Tissue Factor Injection Research Articles

S62798, a TAFIa inhibitor, accelerates endogenous thrombolysis in a murine model of pulmonary thromboembolism

Abstract Pulmonary embolism (PE) is the third leading cause of cardiovascular death in western countries. The enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. In patients, venous thrombosis and thromboembolism risks are associated with increased plasma levels of TAFI (Thrombin Activatable Fibrinolysis Inhibitor) antigen as well as the active form TAFIa. S62798 is a competitive, selective and potent human TAFIa inhibitor (IC50±SD=11.2±0.4nM). It is however less potent on mouse TAFIa (IC50±SD=270±39nM). Here, we tested the ability of S62798 to enhance endogenous fibrinolysis in a mouse model of pulmonary thromboembolism. Human Tissue Factor (TF) was injected in C57Bl6 male mice. Ten minutes later, mice (n=4 to 14 per group) were treated (IV) with S62798 (from 0.01 to 100mg/kg) or vehicle (0.9% NaCl). Ten or twenty minutes (min) later, mice were anesthetized and lungs were collected, homogenized and pulmonary fibrin was quantified by ELISA. Results are expressed as ratio of geometric mean of pulmonary fibrin (μg/mL): tested treatment/ vehicle [95% confidence interval (CI)]. Ten minutes after S62798 treatment, pulmonary fibrin deposition was dose-dependently decreased with a Minimal Effective Dose of 0.04mg/kg [90% prediction interval 0.037 - 0.051] and an ED50 of 0.03mg/kg [95% CI: 0.01; 0.06]. Mice were then treated with 0.1mg/kg S62798 or vehicle (10 min after TF induction) and fibrin deposition in lungs was quantified 10 and 20 minutes post S62798 treatment. The level of pulmonary fibrin deposition was significantly decreased (p<0.0001) compared to vehicle group (ratio 0.31 [0.21; 0.45] at 10 min; 0.35 [0.24; 0.51] at 20 min). Finally, the effect of S62798 (1mg/kg) in combination with heparin was evaluated (n=10/group). When administered 10 min before TF injection, heparin (2000IU/kg) significantly (p<0.0001) decreased pulmonary fibrin level (20 min post TF: ratio 0.03 [0.01; 0.05]). When treatment was done in a curative setting (10 min post TF), heparin alone had no effect (p=0.85) on fibrin deposition (ratio 0.96 [0.65; 1.43]) whereas a similar significant (p<0.0001) decreased pulmonary fibrin deposition was observed in response to S62798 alone or associated with heparin (ratio 0.27 [0.18; 0.40] (S62798 alone) and 0.29 [0.20; 0.43] (S62798+heparin)). In this model, curative S62798 treatment, alone or associated to heparin, accelerated clot degradation by potentiating endogenous fibrinolysis and thus decreased pulmonary fibrin deposition. Due to its capacity to enhance endogenous fibrinolysis, S62798, which has completed phase I studies, is expected to be a therapeutic option for intermediate high risk PE patients on top of anticoagulants. With early recanalization, S62798 should rapidly reduce pulmonary artery pressure and resistance, with concomitant improvement in right ventricular function, preserving cardiac function, and reducing acute PE-related morbidity and mortality in these patients. Funding Acknowledgement Type of funding source: None

Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor

AbstractThe intrinsic tenase complex (FIXa-FVIIIa) of the intrinsic coagulation pathway and, to a lesser extent, thrombin-mediated activation of FXI, are necessary to amplify tissue factor (TF)-FVIIa–initiated thrombin generation. In this study, we determined the contribution of murine FIX and FXI to TF-dependent thrombin generation in vitro. We further investigated TF-dependent FIX activation in mice and the contribution of this pathway to hemostasis. Thrombin generation was decreased in FIX- but not in FXI-deficient mouse plasma. Furthermore, injection of TF increased levels of FIXa-antithrombin complexes in both wild-type and FXI−/− mice. Genetic studies were used to determine the effect of complete deficiencies of either FIX or FXI on the survival of mice expressing low levels of TF. Low-TF;FIX−/y male mice were born at the expected frequency, but none survived to wean. In contrast, low-TF;FXI−/− mice were generated at the expected frequency at wean and had a 6-month survival equivalent to that of low-TF mice. Surprisingly, a deficiency of FXI, but not FIX, exacerbated the size of blood pools in low-TF placentas and led to acute hemorrhage and death of some pregnant dams. Our data indicate that FIX, but not FXI, is essential for survival of low-TF mice after birth. This finding suggests that TF-FVIIa–mediated activation of FIX plays a critical role in murine hemostasis. In contrast, FXI deficiency, but not FIX deficiency, exacerbated blood pooling in low-TF placentas, indicating a tissue-specific requirement for FXI in the murine placenta under conditions of low TF.

OC-04 - Tissue factor positive microvesicles activate platelets in vitro and in vivo and enhance thrombosis in mice

Cancer patients have a 4- to 7- fold increased risk of venous thromboembolism (VTE) compared with general population. Most tumor cells express tissue factor (TF) and constitutively release small membrane microvesicles called tumor microvesicles (TMVs). Clinical studies have shown that circulating MP-TF activity is associated with VTE in pancreatic cancer but not in other types of cancer. Thrombin is a potent platelet agonist and activates platelets via protease activated receptors (PARs). To determine the contribution of the TF+ TMV-thrombin-platelet pathway to cancer-associated thrombosis. A human pancreatic adenocarcinoma cell line expressing high levels of TF (BxPc-3) was selected to study the effect of TF+ TMVs on platelet activation and thrombosis. TF+ TMVs induced platelet activation in vitro in a thrombin-dependent manner. The presence of orthotopically grown BxPc-3 tumors in mice was associated with increased levels of thrombin-antithrombin III complexes (TATc) and larger thrombi in an inferior vena cava stenosis model compared with control mice. Furthermore, injection of BxPc-3 TF+ TMVs into mice triggered platelet activation and enhanced venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and wild-type mice treated with the platelet inhibitor clopidogrel, suggesting that platelet activation was required for the enhanced thrombosis. These studies suggest that platelet inhibitors may reduce thrombosis in cancer patients with elevated levels of TF+ TMVs.

Effect of hydrogen sulfide on tissue factor-induced disseminated intravascular coagulation in rabbits

To investigate the protective effect of hydrogen sulfide (H2S) on tissue factor-induced disseminated intravascular coagulation (DIC) in rabbits and its mechanism. Thirty-two healthy rabbits were randomly divided into four groups: normal control group, NaHS control group, DIC model group, NaHS pretreatment group (each, n = 8). Ten minutes before model reproduction, rabbits in NaHS control and pretreatment groups were given 3.4 mg/kg NaHS (dissolved in normal saline to 5 mL) via ear vein, while rabbits in normal control and DIC model groups were given an equivalent volume of normal saline. Ten minutes later, rabbits in DIC model and NaHS pretreatment groups were intravenously given tissue factor (TF) 2 mL/kg (dissolved in normal saline to 30 mL, at the speed of 1 mL/min for 5 minutes, 2 mL/min for 5 minutes, and 3 mL/min for 5 minutes), and rabbits in normal control and NaHS control groups were given normal saline. 3 mL of blood was collected 10 minutes before TF injection, and 3, 5, 8, 10, 13, 15, 45, 85, 135 minutes after TF injection for determination of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen content (FIB), fibrin degradation products (FDP), and platelet count (PLT). Microcirculation in the mesentery was also observed under microscope. Compared with normal control group, PT and APTT became shorter at 5 minutes after TF injection, and the rate of their change was increased [PT: -8.3 (-11.7 to -5.3)% vs. 1.3 (-2.5 to 3.8)%, P < 0.01; APTT: -19.1 (-30.4 to -9.4)% vs. -2.6 (-6.2 to 3.0)%, P < 0.05]. PT and APTT were prolonged 15 minutes after TF injection, and their changes were more significant [PT: 31.0 (25.0 to 36.9)% vs. -1.3 (-6.3 to 5.0)%, APTT: 61.3 (50.0 to 72.9)% vs. 0.0 (-10.0 to 10.0)%, both P < 0.01] in DIC model group. TT was gradually reduced after TF injection, FIB and PLT were gradually decreased, and their changes were more obvious at 15 minutes in DIC model group compared with those in normal control group [TT: -9.5 (-12.0 to -6.2)% vs. -2.0 (-4.0 to 0.7)%, FIB: -4.3 (-9.9 to -2.2)% vs. -1.0 (-5.8 to 4.3)%, PLT: -90.0 (-93.4 to -86.5)% vs. -1.0 (-3.9 to 2.6), all P < 0.01]. After TF injection, it appeared latex-like particles in FDP test board, and it was gradually increased within 3-15 minutes, and then it gradually became less marked. The rate of blood flow in mesenteric capillaries was decreased obviously within 10 minutes, and it became faster accompanying with obvious hemorrhage. PT and APTT in NaHS pretreatment group became shortened 5 minutes after TF injection, and their rate of change was significantly decreased compared with that of DIC model group [PT: -6.3 (-8.6 to 0.0)% vs. -8.3 (-11.7 to -5.3)%, APTT: -13.6 (-24.2 to -2.3)% vs. -19.1 (-30.4 to -9.4)%, both P < 0.05], and prolonged at 15 minutes, and their rate of change was significantly decreased compared with that of DIC model group [PT: 10.1 (3.8 to 15.2)% vs. 31.0 (25.0 to 36.9)%, P < 0.01; APTT: 27.8 (-15.8 to 39.7)% vs. 61.3 (50.0 to 72.9)%, P < 0.05]. TT, FIB, and PLT were reduced at 15 minutes in NaHS pretreatment group, and their rate of change was markedly decreased compared with that of DIC model group [TT: -4.5 (-7.8 to -1.3)% vs. -9.5 (-12.0 to -6.2)%, P < 0.01; FIB: -3.3 (-8.0 to 1.9)% vs. -4.3 (-9.9 to -2.2)%, P < 0.05; PLT: -58.8 (-53.0 to 64.0)% vs. -90.0 (-93.4 to -86.5)%, P < 0.01]. The rate of agglutination of latex particles in NaHS pretreatment group was decreased significantly at each time point compared with DIC model group; mesenteric capillary blood flow slowed down gradually within 10 minutes, but it was faster as compared with the DIC model group. It became faster later, but bleeding was obviously less. These results show that H2S protects against TF-induced DIC by inhibiting the activity of coagulation system and platelet aggregation.

Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats

There are concerns that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. We have shown that low-dose administration of a direct thrombin inhibitor, melagatran, significantly worsens the coagulation status induced by tissue factor injection in rats. We compared the effect of inhibition of thrombin and factor Xa for their potential to aggravate tissue factor-induced coagulation in rats. Hypercoagulation was induced by the injection of 2.8 U/kg tissue factor after administration of melagatran, heparin and edoxaban in rats. Blood samples were collected 10min after tissue factor injection. Platelet numbers, thrombin–antithrombin complex concentrations and plasma compound concentrations were measured. Though a high dose of melagatran (1mg/kg, i.v.) suppressed platelet consumption and thrombin–antithrombin complex generation induced by tissue factor, lower doses of melagatran (0.01, 0.03 and 0.1mg/kg, i.v.) significantly enhanced platelet consumption and thrombin–antithrombin complex generation. In addition, although melagatran (3mg/kg, i.v.) improved coagulation status when tissue factor was given 5min after the drug administration, and 2, 4 and 8h after melagatran dosing, it deteriorated coagulation status. These results were well explained by the plasma melagatran concentration. Low concentrations (15–234ng/ml) of melagatran aggravated coagulation status whereas it was mended by high concentrations (1190ng/ml or more) of the compound. In contrast, edoxaban and heparin did not show any exacerbation under these examination conditions. These results show that subtherapeutic concentrations of melagatran are associated with coagulation pathway activation, whereas factor Xa inhibition with edoxaban has a low risk of paradoxical hypercoagulation.

Effect of the Direct Factor Xa Inhibitor Apixaban in Rat Models of Thrombosis and Hemostasis

Apixaban is an oral, direct, and highly selective factor Xa inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. Apixaban was evaluated in rat thrombosis and hemostasis models. Thrombosis was produced in the carotid artery by FeCl2 application, in the vena cava by either FeCl2 application or tissue factor injection, and in an arterial-venous shunt. Hemostasis was assessed using cuticle, renal cortex, and mesenteric artery bleeding times. Intravenous apixaban infusions of 0.1, 0.3, 1, and 3 mg/kg per hour increased the ex vivo prothrombin time to 1.24, 1.93, 2.75, and 3.98 times control, respectively. The 0.3, 1, and 3-mg/kg per hour doses inhibited thrombosis in all models. Concentrations for 50% thrombus reduction ranged from 1.84 to 7.57 microM. The 3-mg/kg per hour dose increased cuticle, renal, and mesenteric bleeding times to 1.92, 2.13, and 2.98 times control, respectively. Lower doses had variable (1 mg/kg per hour) or no effect (0.1, 0.3 mg/kg per hour) on hemostasis. Heparin's prolongation of renal and cuticle bleeding time was twice that of apixaban when administered at a dose that approximated apixaban (3 mg/kg per hour) efficacy in arterial thrombosis. In summary, apixaban was effective in a broad range of thrombosis models at doses producing modest increases in multiple bleeding time models.

A Thrombin Inhibitor, but Not a Factor Xa Inhibitor, DU-176b, Aggravates Tissue Factor-Induced Hypercoagulation in Rats.

There is rising concern about recurrent thrombotic events after cessation of anticoagulant therapies. Withdrawal of heparins and a direct thrombin inhibitor is reported to be associated with evidence of rebound coagulation phenomenon in patients with coronary artery diseases (Ref 1). Previously we have shown that low-dose administration of a direct thrombin inhibitor, melagatran, worsens consumption of platelet induced by tissue factor (TF) injection (Ref 2).Objective: To determine whether cessation of melagatran, but not a factor Xa (FXa) inhibitor, DU-176b, aggravates TF-induced hypercoagulation in a rat in vivo model.Methods: Under halothane anesthesia, melagatran (2 mg/kg, i.v. bolus) and DU-176b (0.3 mg/kg, i.v. bolus) were injected into the jugular vein of male Wistar rats. Five min, 2, 4, 8, and 16 hr after dosing, hypercoagulation was induced by injection of 2.8 U/kg TF into the femoral vein of rats anesthetized with thiopental. Blood samples were collected 10 min after TF injection. Platelet numbers and TAT concentrations were measured.Results: Both melagatran and DU-176b inhibited platelet consumption and thrombin-antithrombin complex (TAT) generation when hypercoagulation was induced 5 min after the drug administration. This indicated that these compounds effectively inhibited hypercoagulation at this time point. Melagatran, however, 2 and 4 hr after dosing enhanced platelet consumption and TAT generation, indicating that the thrombin inhibitor aggravated hypercoagulation at these time points. Much later (8 and 16 hr), the effects of melagatran towards inhibition or enhancement disappeared. In contrast, DU-176b did not show any exacerbation of TF-induced hypercoagulation.Conclusion: Cessation of a direct thrombin inhibitor, melagatran, is associated with aggravation of coagulation status. This phenomenon may be implicated in rebound coagulation activation observed in clinical settings. On the other hand, the FXa inhibitor, DU-176b, may be beneficial because of a low risk of recurrent activation of coagulation pathway after cessation.

Different antithrombotic properties of factor Xa inhibitor and thrombin inhibitor in rat thrombosis models

We compared the antithrombotic properties of a factor Xa inhibitor (DX-9065a) with those of a thrombin inhibitor (melagatran) in a rat disseminated intravascular coagulation model and a rat venous thrombosis model. Rat disseminated intravascular coagulation and venous thrombosis models were produced by injection of tissue factor and platinum wire placement, respectively. DX-9065a exerted antithrombotic effects dose dependently in both models. Melagatran was also effective in the venous thrombosis model, whereas it showed an aggravation in the disseminated intravascular coagulation model at low but not high doses. In the in vitro study, DX-9065a decreased the C max of the thrombin generation curve in plasma irrespective of whether protein C was present or not. However, melagatran increased the C max at low concentrations when protein C was present. This increase was not detected in protein C-deficient plasma. These results suggest that, unlike DX-9065a, melagatran in low doses aggravates disseminated intravascular coagulation by increasing thrombin generation, which may be partly due to suppression of negative feedback by activated protein C.

Beneficial effect of JTV-803, a new synthetic inhibitor of activated factor X, against both lipopolysaccharide-induced and tissue factor-induced disseminated intravascular coagulation in rat models.

We examined whether JTV-803, a specific activated factor X inhibitor independent of antithrombin III (ATIII), is effective against disseminated intravascular coagulation (DIC) in rat models induced by tissue factor (TF) or lipopolysaccharides (LPS). In male Wistar rats, DIC was induced by a 4 h infusion of thromboplastin (3.75 U/kg) or LPS (50 mg/kg). The rats were given JTV-803 (0.3 or 3 mg/kg, bolus intravenously) (JTV-803 groups) or low molecular weight heparin (LMWH groups) (200 U/kg, bolus intravenously) prior to an injection of TF or LPS. The results showed that JTV-803 was dose-dependently effective against DIC in both TF-induced and LPS-induced rat models. This anti-DIC effect of JTV-803 at higher doses was almost equivalent to that of LMWH in both types of DIC. Plasma ATIII activity was more prominent in the group treated with JTV-803 than in that treated with LMWH. None of rats died in the TF-induced DIC model with or without drug administration. On the contrary, seven of 22 rats died (mortality rate, 31.8%) in the LPS-induced DIC model without drug administration. Although the mortality rate of rats induced with LPS and treated with LMWH was quite high (6/16, 37.5%), none of the LPS-induced rats treated with JTV-803 died. These findings suggested that JTV-803 can treat both TF-induced and LPS-induced DIC models, and that this drug has greater potential in preserving ATIII and in improving the prognosis of DIC.

All-trans retinoic acid is partially effective against lipopolysaccharide-induced but not against tissue-factor-induced disseminated intravascular coagulation in rat models.

All-trans retinoic acid (ATRA) has been introduced to the management of acute promyelocytic leukemia (APL) as a differentiation treatment. This drug not only causes complete remission, but also improves disseminated intravascular coagulation (DIC) without adding anticoagulants in APL. We have attempted to determine whether ATRA is effective against DIC in rat models induced by tissue factor (TF) or lipopolysaccharide (LPS), because the anticoagulant effect of ATRA has been considered to induce thrombomodulin upregulation and TF downregulation on endothelial cells as well as on APL cells. In male Wistar rats, DIC was induced by a 4-h infusion of thromboplastin (3.75 U/kg) or lipopolysaccharide (30 mg/kg). The rats were given ATRA orally each day at a dose of 100 mg/kg per day for 1 week before the injection of TF or LPS in ATRA treatment groups, or given low molecular weight heparin (LMWH) 10 min before the injection of TF or LPS (200 U/kg, bolus intravenously) in LMWH treatment groups. No significant changes in hemostatic parameters or markers of organ dysfunction were caused by the ATRA administration, while DIC was significantly improved by LMWH in the TF-induced model. DIC was significantly improved by both ATRA and LMWH in the LPS-induced model. These findings suggested that ATRA was useful for treating DIC only in the LPS-induced model, and that drug efficacy should be carefully assessed because the agents used to induce DIC considerably influenced the outcome.

Beneficial Effect of the Active Form of Vitamin D3 against LPS-induced DIC but not against Tissue-factor-induced DIC in Rat Models

1Alpha,25-dihydroxyvitamin D3 (active form of vitamin D3; vitamin D3) has been reported to induce the upregulation of thrombomodulin and downregulation of tissue factor (TF) on monocytes. The possibility exists that vitamin D3 prevents the development of disseminated intravascular coagulation (DIC). In particular, monocyte TF production plays an important role in the pathophysiology of DIC in septic patients. We have attempted to determine whether vitamin D3 is effective against DIC in a rat model induced by lipopolysaccharides (LPS) (30 mg/kg, 4 h) or TF (3.75 U/kg, 4 h) using selective hemostatic parameters, markers of organ dysfunction and pathological findings (assessment of glomelular fibrin deposition). Vitamin D3 was administered orally each day at a dose of 2.0 mg/kg/day for 3 days, or low molecular weight heparin (LMWH 200 u/kg; i.v.) was given 10 min before the injection of TF or LPS in each treatment group. Vitamin D3 was effective against DIC in the rat model induced by LPS only, whereas LMWH was effective against DIC in both rat models induced by either TF or LPS. The anti-DIC effect of vitamin D3 was equal to (or more potent than) that of LMWH. The results suggested that vitamin D3 was useful for the treatment of LPS-induced DIC, and that the assessment of a drug's efficacy should be done carefully given the markedly different results obtained according to the agents used to induce DIC.

Tissue Factor Reduction and Tissue Factor Pathway Inhibitor Release after Heparin Administration

Elevated plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) and large amounts of monocyte procoagulant activity (PCA) have been documented in unstable angina (UA) patients. In in vitro experiments heparin is able to blunt monocyte TF production by inhibiting TF and cytokine gene expression by stimulated cells and after in vivo administration it reduces adverse ischemic outcomes in UA patients. TF and TFPI plasma levels and monocyte PCA have been investigated in 28 refractory UA patients before and during anticoagulant subcutaneous heparin administration (thrice daily weight- and PTT-adjusted for 3 days) followed by 5000 IU X 3 for 5 days. After 2-day treatment, immediately prior to the heparin injection, TF and TFPI plasma levels [(median and range): 239 pg/ml, 130-385 pg/ ml and 120 ng/ml, 80-287 ng/ml] were lower in comparison to baseline samples (254.5 pg/ml, 134.6-380 pg/ml and 135.5 ng/ml, 74-306 ng/ml). Four h after the heparin injection TF furtherly decreased (176.5 pg/ml, 87.5-321 pg/ml; -32.5%. p<0.001) and TFPI increased (240.5 ng/ml, 140-450 ng/ml; +67%, p<0.0001). After 7-day treatment, before the injection of heparin, TF and TFPI plasma levels (200 pg/ml, 128-325 pg/ml and 115 ng/ml, 70-252 ng/ml) significantly decreased (p<0.05) in comparison to the pre-treatment values. On the morning of the 8th day, 4 h after the injection of heparin TF plasma levels and monocytes PCA significantly decreased (156.5 pg/ml, 74-259 pg/ml and from 180 U/105 monocytes, 109-582 U/10(5) monocytes to 86.1 U/10(5) monocytes, 28-320 U/10(5) monocytes; - 38% and -55% respectively) and TFPI increased (235.6 ng/ml, 152-423 ng/ ml; +70%, p<0.001). In conclusion, heparin treatment is associated with a decrease of high TF plasma levels and monocyte procoagulant activity in UA patients. These actions of heparin may play a role in determining the antithrombotic and antiinflammatory properties of this drug.