in insect cells yielded similar K(d) and k(cat) values following reconstitution as WT A2 purified from baby hamster kidney cell-expressed factor VIII. All A2 variants exhibited modest if any increases in K(d) values for factor VIIIa assembly. However, variants S558A, V559A, D560A, G563A, and I566A showed >9-fold increases in K(d) for factor Xase assembly, implicating these residues in stabilizing A2 association with factor IXa. Furthermore, variants Y555A, V559A, D560A, G563A, I566A, and D569A showed >80% reduction in k(cat) for factor Xa generation. These results identify residues in the 558-loop critical to interaction with factor IXa in Xase.

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg(98) and Arg(101) residues, whereas plasmin only cleaved the propeptide downstream of Arg(101). Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn(109)-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation.

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.