Factor Xa Production Research Articles

Spike Protein Subunits of SARS-CoV-2 Alter Mitochondrial Metabolism in Human Pulmonary Microvascular Endothelial Cells: Involvement of Factor Xa.

Mitochondria have been involved in host defense upon viral infections. Factor Xa (FXa), a coagulating factor, may also have influence on mitochondrial functionalities. The aim was to analyze if in human pulmonary microvascular endothelial cells (HPMEC), the SARS-CoV-2 (COVID-19) spike protein subunits, S1 and S2 (S1+S2), could alter mitochondrial metabolism and what is the role of FXA. HPMEC were incubated with and without recombinants S1+S2 (10 nmol/L each). In control conditions, S1+S2 failed to modify FXa expression. However, in LPS (1 μg/mL)-incubated HPMEC, S1+S2 significantly increased FXa production. LPS tended to reduce mitochondrial membrane potential with respect to control, but in higher and significant degree, it was reduced when S1+S2 were present. LPS did not significantly modify cytochrome c oxidase activity as compared with control. Addition of S1+S2 spike subunits to LPS-incubated HPMEC significantly increased cytochrome c oxidase activity with respect to control. Lactate dehydrogenase activity was also increased by S1+S2 with respect to control and LPS alone. Protein expression level of uncoupled protein-2 (UCP-2) was markedly expressed when S1+S2 were added together to LPS. Rivaroxaban (50 nmol/L), a specific FXa inhibitor, significantly reduced all the above-mentioned alterations induced by S1+S2 including UCP-2 expression. In HPMEC undergoing to preinflammatory condition, COVID-19 S1+S2 spike subunits promoted alterations in mitochondria metabolism suggesting a shift from aerobic towards anaerobic metabolism that was accompanied of high FXa production. Rivaroxaban prevented all the mitochondrial metabolic changes mediated by the present COVID-19 S1 and S2 spike subunits suggesting the involvement of endogenous FXa.

Inhibition of factor Xa activity, platelet aggregation, and experimentally induced thrombosis by Sparstolonin B

Background: Sparstolonin B (SsnB) is an isocumarin compound extracted from medicinal plants such as Sparganium stoloniferum and Scirpus yagara with well documented anti-inflammatory activity. Here we examined if SsnB also possesses antithrombotic activity and the underlying mechanisms. Methods: Anti-thrombotic effects of SsnB were determined by measuring in vitro/ex vivo/in vivo clotting times, platelet aggregation assay, production and activity of factor Xa, nitric oxide, and expressions of relative proteins. Results: Treatment with SsnB prolonged the clotting time of human platelet-poor serum at concentrations comparable to the clinical anticoagulant rivaroxaban (as a positive control) and inhibited human platelet aggregation induced by adenosine diphosphate (ADP) or the thromboxane A2 analog U46619. SsnB also inhibited U46619-induced and ADP-induced phosphorylation of phospholipase C (PLC)γ2/protein kinase C (PKC) and intracellular calcium mobilization, both of which are required for platelet aggregation. In addition, SsnB inhibited expression of the cell adhesion factors P-selectin and PAC-1. SsnB increased production of the vasodilator nitric oxide and suppressed secretion of the vasoconstrictor endothelin-1 from ADP- or U46619-treated human umbilical vein endothelial cells. Further, SsnB reduced coagulation factor Xa (FXa) catalytic activity and production by endothelial cells as well as FXa-induced platelet aggregation. Conclusion: Finally, SsnB injection reduced thrombus formation time, number, size, and related mortality in mouse models of thromboembolism. SsnB is a promising antithrombotic agent targeting both FXa and platelet aggregation pathways, which can overcome the side effects of existing antithrombotic agents.

Heparanase procoagulant activity in cancer progression

Heparanase is an endo-β-D-glucuronidase that is capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans on cell surfaces and the extracellular matrix. This activity is strongly implicated in tumor metastasis and angiogenesis. We have earlier demonstrated that apart of its well characterized enzymatic activity, heparanase may also affect the hemostatic system in a non-enzymatic manner. We showed that heparanase up-regulated the expression of the blood coagulation initiator-tissue factor (TF) and interacted with the tissue factor pathway inhibitor (TFPI) on the cell surface membrane of endothelial and tumor cells, leading to dissociation of TFPI and resulting in increased cell surface coagulation activity. Moreover, we demonstrated that heparanase directly enhanced TF activity, which led to increased factor Xa production and subsequent activation of the coagulation system. In patients with cancer, increased heparanase procoagulant activity appeared to be a potential predictor of survival. We have also shown that JAK-2 is involved in heparanase up-regulation via the erythropoietin receptor, a finding that may point to a new mechanism of thrombosis in JAK-2 positive patents with essential thrombocytosis. Recently, we found that the solvent accessible surface of TFPI-2 first Kunitz domain had a role in TF/heparanase complex inhibition. Peptides derived from TFPI-2 inhibitory site were shown to reduce coagulation activation induced by heparanase and to attenuate sepsis severity and tumor growth in a mouse model, without predisposing to significant bleeding tendency. These data imply that inhibition of heparanase procoagulant domain is potentially a good target for sepsis and cancer therapy.

JAK-2 V617F mutation increases heparanase procoagulant activity.

Patients with polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are at increased risk of arterial and venous thrombosis. In patients with ET a positive correlation was observed between JAK-2 V617F mutation, that facilitates erythropoietin receptor signalling, and thrombotic events, although the mechanism involved is not clear. We previously demonstrated that heparanase protein forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF) which leads to increased factor Xa production and subsequent activation of the coagulation system. The present study was aimed to evaluate heparanase procoagulant activity in myeloproliferative neoplasms. Forty bone marrow biopsies of patients with ET, PV, PMF and chronic myelogenous leukaemia (CML) were immunostained to heparanase, TF and TF pathway inhibitor (TFPI). Erythropoietin receptor positive cell lines U87 human glioma and MCF-7 human breast carcinoma were studied. Heparanase and TFPI staining were more prominent in ET, PV and PMF compared to CML. The strongest staining was in JAK-2 positive ET biopsies. Heparanase level and procoagulant activity were higher in U87 cells transfected to over express JAK-2 V617F mutation compared to control and the effect was reversed using JAK-2 inhibitors (Ruxolitinib, VZ3) and hydroxyurea, although the latter drug did not inhibit JAK-2 phosphorylation. Erythropoietin increased while JAK-2 inhibitors decreased the heparanase level and procoagulant activity in U87 and MCF-7 parental cells. In conclusion, JAK-2 is involved in heparanase up-regulation via the erythropoietin receptor. The present findings may potentially point to a new mechanism of thrombosis in JAK-2 positive ET patients.

Heparanase procoagulant activity, factor Xa, and plasminogen activator inhibitor 1 are increased in shift work female nurses.

Epidemiologic studies indicate on an increased risk of cardiovascular disease and cancer in shift workers, although the underlying mechanism is obscure. Heparanase directly enhances tissue factor (TF) activity leading to increased factor Xa production and subsequent activation of the coagulation system. In the present study, a comparison of coagulation markers among healthy shift working (SW) vs. healthy daytime working (DW) female nurses was performed. Thirty SW and 30 DW female nurses were enrolled. For each of the 60 participants, blood was drawn between 7:00 and 8:00 a.m. and at least 8 h after the last work shift. Plasma was studied for coagulation marker that included TF/heparanase procoagulant activity, TF activity, heparanase procoagulant activity, heparanase level, factor Xa level, plasminogen activator inhibitor 1 (PAI-1), plasminogen, α2-antiplasmin, fibrinogen, global protein C, von Willebrand factor, and D-dimer by chromogenic assays and enzyme-linked immunosorbent assays (ELISAs). Sleep quality was assessed by self-report according to the Pittsburgh Sleep Quality Index. The heparanase procoagulant activity increased by 2-fold and the TF/heparanase procoagulant activity increased by 1.5-fold in SW nurses compared to DW nurses (P < 0.05). Factor Xa levels and PAI-1 levels were significantly higher among SW nurses compared to the DW group (22 vs. 18 ng/ml, P < 0.05, and 32 vs. 22 ng/ml, P < 0.005, respectively). No significant differences were found in the other tested coagulation markers between the study groups. Heparanase procoagulant activity, factor Xa level, and PAI-1 level were significantly higher in SW nurses compared to the DW group. These alterations of blood coagulation activation may potentially contribute to cardiovascular and cancer morbidity.

In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia

AbstractSuccessful competition of activated factor VII (FVIIa) with zymogen factor VII (FVII) for tissue factor (TF) and loading of the platelet surface with FVIIa are plausible driving forces behind the pharmacological effect of recombinant FVIIa (rFVIIa) in hemophilia patients. Thrombin generation measurements in platelet-rich hemophilia A plasma revealed competition for TF, which potentially could reduce the effective (r)FVIIa:TF complex concentration and thereby attenuate factor Xa production. However, (auto)activation of FVII apparently counteracted the negative effect of zymogen binding; a small impact was observed at endogenous concentrations of FVII and FVIIa but was virtually absent at pharmacological amounts of rFVIIa. Moreover, corrections of the propagation phase in hemophilia A required rFVIIa concentrations above the range where a physiological level of FVII was capable to downregulate thrombin generation. These data strongly suggest that rFVIIa acts independently of TF in hemophilia therapy and that FVII displacement by rFVIIa is a negligible mechanistic component.

Heparanase and coagulation-new insights.

Heparanase, a β-D-endoglucuronidase abundant in platelets that was discovered 30 years ago, is an enzyme that cleaves heparan sulfate side chains on the cell surface and in the extracellular matrix. It was later recognized as being a pro-inflammatory and pro-metastatic protein. We had earlier demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. We had shown that heparanase up-regulated the expression of the blood coagulation initiator tissue factor (TF) and interacted with the tissue factor pathway inhibitor (TFPI) on the cell surface membrane of endothelial and tumor cells, leading to dissociation of TFPI and resulting in increased cell surface coagulation activity. Moreover, we have demonstrated that heparanase directly enhanced TF activity which led to increased factor Xa production and subsequent activation of the coagulation system. Recently, heparanase inhibitory peptides derived of TFPI-2 were demonstrated by us to inhibit heparanase procoagulant activity and attenuate sepsis in mouse models.

Heparanase multiple effects in cancer

Heparanase is an endo-β-D-glucuronidase that is capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Among these are the up-regulation of vascular endothelial growth factor (VEGF)-A, VEGF-C and activation of intra-cellular signaling involved in cell survival and proliferation. We had earlier demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. We had shown that heparanase up-regulated the expression of the blood coagulation initiator- tissue factor (TF) and interacted with the tissue factor pathway inhibitor (TFPI) on the cell surface membrane of endothelial and tumor cells, leading to dissociation of TFPI and resulting in increased cell surface coagulation activity. Moreover, we have demonstrated that heparanase directly enhanced TF activity which led to increased factor Xa production and subsequent activation of the coagulation system. Taking into account the prometastatic, pro-angiogenic and pro-coagulant functions of heparanase, over-expression in human malignancies and abundance in platelets, implies that heparanase is potentially a good target for cancer therapy.

The Procoagulant Envelope Virus Surface: Contribution to Enhanced Infection

Many virus types are covered by a lipid bilayer. This structure called an envelope, is derived from the host cell and includes host- and virus-encoded proteins. Because envelope components first interact with the host, it is the trigger for infection, immunity and pathology. The roles of especially host-derived constituents are poorly understood. Focusing on herpes simplex type 1 (HSV1) as a model, we have shown that the envelope acquires the physiological initiators of coagulation from the host cell; tissue factor (TF) and procoagulant phospholipid (proPL). Unlike resting cells, where TF and proPL accessibility is carefully restricted, their expression is constitutive on the purified virus enabling factor VIIa (FVIIa)-dependant factor Xa (FXa) and thrombin generation. Interestingly, HSV1-encoded glycoprotein C (gC) on the virus enhances FXa production. In addition to coagulation proteases, HSV1 also facilitates fibrinolytic plasmin generation. HSV1 TF and gC combine to optimally enhance cultured cell infection when both FVIIa and FXa are available through protease activated receptor (PAR) 2. Plasmin also increases infection through PAR2, whereas thrombin provides an additive effect via PAR1. Thus, depending on the host cell, TF and proPL may be a general feature of enveloped viruses, enabling coagulation protease activation and PAR-mediated effects on infection.

Procoagulant activity and cellular origin of microparticles in human amniotic fluid

Amniotic fluid contains various procoagulant factors involved in intro-vascular amniotic fluid-induced coagulopathies. During the progression of normal pregnancy, microparticles would be shed off from cells and accumulate in amniotic fluid over time. In this study, our aims were to investigate the cellular origin and procoagulant entity of these microparticles. Twenty amniotic fluid samples from healthy parturient women were collected, and the microparticles were isolated and stained with phycoerythrin-labeled antibodies to CD138, CD41a, CD144 and CD11b to identify their cellular origin. Their phosphatidylserine and tissue factor expression levels were quantified with fluorescein isothiocyanate-labeled annexin V and anti-tissue factor antibody staining. Their procoagulant activity was tested with plasma coagulation assay and factor Xase and prothrombinase assays. Phenotypic analysis showed 36.8% and 33.8% of amniotic fluid microparticles positive for CD138 and CD11b, respectively, indicating their epithelial cell or leukocyte origin. Of these microparticles, 66.3±5.9% expressed phosphatidylserine while 37.4±4.1% expressed tissue factor. In addition, amniotic fluid microparticles could significantly shorten the plasma coagulation time and increase the production of factor Xa and thrombin. Inhibition assays with annexin V and anti-tissue factor antibody confirmed the coagulation effects of amniotic fluid microparticles. The microparticles derived from epithelial and leukocytes may be a mechanism of amniotic fluid-induced coagulopathies.

Shiga toxin downregulates tissue factor pathway inhibitor, modulating an increase in the expression of functional tissue factor on endothelium

IntroductionEndothelial expression of tissue factor (TF) may play a major role in (Stx)-related hemolytic uremic syndrome. We examined human umbilical vein endothelial cell (HUVEC) monolayers to determine the interaction between TF and TF pathway inhibitor (TFPI), hypothesizing that changes in TFPI modulate TF expression. Materials and MethodsWe studied 1) cell surface expression of globotriasylceramide (Gb3, the receptor for Stx) with Stx-1 (10 pM), TNFα (20Ng/ml), or Stx-1 plus TNFα compared to control, 2) gene expression of TF and TFPI, 3) total cellular and cell surface antigenic TF and TFPI, 4) TFPI secretion into supernatant, and 5) factor Xa production. Results and ConclusionsGb3 expression, negligible with control and Stx-1 alone, increased significantly with TNFα and with Stx-1 plus TNFα. TF mRNA increased 1.25±0.32- fold (N=9; p=0.041) with Stx-1 alone vs. 2.82±0.92-fold (N=13; p<0.0005) with TNFα alone. However, Stx-1 plus TNFα yielded a 6.51±3.48-fold increase (N=17; p<0.0005). TFPI mRNA decreased with TNFα (p<0.001) and Stx-1 plus TNFα (p<0.0005). Total cellular and cell surface TF antigen increased significantly with TNFα, but no further with Stx-1 plus TNFα. Total TFPI cellular and cell surface antigen levels, and TFPI secretion decreased significantly with Stx-1 plus TNFα. Median factor Xa production for Stx-1 plus TNFα vs TNFα alone increased (p<0.001) 3.24-fold. Our results indicate that a subinhibitory concentration of Stx-1 plus TNFα impairs TFPI gene expression, synthesis, cell-surface association, and secretion, leading to augmented functional TF.

Shiga Toxin Upregulates Tissue Factor Gene Expression in and Functional Tissue Factor On Endothelium in the Presence of Complement Activation

Abstract 1075The pathophysiology of Shiga toxin (Stx)-related hemolytic uremic syndrome remains poorly understood. We hypothesize that tissue factor (TF) expression on vascular endothelium is central to the genesis of this disorder and is driven both by direct effects of Stx on endothelium activated by inflammatory cytokines derived from the action of Stx in the gastrointestinal tract, and by Stx-induced complement activation, which further augments TF expression. We used human umbilical vein endothelial cell monolayers to examine 1) gene expression of TF and TF pathway inhibitor (TFPI) with TNFα (20 ng/ml) ± Stx-1 (10 pM) compared to control, 2) total cellular and cell surface antigenic TF and TFPI, 3) TFPI secretion into supernatant, 4) factor Xa production, and 5) soluble levels of C5b-9 in complete media in response to 3–30 pM Stx-1 in the absence of cells. TF mRNA increased 2.82 ± 0.92-fold (N=13; p <0.0005) with TNFα alone vs. 1.25 ± 0.32-fold (N= 9; p =0.041) for Stx-1 alone (Fig. 1). TNFα plus Stx-1 yielded a 6.51 ± 3.48-fold increase (N=17; p <0.0005; Fig. 1). TFPI mRNA decreased with TNFα (p <0.001) andTNFα plus Stx-1 (p < 0.0005). Total cellular (p = 0.048) and cell surface (p = 0.027) TF antigen increased with TNFα, and no further with TNFα plus Stx-1. Total TFPI cellular (p = 0.0017) and cell surface (p = 0.030) antigen levels, and secretion of TFPI (p = 0.018) decreased with TNFα plus Stx-1. Median factor Xa production for TNFα plus Stx-1 vs. TNFα alone increased (p < 0.001) 3.24-fold. In the absence of cells, M199 with 10% human serum to which was added Stx-1 yielded levels of C5b-9 of 648 and 1102 ng/ml vs. 106 and 313 ng/ml in the presence of 25 mM EDTA (to block the classical and alternative pathways of complement), and intermediate levels in the presence of 15 mM EGTA and 7.5 mM Mg++ (to block the classical pathway). Similar results were found with M199 with 10% human plasma, collected using 0.2 U/ml FC in media of dalteparin. C5b-9 levels increased between 3 and 10 pM Stx-1, but did not increase further at 30 pM Stx-1 (Fig. 2). Finally, by means of immunofluorescence we also detected and measured C5b-9 on fixed cell monolayers, whether treated prior to fixation with TNFα alone or TNFα plus Stx-1. We conclude that Stx-1 augments functional TF on endothelium by increasing TF mRNA, and impairing synthesis, cell-surface association, and secretion of TFPI, while at the same time activating both the alternative and classical complement pathways. These findings support the development of therapeutic approaches to epidemic HUS that incorporate inhibitors of the TF pathway, inhibitors of the alternative and classical complement pathways, or both. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

PL-14 Heparanase procoagulant activity

Heparanase that was cloned from and is abundant in the placenta is implicated in cell invasion, tumor metastasis and angiogenesis. We have recently demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. Heparanase was shown to up-regulate tissue factor (TF) expression and interact with tissue factor pathway inhibitor (TFPI) on cell surface, leading to dissociation of TFPI from cell membrane of endothelial and tumor cells, resulting in increased cell surface coagulation activity. We have lately shown that heparanase directly enhances TF activity resulting in increased factor Xa production and activation of the coagulation system. Data indicate increased plasma levels of heparanase suggesting its possible involvement in pregnancy vascular complications. Elevation in heparanase levels and procoagulant activity was also documented in orthopedic surgery patients receiving prophylactic doses of enoxaparin. Taking into account the pro-metastatic and pro-angiogenic functions of heparanase, with over-expression in human malignancies and abundance in platelets and placenta, its involvement in the coagulation machinery is an intriguing novel platform for further research.

Heparanase – A Link Between Coagulation, Angiogenesis and Cancer

Heparanase that was cloned from and is abundant in the placenta is implicated in cell invasion, tumor metastasis, and angiogenesis. Recently we have demonstrated that heparanase may also affect the hemostatic system in a non-enzymatic manner. Heparanase was shown to up-regulate tissue factor (TF) expression and interact with tissue factor pathway inhibitor (TFPI) on the cell surface, leading to dissociation of TFPI from the cell membrane of endothelial and tumor cells, resulting in increased cell surface coagulation activity. More recently, we have shown that heparanase directly enhances TF activity, resulting in increased factor Xa production and activation of the coagulation system. Data indicate increased levels and possible involvement of heparanase in vascular complications in pregnancy. Taking into account the prometastatic and proangiogenic functions of heparanase, overexpression in human malignancies, and abundance in platelets and placenta, its involvement in the coagulation machinery is an intriguing novel arena for further research.

Heparanase Activation of the Coagulation System in a Mice Sepsis Model

Abstract 378 Background:Heparanase that is abundant in platelets and neutrophils is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is directly involved in the regulation of the hemostatic system. Heparanase was shown to form a complex and enhance tissue factor (TF) activity, resulting in increased factor Xa production (Nadir et al, Haematologica, 2010). In the present study, the effect of heparanase on sepsis was investigated. Methods: ICR mice, heparanase knock-out mice and heparanase over-expression mice were studied. Sepsis was induced by intra-peritoneal injection of lipopolysaccharides (LPS). Four hours after injection, blood was obtained via temple plexus puncture followed by mice sacrificing. Levels of thrombin-antithrombin (TAT), D-Dimer and IL-6 were tested using ELISA. Plasma factor VII levels were evaluated using the Western blot analysis and fibrinogen levels in the lung, kidney and spleen were estimated by immunostaining. Results: Intra-peritoneal injection of heparanase (0.5μg/mg) compared to PBS injection, increased the TAT (p<0.05) and D-Dimer (p<0.05) levels, which were similar to the ones induced by LPS (5μg/mg), although the IL-6 levels did not rise and mice did not show signs of illness. Moreover, heparanase injection half an hour following LPS administration resulted in reduced levels of TAT, D-Dimer, fibrinogen and of factor VII (p<0.001), increased levels of IL-1 (p<0.001) and profuse blood leak from puncture, compatible with severe disseminated intravascular coagulation (DIC). Similarly, injection of LPS to heparanase knock-out mice led to lower levels of TAT (p<0.001), D-Dimer (p<0.001) and IL-6 (p<0.001) compared to control mice, while injection of LPS to heparanase over-expression mice resulted in DIC. Conclusions: Heparanase is found to induce activation of the coagulation system without inflammatory response. It also contributes to laboratory enhancement of sepsis resulting in DIC. Inhibitors of heparanase may potentially attenuate morbidity and mortality in sepsis. Disclosures:No relevant conflicts of interest to declare.

An assay to evaluate heparanase procoagulant activity

BackgroundHeparanase that was cloned from and is abundant in the placenta is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is directly involved in the regulation of the hemostatic system. Heparanase was shown to form a complex and enhance tissue factor (TF) activity, resulting in increased factor Xa production (Nadir et al. Haematologica, 2010). The present work suggests a novel assay to evaluate heparanase procoagulant activity. MethodsHeparanase procoagulant activity was studied using purified proteins of heparanase, TF, factor VIIa and factor X. The assay was verified in 55 plasma samples and compared to heparanase and tissue factor pathway inhibitor (TFPI) levels by ELISA and factor Xa, thrombin levels and antithrombin activity by chromogenic substrates. Thirty five samples were of third-trimester pregnant women (weeks 36–41) who were in labor or came for appointed elective cesarean section and 20 control samples were of non-pregnant healthy women. Resultsheparanase procoagulant activity assay was shown to differentiate heparanase procoagulant effect from TF activity, in purified proteins. Heparanase procoagulant activity was significantly higher in the plasma of pregnant women compared to non-pregnant (p<0.005). Heparanase relative contribution to the TF / heparanase complex activity was significantly higher in the plasma of pregnant women compared to non-pregnant (29% increase, p<0.0001). Differences in heparanase procoagulant activity were more prominent than changes in heparanase levels by ELISA, TF activity, factor Xa, thrombin and free TFPI levels. ConclusionsHeparanase procoagulant activity can be determined by the suggested assay. The assay revealed a significant contribution of heparanase to the procoagulant state in late third-trimester pregnancy and at delivery.

Activation of Protease-Activated Receptors 3 and 4 Accelerates Tissue Factor–Induced Thrombin Generation on the Surface of Vascular Smooth Muscle Cells

To determine factors regulating human aortic smooth muscle cells (HASMC) supported tissue factor-induced thrombin generation. The addition of nonlipidated tissue factor and Ca(2+) to HASMCs maintained in reptilase-treated platelet-poor plasma resulted in the robust formation of thrombin after a lag phase of approximately 6 minutes. Pretreatment with low concentrations of α-thrombin before the addition of tissue factor and Ca(2+) accelerated the rate of thrombin generation (time to reach half of peak thrombin was reduced by [mean ± SD] 42.0 ± 2.2%; P<0.05) but had no effect on the amount of peak thrombin generated. Protease-activated receptor (PAR) 3 activating peptides (APs) or PAR-4 APs accelerated thrombin generation without affecting peak thrombin levels (time to half of peak thrombin decreased by 17.4 ± 5.6% and 21.7 ± 3.5%; P<0.05 with PAR-3 AP and PAR-4 AP, respectively). The addition of PAR-3 AP and PAR-4 AP together had an additive effect, with a reduction in time to half of peak thrombin of 43.9 ± 4.0%. PAR-3 AP or PAR-4 AP enhanced tissue factor-induced factor Xa production and phosphatidylserine exposure on the surface of HASMCs. PAR-1 activation had no effect on thrombin generation, factor Xa production, or phosphatidylserine exposure. Low concentrations of α-thrombin accelerate tissue factor-induced thrombin generation on the surface of HASMCs, and this effect is mediated by PAR-3 and PAR-4.

Recombinant Factor VIIa in Cirrhosis

Purpose: A 49-year-old woman with newly diagnosed cirrhosis was admitted to outside hospital with massive hematochezia. She received 13 units of blood transfusion and 7 units of fresh frozen plasma. Her EGD there showed non-bleeding esophageal varices and portal hypertensive gastropathy, and her colonoscopy showed two small non-bleeding ulcers in sigmoid colon and rectum. Etiology of her hematochezia remained unclear. She was transferred for further management. Repeat EGD and colonoscopy showed same findings. Tagged-RBC scan was negative. Over the next 24h, she received another 7 units of blood transfusion and other blood products. She was deemed not a surgical candidate. Due to recurrent hemorrhage, the patient received one dose of recombinant factor VIIa (rFVIIa), resulting in complete resolution. Four days later, she developed abdominal pain and lower extremity swelling. CT scan showed pneumatosis intestinalis suggestive of ischemic bowel. Doppler ultrasound showed bilateral deep vein thromboses. MRA showed 50% narrowing of celiac axis with visualization of only the proximal superior mesenteric artery but not the inferior mesenteric artery. rFVIIa is approved for use in bleeding episodes due to hemophilia A or B and factor VII deficiency. It enhances thrombin generation by increasing factor Xa production or direct binding to activated platelets. rFVIIa is used off-label in other emergencies, including intracerebral hemorrhage and bleeding complications due to surgery or trauma, gynecological disease, anticoagulation use, thrombocytopenia, or liver failure. There is limited data on efficacy and safety of rFVIIa use in cirrhotic patients. Studies have shown its ability to normalize prothrombin time in variceal hemorrhage, prevent bleeding in cirrhotic patients undergoing liver biopsies, and reduce transfusion requirement in orthotopic liver transplantation. However, recent study shows no reduction of mortality in variceal hemorrhage in patients with Child-Pugh class B or C at 24h. Consensus European Guidelines do not recommend use of rFVIIa in patients with Child-Pugh class A and indicate uncertain efficacy in patients with Child-Pugh class B or C. Cochrane Review shows no evidence of reduced risk of death in patients with liver disease and upper gastrointestinal tract hemorrhage. Thromboembolic complications, including strokes, myocardial infarctions, and deep vein thromboses, have been reported, but without increased occurrence rate. This case demonstrates the dilemma in management of decompensated cirrhotic patients with uncontrolled gastrointestinal hemorrhage and potential complications associated with rFVIIa use.

Development of an inhibitory antibody fragment to human tissue factor using phage display technology

AbstractTissue factor is involved in the etiology of thrombotic diseases initiating the thrombosis associated with the inflammation that occurs during infection. The prevention of blood coagulation and inflammation is of primary importance in a number of pathological situations. A single‐chain variable antibody fragment of molecular weight of 26 kD that inhibits the action of human tissue factor was selected by phage display technology, purified and tested for its tissue factor inhibitory effect, purified on a protein A column, and its purity evaluated on SDS‐PAGE. The effects of the antibody fragment on prothrombin times, Factor Xa production, and thrombin generation were assessed with increasing fragment concentrations, using chromogenic and fluorometric substrates. The antibody fragment dose‐dependently prolonged the prothrombin time (IC50=0.5 μM) and delayed the lag phase before the thrombin generation burst and the peak thrombin concentration in the thrombin generation assay. The effect on thrombin generation was more pronounced in thrombophilic plasma than in normal plasma. Antibody‐based tissue factor inhibitors therefore may provide an effective treatment for thrombotic disease without serious bleeding complications. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.

Structural Determinants of Factor IX(a) Binding in Nitrophorin 2, a Lipocalin Inhibitor of the Intrinsic Coagulation Pathway

Nitrophorin 2 (NP2) is a salivary lipocalin from Rhodnius prolixus that binds with coagulation factors IX (fIX) and IXa (fIXa). Binding of NP2 with fIXa results in potent inhibition of the intrinsic factor Xase complex. A panel of site-directed surface mutants of NP2 was generated to locate determinants of high affinity fIX(a) binding. The locations of the mutations were based on comparisons with the related, but less potent, inhibitor nitrophorin 3 (NP3). Three point mutants (K21A, K92A, and V94A) were found that clearly reduced the inhibitory potency as measured by the activity of a reconstituted factor Xase system. Binding of NP2 with fIXa and fIX as measured by surface plasmon resonance and isothermal titration calorimetry was reduced in a similar manner. Of the three mutants, two (K92A and V94A) were located on the loop connecting beta-strands E and F of the lipocalin beta-barrel. The largest changes were seen with the K92A mutation, which lies at the apex of the loop, with a smaller effect being seen with mutation of Val(94). Combination of four E-F loop mutations (K92A, A93K, V94A, E97A) in a single mutant reduced the inhibitory potency and binding to levels similar to those seen with NP3 without affecting heme or histamine binding.