Low Tissue Factor Research Articles

Early Vascular Alterations in SLE and RA Patients—A Step towards Understanding the Associated Cardiovascular Risk

Accelerated atherosclerosis represents a major problem in both systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients, and endothelial damage is a key feature of atherogenesis. We aimed to assess early endothelial changes in SLE and RA female patients (127 SLE and 107 RA) without previous CV events. Biomarkers of endothelial cell activation (intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), thrombomodulin (TM), and tissue factor (TF)) were measured and endothelial function was assessed using peripheral artery tonometry. Reactive hyperemia index (RHI), an indicator of microvascular reactivity, and augmentation index (AIx), a measure of arterial stiffness, were obtained. In addition, traditional CV risk factors, disease activity and medication were determined. Women with SLE displayed higher sICAM-1 and TM and lower TF levels than women with RA (p = 0.001, p<0.001 and p<0.001, respectively). These differences remained significant after controlling for CV risk factors and medication. Serum levels of vascular biomarkers were increased in active disease and a moderate correlation was observed between sVCAM-1 levels and lupus disease activity (rho = 0.246) and between TF levels and RA disease activity (rho = 0.301). Although RHI was similar across the groups, AIx was higher in lupus as compared to RA (p = 0.04). Also in active SLE, a trend towards poorer vasodilation was observed (p = 0.06). In conclusion, women with SLE and RA present with distinct patterns of endothelial cell activation biomarkers not explained by differences in traditional CV risk factors. Early vascular alterations are more pronounced in SLE which is in line with the higher CV risk of these patients.

Whole-Blood Thrombin Generation Monitored with a Calibrated Automated Thrombogram-Based Assay

The calibrated automated thrombogram (CAT) assay in plasma is a versatile tool to investigate patients with hypo- or hypercoagulable phenotypes. The objective was to make this method applicable for whole blood measurements. Thin-layer technology and the use of a rhodamine 110-based thrombin substrate appear to be essential for a reliable thrombin generation (TG) assay in whole blood. Using this knowledge we developed a whole blood CAT-based assay. We demonstrated that the whole blood CAT-based assay is a sensitive and rapid screening test to assess function of the hemostatic system under more nearly physiological conditions than the TG assay in plasma. Under conditions of low tissue factor concentration (0.5 pmol/L) and 50% diluted blood, the intraassay CV of the thrombogram parameters, endogenous thrombin potential and thrombin peak height, were 6.7% and 6.5%, respectively. The respective interassay CVs were 12% and 11%. The mean interindividual variation (SD) of 40 healthy volunteers was 633 (146) nmol · min/L for the endogenous thrombin potential and 128 (23) nmol/L for the thrombin peak. Surprisingly, erythrocytes contributed more than platelets to the procoagulant blood cell membranes necessary for optimal TG. Statistically significant (P < 0.001) and potentially clinically significant correlations were observed between circulating factor-VIII concentrations in blood of hemophilia A patients and endogenous thrombin potential (r = 0.62) and thrombin peak height (r = 0.58). We have developed a reliable method to measure TG in whole blood. The assay can be performed with a drop of blood and may provide a useful measurement of TG under more physiological conditions than plasma.

Abstract 264: Differential Profiles of Thrombin Inhibitors (Dabigatran, Hirudin, Bivalirudin and Heparin) in the Thrombin Generation Assay (TGA) and Thromboelastography (TEG) in Vitro

Thrombin is a central enzyme in haemostasis and thrombosis, and a proven target for anticoagulant therapies. Different classes of thrombin inhibitors, while exerting therapeutic benefits in most clinical trials, have different indications, dosing regimens, and bleeding complications. To gain more insight into the underlying mechanisms for their differential clinical profiles, we compared four marketed and representative agents, including dabigatran, hirudin, bivalirudin (direct thrombin inhibitors, DTIs), and heparin (an indirect thrombin inhibitor), in two in vitro spike-in assays with concentration titrations covering their therapeutic ranges. The two assays were the Thrombinoscope TGA with plasma, triggered by low tissue factor (1 nM TF), and TEG with whole blood, triggered by 1:8000 Recombiplastin (equivalent to low TF), with or without a threshold level of tPA to induce fibrinolysis. In TGA, the largest effect was prolongation of lag time, with the potency of the three DTIs rank-ordered as hirudin&gt;dabigatran&gt;bivalirudin; regarding peak, slope, and ETP, while complete inhibition was achieved with 1-2 μM dabigatran or hirudin, bivalirudin had no effect even at 4 μM, possibly due to its short half life in plasma. In TEG, the three DTIs prolonged clotting time (R) in the same rank order as TGA; for clot strength (MA), while all four agents reduced MA in synergy with tPA, only hirudin reduced MA without tPA, likely due to its highest potency. With tPA-induced fibrinolytic activity (Ly30), dabigatran and bivalirudin enhanced Ly30 (dabigatran&gt;bivalirudin), but hirudin and heparin did not. This contrast might involve differential access to clot-bound thrombin. Heparin had a steep dose-response curve for both lag time in TGA and R in TEG, which is in line with its very narrow therapeutic index. All three DTIs, but not heparin, displayed the previously reported paradoxical increase in peak and slope in TGA in the low concentration range, suggesting this is indeed a class effect of DTI. In summary, our observations highlight the distinct features of each agent in thrombin generation, coagulation, and fibrinolysis. These results in combination with known clinical properties are informative on efforts to define the optimal profiles of new anticoagulants.

Inducible expression of tissue factor in small-cell lung cancer: impact on morphology and matrix metalloproteinase secretion

Tissue factor (TF), the transmembrane receptor for factor VIIa (FVIIa), has key regulatory functions in coagulation as well as in tumour progression and metastasis. Small-cell lung cancer (SCLC) metastasises more aggressively than non-small-cell lung cancer (NSCLC). Previously, we described the transition of SCLC cell line H69 to adherent growth and TF expression. Here, we explored the differential expression of TF and its functional impact on morphology and matrix metalloproteinase (MMP) secretion. The constitutional TF expression was evaluated in a panel of established NSCLC and SCLC cell lines. Furthermore, in three stress-selected adherent SCLC H69 cells, TF and MMP expressions were determined by mRNA, protein, and activity measurements. RNA interference-mediated TF down-regulation and FVIIa stimulation were used to study the impact of TF on cellular functions. NSCLC cells expressed high TF antigen (median 3.75 ng/mg; range 0.31-65.2 ng/mg protein, n = 8), while SCLC expressed none or low TF (median 0.07 ng/mg; range 0-0.39 ng/mg protein, n = 6). However, selected H69 adherent cells markedly expressed TF (range: 4.8-44.3 ng/mg protein, n = 3) and secreted MMP-2 and MMP-9. FVIIa stimulated MMP-2 and MMP-9 secretion in H69adh cells, whereas TF down-regulation diminished MMP-2 and MMP-9 expression and promoted reversion to suspension growth. Our data show the significance of TF expression in the reversible growth phenotype of H69. Because TF, MMP expression, and adherence are highly relevant to cancer metastasis, this study suggests a novel mechanism of adaptation, thereby adding to the understanding of SCLC biology and its aggressiveness.

Peroxisome Proliferator-Activated Receptor-Gamma Agonists Suppress Tissue Factor Overexpression in Rat Balloon Injury Model with Paclitaxel Infusion

The role and underlying mechanisms of rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist, on myocardial infarction are poorly understood. We investigated the effects of this PPAR-γ agonist on the expression of tissue factor (TF), a primary molecule for thrombosis, and elucidated its underlying mechanisms. The PPAR-γ agonist inhibited TF expression in response to TNF-α in human umbilical vein endothelial cells, human monocytic leukemia cell line, and human umbilical arterial smooth muscle cells. The overexpression of TF was mediated by increased phosphorylation of mitogen-activated protein kinase (MAPK), which was blocked by the PPAR-γ agonist. The effective MAPK differed depending on each cell type. Luciferase and ChIP assays showed that transcription factor, activator protein-1 (AP-1), was a pivotal target of the PPAR-γ agonist to lower TF transcription. Intriguingly, two main drugs for drug-eluting stent, paclitaxel or rapamycin, significantly exaggerated thrombin-induced TF expression, which was also effectively blocked by the PPAR-γ agonist in all cell types. This PPAR-γ agonist did not impair TF pathway inhibitor (TFPI) in three cell types. In rat balloon injury model (Sprague-Dawley rats, n = 10/group) with continuous paclitaxel infusion, the PPAR-γ agonist attenuated TF expression by 70±5% (n = 4; P<0.0001) in injured vasculature. Taken together, rosiglitazone reduced TF expression in three critical cell types involved in vascular thrombus formation via MAPK and AP-1 inhibitions. Also, this PPAR-γ agonist reversed the paclitaxel-induced aggravation of TF expression, which suggests a possibility that the benefits might outweigh its risks in a group of patients with paclitaxel-eluting stent implanted.

The Utility of Thromboelastography As a Screening Test for Bleeding Disorders

Abstract 4352 Introduction:Accurate assessment of bleeding disorders requires a thorough clinical evaluation and appropriate laboratory testing process. These patients comprise a substantial proportion of consultations carried out by hematologists. The limitations of each screening test, and the heterogeneity of these disorders continue to make the diagnosis cumbersome for the practicing clinician. Plasma clotting times, such as the prothrombin time and activated partial thromboplastin time are the most frequently used screening tests to assess adequacy of hemostasis. Thromboelastography (TEG) lends the ability to assess hemostasis globally while also assessing the effects of platelets, leucocytes and red cells on coagulation. Aims:To determine the utility of TEG as an effective screening test for bleeding disorders Methods:Medical records of patients referred to Hematology Service from August 2006 through July 2011 were retrospectively reviewed after institutional review board approval. Results:One hundred ninety-five patients (125 females, 70 males: ages 0.1 to 20 years) were evaluated for a bleeding disorder based on either bleeding symptoms or abnormal routine clotting tests and had TEG performed with low dose tissue factor (1:190 000 concentration). Most common symptoms were epistaxis (83/195), bruising (67/195), menorrhagia (48/195), surgical bleeding (16/195). Twenty-nine patients were diagnosed with a bleeding disorder based on clinical evaluation and laboratory testing. Type 1 vonWillebrand disease (vWD) was diagnosed in 16 patients, heterozygous factor VII deficiency in 6 patients, factor XII deficiency in 3 patients, factor XI deficiency in 2 patients and platelet delta storage pool disease in 1 patient. One patient was a symptomatic hemophilia carrier.Preliminary analysis revealed that among the vWD patients, only 1 patient demonstrated abnormalities in all parameters of TEG [Prolonged Reaction time (R Time), k time (rate of clot formation) and decreased Maximum amplitude (MA)] and 2 showed prolonged R and k time without concomitant decrease in MA. In patients with heterozygous factor VII deficiency, only 2 of 4 patients showed prolonged R and k times. The symptomatic hemophilia carrier, 1 of 3 with factor XII and 1 of 2 patients with factor XI deficiency had prolonged R as their sole abnormality. TEG was completely normal in the patient with platelet delta storage pool disease. The sensitivity of the R time to diagnose a clotting factor (including low factor VIII with vWD) deficiency was only 58% with a specificity of 78%. R time correlated with PTT and PT in up to 50% (vWD: 12%, FVII deficiency: 33%, FXII: 33%, FXI: 50%) of the patients. R time was also prolonged in 46/166 (28%) patients without a definitive bleeding disorder, however 10 of these patients had a lupus anticoagulant. Conclusion:In our study of 195 patients referred for evaluation of bleeding symptoms or abnormal coagulation tests, TEG was of limited value in identifying congenital coagulation defects with both poor sensitivity and specificity. Future studies could examine different agonists or conditions for TEG that may improve its sensitivity for detection of congenital bleeding disorders. Disclosures:No relevant conflicts of interest to declare.

Factor XI Activation In Tissue Factor-Initiated Blood Coagulation

Abstract 2239 Introduction.In in vitro models of tissue factor (TF)-initiated coagulation, FXI activation has been linked to increased thrombin generation. However the effects of FXI in experimental models of normal hemostasis have often been subtle, prompting ongoing investigations to define contributing cofactors, potential collaborating activators and/or reaction conditions (e.g. low TF concentrations). In this study, predictions from a computational model of TF-initiated thrombin generation that includes thrombin dependent FXI activation are used to direct an investigation of the role of FXI in two empirical models of TF-initiated coagulation. Methods. FXI activation and FXIa interactions were computationally modeled by adding the appropriate sets of equations describing thrombin activation of FXI, FXIa activation of FIX, antithrombin inhibition of FXIa, and high molecular weight kininogen binding to FXI to the existing framework of differential equations. The efficacy of FXIa in promoting thrombin generation (α- thrombin-antithrombin, αTAT) was assessed via titration in contact pathway inhibited whole blood and compared to activation by TF, FIXa, FXa and α-thrombin. TF-initiated reactions and their resupply were performed as described previously (Orfeo T et al. J. Biol. Chem., 2008) in either contact pathway inhibited blood ± an inhibitory anti-FXI antibody) or in synthetic coagulation proteome (SCP) mixtures (± FXI), allowing FXI effects on both the TF dependent phase and the resulting procoagulant pool of catalysts to be evaluated. Results. The computational model (± FXI pathway) was validated by showing congruence between computational thrombin generation profiles initiated with 5 pM TF and corresponding SCP reconstructions, which showed the lag phase shortened by 30 to 60 s and maximum thrombin levels increased when FXI was present. Similarly there was good correspondence between computational and SCP thrombin generation when FXIa (4 pM by active site) was the initiator. A computational analysis provided the following ranking of effectiveness in initiating thrombin generation: FXIa>TF>FIXa>FXa>α-thrombin. When tested in contact pathway inhibited blood, addition of 5 pM FXIa (by active site) resulted in clot times and 20 min αTAT levels similar to those observed with 5 pM TF, while similar outcomes required 25 pM FIXa, 100 pM FXa or between 10–100 nM α-thrombin. The computational modeling made clear two consequences of FXI feedback activation. The first was mechanistic, demonstrating that the amplification of thrombin generation was achieved by better coordinating the initial activations of FIX and FVIII; limited early initiation phase activation of FXI (<0.01% zymogen activation) increases the amount of FIXa available during the activation of FVIII, yielding higher concentrations of intrinsic tenase earlier in the initiation phase. The second consequence was increased FIXa accumulation as the reaction proceeds. This prediction was confirmed in two ways: 1) Western blot analysis of SCP reactions containing FXI showed ∼20% consumption of FIX over 20 min compared to no detectable FIX consumption in reactions without FXI; and 2) Resupply of TF-initiated SCP reactions (± 30 nM FXI) demonstrated that both the FVIII dependence and the long-term stability of the observed re-initiation of thrombin generation depended on the presence of FXI. Resupply studies were then performed in contact pathway inhibited blood (N=4 individuals) by using an inhibitory anti-FXI antibody to negate FXI contributions to both the TF-initiated and resupply dynamics. No effect of blocking FXI function on the time courses of αTAT formation was observed in the TF initiated phase. Two of the four individuals showed attenuated αTAT formation upon resupply when FXI function was blocked from the onset of TF initiation. Conclusions. FXIa is a potent initiator of coagulation, and the computational and SCP analyses predict the potential for feedback activation by thrombin after TF initiation to somewhat enhance the propagation phase of thrombin generation and more dramatically enhance the resupply response. However in contact pathway inhibited blood, FXI contributions to Tf-initiated thrombin generation are not discernible and the effect on the resupply response is variable between individuals suggesting additional mechanisms suppressing FXI activation in blood. Disclosures:Mann:Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

Increased tissue factor pathway inhibitor activity is associated with myocardial infarction in young women: results from the RATIO study.

The tissue factor pathway inhibitor (TFPI)/protein S anticoagulant system is a potent inhibitor of blood coagulation. TFPI and protein S are major determinants of thrombin generation (TG) tests determined at low tissue factor (TF) and at high TF concentrations in the presence of activated protein C (APC). Both TFPI and protein S protect against venous thrombosis, but the importance of the TFPI/protein S system in arterial thrombosis remains unclear. To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. The RATIO study is a case-control study in women under 50 years of age, including 205 patients and 638 controls. TFPI and protein S were quantified using ELISA. The TFPI/protein S activity (nTFPIr) and the APC sensitivity ratio (nAPCsr) were determined using TG tests. Odds ratios (ORs) adjusted for putative confounders and corresponding 95% confidence intervals (95% CI) were determined. Women with MI had higher TFPI levels than controls (135.9 ± 40% vs. 124.2 ± 41%), resulting in increased TFPI/protein S activities and increased APC sensitivity. Furthermore, an increased TFPI activity was associated with MI [nTFPIr: adjusted OR Q1 vs. Q4 = 2.1 (95%CI 1.1-4.1)]. Additionally, an increased APC sensitivity was associated with MI [nAPCsr: adjusted OR Q1 vs. Q4 = 1.7 (95% CI 0.9-3.2)] Women with MI had increased TFPI levels compared with controls. Consequently, the TFPI/protein S activity and APC sensitivity are increased in women with MI. Whether this increase in TFPI activity acts as a compensating mechanism for an increased procoagulant state or is a marker of endothelial damage remains to be investigated.

Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice

AbstractStudies have correlated elevated plasma factor VIII (FVIII) with thrombosis; however, it is unclear whether elevated FVIII is a proinflammatory biomarker, causative agent, or both. We raised FVIII levels in mice and measured the time to vessel occlusion (TTO) after ferric chloride–induced injury. Compared with control (saline-infused) mice, elevated FVIII had no effect after longer (3-minute) carotid artery injury, but it shortened the TTO after shorter (2-minute) injury (P < .008). After injury, circulating thrombin-antithrombin (TAT) complexes were lower after short versus long injury (P < .04), suggesting short treatment produced less coagulation activation. TAT levels in FVIII-infused mice were higher than in controls after short, but not longer, injury. Accordingly, elevated FVIII had no effect on in vitro thrombin generation or platelet aggregation triggered by high tissue factor, but it increased thrombin generation rate and peak (2.4- and 1.5-fold, respectively), and it accelerated platelet aggregation (up to 1.6-fold) when initiated by low tissue factor. Compared with control mice, elevated FVIII stabilized thrombi (fewer emboli) after short injury, but it had no effect after longer injury. TTO and emboli correlated with TATs. These results demonstrate dependence of FVIII activity on extent of vascular injury. We propose elevated plasma FVIII is an etiologic, prothrombotic agent after moderate but not extensive vascular damage.

Protective Roles for Fibrin, Tissue Factor, Plasminogen Activator Inhibitor-1, and Thrombin Activatable Fibrinolysis Inhibitor, but Not Factor XI, during Defense against the Gram-Negative BacteriumYersinia enterocolitica

Septic infections dysregulate hemostatic pathways, prompting coagulopathy. Nevertheless, anticoagulant therapies typically fail to protect humans from septic pathology. The data reported in this work may help to explain this discrepancy by demonstrating critical protective roles for coagulation leading to fibrin deposition during host defense against the Gram-negative bacterium Yersinia enterocolitica. After i.p. inoculation with Y. enterocolitica, fibrinogen-deficient mice display impaired cytokine and chemokine production in the peritoneal cavity and suppressed neutrophil recruitment. Moreover, both gene-targeted fibrinogen-deficient mice and wild-type mice treated with the anticoagulant coumadin display increased hepatic bacterial burden and mortality following either i.p. or i.v. inoculation with Y. enterocolitica. Mice with low tissue factor activity succumb to yersiniosis with a phenotype similar to fibrin(ogen)-deficient mice, whereas factor XI-deficient mice show wild-type levels of resistance. Mice deficient in plasminogen activator inhibitor-1 or thrombin-activatable fibrinolysis inhibitor display modest phenotypes, but mice deficient in both plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor succumb to yersiniosis with a phenotype resembling fibrin(ogen)-deficient mice. These findings demonstrate critical protective roles for the tissue factor-dependent extrinsic coagulation pathway during host defense against bacteria and caution that therapeutics targeting major thrombin-generating or antifibrinolytic pathways may disrupt fibrin-mediated host defense during Gram-negative sepsis.

P.1.d.020 Sensitisation and tolerance-like effects of diazepam after repeated administration in rats

It has been demonstrated that tissue factor (TF) may be involved in the tumor-derived procoagulatory status and angiogenic modulation in certain solid tumors. In the present study, we examined immunohistochemical localisation of TF in esophageal squamous cell carcinomas (ESCC) from 103 patients. TF immunopositivity was found in 91.3% of all tumor sections, while normal esophageal tissues were immunonegative. Patients were divided into a low TF immunoreactivity group (9 cases of negative and 48 cases of weak positive) and a high TF immunoreactivity group (35 cases of moderate positive and 11 cases of strong positive). TF immunoreactivity was significantly correlated to the presence of distant metastasis (P=0.0014), while it was not correlated to patient's gender, age, tumor size, depth of tumor invasion or lymph node metastasis. Survival analysis revealed that the overall survival rate in the patients that had high TF immunoreactivity was significantly poorer than those with low TF immunoreactivity (P=0.0094). Univariate analysis demonstrated that tumor size (P=0.0095), depth of tumor invasion (P=0.0050), lymph node metastasis (P=0.0045) and distant metastasis (P<0.0001) were effective predictors of prognosis in patients. However, only distant metastasis could independently predict patients’ outcomes by the analysis of multivariate proportional hazards regression (P=0.0043). Furthermore, the intratumoral microvessel density (MVD), evaluated by CD34 immunolabeling, indicated that MVD was positively correlated to the TF immunoreactivity (P=0.0056). It is concluded that TF immunopositivity in ESCC tissues is strongly correlated to the intratumoral angiogenesis and to poor patient prognosis.

Characterization of Human Late Outgrowth Endothelial Progenitor-Derived Cells under Various Flow Conditions

Background: Endothelial progenitor-derived cells (EPC) are a cell therapy tool in peripheral arterial disease and for re-endothelialization of bypasses and stents. Objective: To assess EPC behavior under flow conditions normally found in vivo. Results: EPC were isolated from human cord blood, cultured on compliant tubes and exposed in an in vitro flow system mimicking hemodynamic environments normally found in medium and large arteries. EPC exposed for 24 h to unidirectional (0.3 ± 0.1 or 6 ± 3 dynes/cm²) shear stress oriented along flow direction, while those exposed to bidirectional shear stress (0.3 ± 3 dynes/cm²) or static conditions had random orientation. Under bidirectional flow, tissue factor (TF) activity and mRNA expression were significantly increased (2.5- and 7.0-fold) compared to static conditions. Under low shear unidirectional flow TF mRNA increased 4.9 ± 0.5-fold. Similar flow-induced increases were observed for TF in mature umbilical vein-derived endothelial cells. Expression of tissue-type plasminogen activator (t-PA), urokinase (u-PA) and monocyte chemotactic protein 1 (MCP1) were reduced by 40–60% in late outgrowth endothelial progenitor-derived cells (LO-EPC) exposed to any flow environment, while MCP1, but not t-PA or u-PA, was decreased in HUVEC. Conclusions: Flow, in particular bidirectional, modifies the hemostatic balance in LO-EPC with increased TF and decreased plasminogen activator expression.

Thrombin generation and activated protein C resistance in the absence of factor V Leiden correlates with the risk of recurrent venous thromboembolism in women aged 18–65 years

Identification of patients at high risk of recurrence after a first event of venous thromboembolism (VTE) remains difficult. Resistance to activated protein C (APC) is a known risk factor for VTE, but data on the risk of recurrence is controversial. We wanted to investigate whether APC resistance in the absence of factor V Leiden, determined with global coagulation test such as the thrombin generation assay, could be used as a marker for increased risk of recurrent VTE among women 18-65 years old after a first event of VTE. In a cohort of 243 women with a first event of VTE, plasma was collected after discontinuation of anticoagulant treatment and the patients were followed up for 46 months (median). Thrombin generation was measured via calibrated automated thrombography, at 1 pM and 10 pM of tissue factor (TF). In women without factor V Leiden (n=117), samples were analysed in the absence and in the presence of APC. Increase in ETP (endogenous thrombin potential) and peak height analysed in the presence of APC correlated significantly with higher risk of recurrence. At 1 pM, peak height correlated with increased risk of recurrence. In conclusion, high thrombin generation in the presence of APC, in women after a first event of VTE is indicative for an increased risk of a recurrence. We also found that thrombin generation at low TF (1 pM) is correlated with the risk of recurrence. Our data suggest that APC resistance in the absence of factor V Leiden is a risk factor for recurrent VTE.

Increased risk of gestational vascular complications in women with low free tissue factor pathway inhibitor plasma levels out of pregnancy

Tissue factor pathway inhibitor (TFPI) plays a crucial role in haemostasis by regulating TF-induced initiation of coagulation. Since it is expressed by endothelial and trophoblastic cells, TFPI is of particular importance at the placental level and might be involved in the occurrence of gestational vascular complications (GVC). In the present study, we investigated plasma free TFPI antigen in four groups of women: healthy non-pregnant women without history of pregnancy complications; women at the beginning (<12 weeks) and women during the third trimester of a normal pregnancy; women with late pregnancy complications (pre-eclampsia / HELLP syndrome, intra-uterine fetal death, fetal growth retardation) at the time of obstetrical event and/or at distance from pregnancy. In normal pregnancy, TFPI increased between first trimester and delivery (median 5.0 ng/ml vs. 7.1 ng/ml; p<0.0001) but remained lower than in non-pregnant controls (median 8.2 ng/ml; p<0.0001). In patients, when measured concomitantly to the obstetrical event, TFPI showed no difference with normal late pregnancy levels. In contrast, at distance from pregnancy, in the absence of any hormonal influence, TFPI was significantly lower than in non-pregnant controls (median 5.9 vs. 8.2ng/ml, p < 0.0001). After categorisation into quartiles, an inverse dose-effect relationship was demonstrated between TFPI categories recorded apart from pregnancy and GVC risk, with a crude odds ratio of 43.5 (95% confidence interval 8.2-230) for patients with TFPI values in the lowest quartile (< 5.7 ng/ml). In conclusion, low free TFPI at distance from pregnancy appears to be a strong indicator of GVC risk.

The role of fibrinogen: a new paradigm in the treatment of coagulopathic bleeding

Fibrinogen is involved in both primary and secondary hemostasis, playing an important role in platelet aggregation and the establishment of a fibrin network. Recent evidence suggests that very high levels of fibrinogen act as antithrombin and can reduce endogenous thrombin potential and compromise clot stability, particularly following a low tissue factor stimulus. Several laboratory methods for measuring plasma fibrinogen concentrations are available, but results vary depending on the type of method and the use of artificial colloid plasma expanders. Adopting only the Clauss method can provide erroneously high levels when used in patients who have received colloid plasma expanders. This may contribute to a hazardous delay or complete lack of treatment. Multiple in vitro experiments, animal studies, and proof-of-principle randomized, clinical studies have recently suggested that hemostatic intervention with a fibrinogen concentrate may be efficient and safe in controling perioperative bleeding. In particular, fibrinogen concentrate has a key role in improving clotting function and reducing blood loss in settings such as trauma and cardiothoracic surgery. However, prospective studies are needed to determine the clinical efficacy and safety of fibrinogen concentrate when used as a hemostatic intervention for patients with massive bleeding due to trauma or surgery.

Tissue factor as a novel marker for detection of circulating cancer cells

Tissue factor (TF) is a molecular marker that is up-regulated in cancer cells and aids tumoral dissemination. Our purpose was to develop a nested RT-PCR strategy against TF for detecting blood-borne tumour cells. Our method detected TF expression in a minimum of 1.5 pg total RNA from MCF7 cells. A preliminary study in blood samples from 16 advanced breast carcinoma patients showed that 80% of patients with high TF load progressed and died, while only 18% with low TF load showed the same behaviour. Kaplan-Meier analysis confirmed worse overall survival in patients with high TF load.

A New Procoagulant Mechanism Mediated by Fucoidans

AbstractAbstract 4420Fucoidans are heterogeneous, polyanionic molecules with procoagulant activities in a wide concentration range. They have been described as non-anticoagulant sulfated polysaccharides (NASP) and shown to improve clotting in FVIII- and FIX-deficient plasma. In vitro characterization has suggested that fucoidans exert their procoagulant activity by inhibiting tissue factor pathway inhibitor (Liu et al. Thromb Haemost 2006; 95:68) and by accelerating thrombin-dependent FVa formation (Mutch et al. J Thromb Haemost 2007; 5 Suppl2). In our study we describe a new, previously unrecognized mechanism by which fucoidans act as procoagulant agents. The procoagulant activity of several fucoidans was characterized by calibrated automated thrombography in tissue factor (TF)-dependent experiments and by using coagulation factor-deficient plasmas. Spiking experiments with purified coagulation factors or inhibitory antibodies verified the mechanism.Stimulation of thrombin generation (TG) by fucoidans requires anionic lipid surfaces like synthetic phospholipid vesicles which contain phosphatidylserine and is TF-dependent (0-20pM). However, stimulatory activity was most pronounced in the absence of TF. Control experiments with corn trypsin inhibitor or FXII-deficient plasma excluded any involvement of the contact system. Plasmas from patients with congenital coagulation factor deficiencies were screened for TG to identify the target coagulation factor by which fucoidans exert their procoagulant activities. In the absence of TF, plasmas deficient in coagulation factors from the common pathway do not support fucoidan-mediated thrombin generation, whereas FVII-deficient plasma does. FXI was identified as the most upstream factor of the intrinsic pathway which is required for fucoidan-stimulated thrombin generation, suggesting it to be the target for the procoagulant activities of fucoidan. Spiking 30nM FXI to FXI-deficient plasma restored fucoidan-mediated TG and addition of polyclonal FXI inhibitory antibodies to normal plasma abrogated TG. Fucoidan-dependent TG did not improve when FXIa (60pM) was added to FXI-deficient plasma, suggesting activation of FXI by fucoidan. The relevance of this mechanism in hemophilia A plasma was studied by addition of low levels of FVIII (0.2-10%) resulting in a FVIII concentration-dependent increase in fucoidan-mediated TG. These results highlight the requirement of a functional intrinsic pathway for this new mechanism of fucoidans. Our findings present FXI activation at low TF concentrations as a possible mechanism for fucoidan. Disclosures:Dockal:Baxter Innovations GmbH: Employment. Panholzer:Baxter Innovations GmbH: Employment. Hartmann:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Influence of the F12-4 C&gt;T polymorphism on hemostatic tests

The common F12 -4 C>T polymorphism significantly regulates plasma levels of FXII, the first element of the intrinsic pathway of coagulation. Due to the robust effects that this pathway has on blood coagulation in vitro, the objective of our study was to evaluate the influence of this polymorphism on different hemostatic tests. We studied 46 hemostatic parameters in 566 participants: 280 patients with mucocutaneous bleeding and 286 controls. The F12 -4T allele, associated with reduced levels of FXII (P < 0.001), also significantly delayed the activated partial thromboplastin time (aPTT) expressed as aPTTr (ratio sample plasma/normal pooled plasma). Thus, both patients and controls carrying the T allele had higher aPTTr than C/C homozygous individuals (P < 0.001). Interestingly, 92% of healthy controls who had prolonged aPTTr carried the F12 -4T allele. Moreover, individuals with the F12 -4T allele also had less thrombin generation (assessed by endogenous thrombin potential, thrombin peak and time to achieve the peak of thrombin) using a test with low tissue factor concentration and explicit contact phase activation. Finally, both patients and controls carrying the F12 -4T allele also displayed significantly lower FIXc and FVIIc levels than C/C individuals (P < 0.01). For all associations except for FVIIc, a gene-dosage effect was observed, and homozygous TT individuals had the farthest values. Our study reveals a significant effect of the F12 -4 C>T polymorphism on hemostatic tests widely used in routine clinical practice.

Thrombin generation in haemophilia A patients with mutations causing factor VIII assay discrepancy

Up to 40% of patients with mild haemophilia A have a discrepancy whereby factor VIII (FVIII) measurements by a two-stage chromogenic assay (FVIII:C(CH)) are disproportionately reduced compared with the FVIII one-stage clotting value (FVIII:C). Which assay best reflects the coagulation potential and clinical phenotype in this patient group is of clinical significance, yet remains unclear. We have assessed the global coagulant ability of haemophilia patients with FVIII assay discrepancy using calibrated automated thrombography (CAT). A total of 18 patients with mutations Arg531His/Cys or Arg698Trp causing FVIII discrepancy were investigated, together with 12 haemophilia patients with concordant FVIII values and 15 normal controls. Factor VIII levels in all patients and controls were measured using both one-stage clotting assay and two-stage chromogenic assay. Thrombin generation was assessed in platelet-poor plasma by CAT using a low tissue factor concentration (1 pm). FVIII:C(CH) values were below normal in all patients, and in the discrepant group were between 1.5- and 8-fold lower than FVIII:C values. CAT parameters were affected in all haemophilia patients. The endogenous thrombin potential (ETP) was reduced to 58-67% of the mean normal value (1301 nm min(-1)), whereas peak thrombin was further reduced to 27-30% of the mean normal value (178 nm) in both discrepant and concordant patient groups. Analysis of the discrepant patient group showed the most significant correlation between the one-stage FVIII:C assay and ETP (r(2) = 0.44) and peak thrombin parameters (r(2) = 0.27).

Porcine Endothelial Cells Cocultured with Smooth Muscle Cells Became ProcoagulantIn Vitro

Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts.