Induction Of Thrombosis Research Articles

Selection of Tissue Factor-Deficient Cell Transplants as a Novel Strategy for Improving Hemocompatibility of Human Bone Marrow Stromal Cells.

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation.Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC.Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells.Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.

RGD delivery of truncated coagulase to tumor vasculature affords local thrombotic activity to induce infarction of tumors in mice

Induction of thrombosis in tumor vasculature represents an appealing strategy for combating cancer. Herein, we combined unique intrinsic coagulation properties of staphylocoagulase with new acquired functional potentials introduced by genetic engineering, to generate a novel bi-functional fusion protein consisting of truncated coagulase (tCoa) bearing an RGD motif on its C-terminus for cancer therapy. We demonstrated that free coagulase failed to elicit any significant thrombotic activity. Conversely, RGD delivery of coagulase retained coagulase activity and afforded favorable interaction of fusion proteins with prothrombin and αvβ3 endothelial cell receptors, as verified by in silico, in vitro, and in vivo experiments. Although free coagulase elicited robust coagulase activity in vitro, only targeted coagulase (tCoa-RGD) was capable of producing extensive thrombosis, and subsequent infarction and massive necrosis of CT26 mouse colon, 4T1 mouse mammary and SKOV3 human ovarian tumors in mice. Additionally, systemic injections of lower doses of tCoa-RGD produced striking tumor growth inhibition of CT26, 4T1 and SKOV3 solid tumors in animals. Altogether, the nontoxic nature, unique shortcut mechanism, minimal effective dose, wide therapeutic window, efficient induction of thrombosis, local effects and susceptibility of human blood to coagulase suggest tCoa-RGD fusion proteins as a novel and promising anticancer therapy for human trials.

Tumor vascular infarction: prospects and challenges.

Induction of thrombosis in tumor vasculature represents an appealing strategy for combating cancer. Formation of fibrin clots may be sufficient to occlude the blood vessels that feed tumor cells, contributing to massive ischemia, vascular infarction, and the subsequent necrosis and apoptosis of neoplastic cells. This approach called as tumor vascular infarction was pioneered by Huang et al. (Science 275:547-550, 1997). Since then, different vascular targeting moieties were linked to a truncated form of human tissue factor (tTF), to generate coaguligands with selective thrombotic activities on tumor neovasculature. In contrast to the wide clinical application of angiogenesis inhibitors and tumor vascular disrupting agents, tTF-NGR is the only example of clinically tested coaguligands. Notably, among these three tumor vascular targeting approaches, tumor vascular infarction is the only modality manifesting long-term curative potential in mice. Translation of this worthy approach has been limited, as induction of thrombosis by TF fusion proteins is leaky. In this review, we describe the clinical significance of tumor vascular infarction, highlight its advantages and disadvantages, and propose a novel strategy for expediting its translation to clinical settings.

E-selectin inhibition with GMI-1271 decreases venous thrombosis without profoundly affecting tail vein bleeding in a mouse model.

Selectins, such as E-selectin (CD62E), function in venous thrombosis by binding and activating immune cells to initiate the coagulation cascade. GMI-1271 is a small molecule antagonist that inhibits E-selectin activity. Here we determine whether inhibition of E-selectin is sufficient to decrease acute venous thrombosis and associated inflammatory events in both prophylactic and treatment protocols without significantly affecting haemostasis. Male C57BL/6 mice underwent surgery for experimental thrombosis induction and were harvested at peak thrombus formation in our animal model, two days post induction. Groups included non-thrombosed true controls, shams, controls, and prophylactic or treatment groups of GMI-1271 (10 mg/kg intraperitoneal BID (twice a day) and low-molecular-weight heparin (LMWH, Lovenox 6 mg/kg subcutaneously (SC), once a day (SID). Compared with control animals, prophylaxis or treatment with LMWH and GMI-1271 in a dose-dependent manner significantly decreased thrombosis. GMI-1271 significantly lowered tail bleeding times when compared to LMWH. GMI-1271 and LMWH prophylactically administered significantly decreased vein wall neutrophil cell extravasation. However, all treatment and prophylactic therapies significantly decreased vein wall monocyte extravasation versus controls. GMI-1271 prophylactic therapy significantly decreased intra-thrombus cell counts versus control animals and other treatment groups. Immunohistochemistry confirmed that both treatments with GMI-1271 and LMWH significantly decreased activated leukocyte migration. GMI-1271 therapy significantly decreased thrombus weight and resulted in significantly lower bleeding times than LMWH. GMI-1271 treated mice showed decreased local and systemic inflammatory effects while modulating neutrophil activation, suggesting that GMI-1271 is a viable therapeutic candidate for venous thrombosis prophylaxis and treatment.

Efficacy of Dabigatran Etexilate in a Murine Model of Cancer Associated Thrombosis

Venous thromboembolism (VTE) is a common condition leading to significant patient morbidity and mortality. Thrombosis is a leading direct cause of death in cancer patients with fatal pulmonary embolism being three times more common in cancer patients than in non-cancer patients. In the presence of cancer, recurrent VTE is common and bleeding is particularly problematic due to the use of anticoagulants and the frequent need for surgical interventions. However the therapeutic options for the management of VTE in patients with cancer are limited. The treatment of cancer associated thrombosis (CAT) consists primarily of low molecular weight heparin (LMWH). However, LMWH can only be given parentally and an orally administered medication can have great benefits in improving the quality of life and compliance of patients with malignancies who suffer from VTE. Recently, New Oral Anticoagulants (NOACs) have become available. The advantages of NOACs are that they require less dose adjustment and are given orally. Particularly, during VTE treatment with dabigatran, the incidence of bleeding during VTE treatment is reduced without compromising efficacy. A recent metaanalysis of cancer patients treated with NOACs for VTE demonstrate that NOACs were as effective and safe as warfarin. However, no studies have directly studied NOACs in cancer patients nor compared NOACs with LMWH with selective inclusion of cancer patients only. Dabigatran etexilate (DE), a direct thrombin inhibitor, is one of the NOACs, which has a rapid onset of action and is characterized by its low potential for drug-drug interactions. Treatment with DE can have a significant impact on the burden of care in patients with CAT as it is orally administered and is associated with a lower incidence of bleeding during treatment for VTE. METHODSWe have developed an in vivo murine model of VTE as well as CAT. Thrombus formation is induced in the inferior vena cava (IVC) using the recently described partial flow restriction model and monitored via real time ultrasound using the Vevo770® microimaging system. The importance of our VTE murine model is the non-invasive real-time imaging technique, similar to what is used in clinical practice, making this model an ideal tool for in vivo drug testing. For the CAT model, lymphoma is induced in vivo using murine B lymphoma cells (Eµ-Myc cells) administered intravenously via the tail vein (1 x 106 cells/mouse). ); lymphoma development is attained after six days and inguinal lymph node size is measured via the Vevo770®. Thrombosis is then induced as described above. Anticoagulation is started 24 hours after thrombosis induction, with either dalteparin (LMWH) at a dose of 200 U/kg/day subcutaneously (or vehicle control) or with Dabigatran at a dose of 45 mg/kg/day by oral gavage (or vehicle control). Thrombosis size is then remeasured after 3 days of anticoagulation. RESULTSBoth dalteparin and dabigatran etexilate are effective in reducing the size of the thrombus in our VTE and CAT murine models, although the efficiency differs between cancer and non-cancer associated thrombosis. In our VTE model, daltiparin reduces thrombus size by 32% as compared to 18% for dabigatran, whereas, in our CAT model, dalteparin reduces thrombus size only 11% as compared to 24.9% with dabigatran. CONCLUSIONUsing in vivo murine models, dalteparin is more effective as compared to dabigatran in VTE not associated with cancer whereas the effectiveness of dabigatran is superior in CAT. As a proof of principle, the above described models can be further employed to study the pathophysiology of VTE and CAT and can provide biologic plausibility for pursuing further studies for using NOACs, specifically Dabigatran, in CAT. DisclosuresVanryn:boehringer-Ingelheim: Employment. Blostein:boehringer-ingelheim: Research Funding.

Natural Killer Cells Induce the Formation of Neutrophil Extracellular Traps (NETs) in Venous Thrombosis

Thrombosis is considered to be a pathological deviation of physiologic hemostasis involving similar mechanisms. Interestingly, recent work demonstrates that innate immune cells promote venous thrombosis. Innate immune cells were shown to collaborate to induce the activation of the coagulation cascade and platelets. In particular, neutrophils contribute to venous thrombosis through the release of neutrophil extracellular traps (NETs). However, the mechanism triggering the formation of NETs during venous thrombosis remain unknown. Of interest, a study showed that IFNγ induced the formation of NETs. Thus, we investigated the role of IFNγ-producing cells in the development of thrombosis.We used mice lacking IFNγ, Tbet (the transcription factor regulating the expression of IFNγ) or wild type mice. Venous thrombosis was induced using the flow restriction model in the inferior vena cava , as has been previously published. In Tbet-/-, IFNγ-/- and WT mice, we show that the absence of Tbet or IFNγ decreases the formation of thrombi after venous thrombosis induction, suggesting that the Tbet+/IFNγ producing cells are required for the early development of venous thrombosis. Comparing the composition of the thrombi from Tbet-/-, IFNγ-/- and WT mice, we show that, in all mice, neutrophils are the main cellular component of thrombi followed by monocytes; however, the number of neutrophil extracellular traps (NETs) formed during thrombosis is significantly lower in Tbet-/- and IFNγ-/- mice. Furthermore, NET formation is also decreased in WT mice specifically depleted of IFNγ and increases in Tbet-/- and IFNγ-/- mice injected with recombinant IFNγ. In vitro, we show that stimulation of WT murine neutrophils with recombinant IFNγ triggers the formation of NETs demonstrating that Tbet and IFNγ are crucial for NET formation by neutrophils.Natural killer (NK) cells are the main producers of IFNγ . Thus, we investigated the role of NK cells in venous thrombosis induced by flow restriction. NK cells were specifically depleted with an antibody during the development of venous thrombosis. The absence of NK cells results in smaller thrombi suggesting that NK cells are required for early thrombus development. Additionally, depletion in NK cells results in decreased in-situ IFNγ production and decreased NET formation. To directly link NK cells to the formation of NETs, WT neutrophils were co-cultured with Tbet-/- and IFNγ-/- NK cells. We show that WT neutrophils release less NETs when cultured with Tbet-/- and IFNγ-/- NK cells as compared to WT NK cells. These data suggest that NK cells trigger the formation of NETs by neutrophils through the production of IFNγ.Hence, we demonstrate that, in a partial flow restriction model of venous thrombosis, Tbet and IFNγ are crucial for thrombus development by promoting the formation of NETs by neutrophils and that NK cells are key effector cells in this process. DisclosuresBlostein:boehringer-ingelheim: Research Funding.

Innate Effector-Memory T-Cell Activation Regulates Post-Thrombotic Vein Wall Inflammation and Thrombus Resolution.

Immune cells play an important role during the generation and resolution of thrombosis. T cells are powerful regulators of immune and nonimmune cell function, however, their role in sterile inflammation in venous thrombosis has not been systematically examined. This study investigated the recruitment, activation, and inflammatory activity of T cells in deep vein thrombosis and its consequences for venous thrombus resolution. CD4+ and CD8+ T cells infiltrate the thrombus and vein wall rapidly on deep vein thrombosis induction and remain in the tissue throughout the thrombus resolution. In the vein wall, recruited T cells largely consist of effector-memory T (TEM) cells. Using T-cell receptor transgenic reporter mice, we demonstrate that deep vein thrombosis-recruited TEM receive an immediate antigen-independent activation and produce IFN-γ (interferon) in situ. Mapping inflammatory conditions in the thrombotic vein, we identify a set of deep vein thrombosis upregulated cytokines and chemokines that synergize to induce antigen-independent IFN-γ production in CD4+ and CD8+ TEM cells. Reducing the number of TEM cells through a depletion recovery procedure, we show that intravenous TEM activation determines neutrophil and monocyte recruitment and delays thrombus neovascularization and resolution. Examining T-cell recruitment in human venous stasis, we show that superficial varicose veins preferentially contain activated memory T cells. TEM orchestrate the inflammatory response in venous thrombosis affecting thrombus resolution.

Ultrastructural investigation of the time-dependent relationship between breast cancer cells and thrombosis induction

Thromboembolic complications are a common cause of death in breast cancer patients. The in vivo relationship between coagulation factors and circulating tumours is a multifaceted one, with tumour cells implicated in thrombocytosis and platelets associated with coagulation-mediated metastasis. Platelets and fibrin networks are known to be morphologically altered in patients with cancer, and their relationship with breast cancer cells themselves may increase thrombosis susceptibility. This was investigated in vitro, assessing such morphological alterations through the establishment of a MCF-7 breast cancer cell co-culture system with blood plasma. Co-culture duration ranged from zero to thirty minutes, with five-minute intervals, in order to assess the time-dependent ultrastructural conformations of platelet and fibrin networks, using scanning electron microscopy. It was found that enhanced coagulability was related to co-culture duration. Changes in platelet and fibrin network morphology from normal were visible as early as five minutes in co-culture with MCF-7 cells. With longer co-culture duration thrombosis-linked variation in structural configuration was intensified, including advanced platelet aggregation and adherence characteristics, compression of fibrin networks into plaques of increased density, and upsurge of microparticulate extrusion implicated in amplifying procoagulant events during the metastatic process. These results confirm that cancer cells are stimulators of coagulation even in an in vitro system and breast cancer patients may become increasingly susceptible to thrombotic-related consequences with increased exposure to tumour cells, especially during metastasis.

Abstract A19: Pulmonary microthrombosis enhances tumorigenesis via myeloid hypoxia-inducible factors

Abstract Thrombosis causes local blood flow restriction and tissue hypoxia, and both of these are associated with tumor cell metastasis. To better understand the regulation of thrombosis-induced metastasis, we created a model incorporating elements of both processes. Controlled induction of pulmonary microthrombosis caused an increase in expression of first hypoxia-inducible factor (HIF)1α, and subsequently HIF2α. Induction of thrombosis before the introduction of tumor cells to venous circulation had no effect on pulmonary tumor number or size; but thrombosis at the time of tumor cell seeding increased number and size of tumors in the lung. Thrombosis on the day after seeding of tumor cells caused an even greater increase in tumor number and size, and this effect persisted until even when thrombosis was induced five days after the introduction of tumor cells into the blood. Experiments on myeloid HIF1α or HIF2α knockout mice demonstrated that loss of either HIF1α or HIF2α eliminated the advantage given to pulmonary tumorigenesis by thrombotic insult. In primary human tumours, markers of thrombosis were positively correlated with expression of the 2 HIFα isoforms. These data demonstrate the importance of microthrombosis in a novel model of metastasis and the essential role of the myeloid cell-specific HIFα response in mediating this process. Citation Format: Colin E. Evans, Asis Palazon, Jingwei Sim, Petros A. Tyrakis, Par-Ola Bendahl, Mattias Belting, Helene Rundqvist, Cristina Branco, Randall S. Johnson. Pulmonary microthrombosis enhances tumorigenesis via myeloid hypoxia-inducible factors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A19.

A comparison of the effects of factor XII deficiency and prekallikrein deficiency on thrombus formation

Studies with animal models implicate the plasma proteases factor XIIa (FXIIa) and α-kallikrein in arterial and venous thrombosis. As congenital deficiencies of factor XII (FXII) or prekallikrein (PK), the zymogens of FXIIa and α-kallikrein respectively, do not cause bleeding disorders, inhibition of these enzymes may have therapeutic benefit without compromising hemostasis. The relative contributions of FXIIa and α-kallikrein to thrombosis in animal models are not clear. We compared mice lacking FXII or PK to wild type mice in established models of arterial thrombosis. Wild type mice developed carotid artery occlusion when the vessel was exposed to a 3.5% solution of ferric chloride (FeCl3). FXII-deficient mice were resistant to occlusion at 5% FeCl3 and partially resistant at 10% FeCl3. PK-deficient mice were resistant at 3.5% FeCl3 and partially resistant at 5% FeCl3. Mice lacking high molecular weight kininogen, a cofactor for PK activation and activity, were also partially resistant to thrombosis at 5% FeCl3. Induction of carotid artery thrombosis with Rose Bengal was delayed in FXII-deficient mice compared to wild type or PK-deficient animals. In human plasma supplemented with silica, DNA or collagen to induce contact activation, an antibody to the FXIIa active site was more effective at preventing thrombin generation than an antibody to the α-kallikrein active site. Similarly, the FXIIa antibody was more effective at reducing fibrin formation in human blood flowing through collagen coated-tubes. The findings suggest that inhibitors of FXIIa will have more potent anti-thrombotic effects than inhibitors of α-kallikrein.

Murine model of large-vein electrolytic injury induction of thrombosis with slow resolution

Deep vein thrombosis (DVT) and its sequela, pulmonary embolism, occur at a rate of 1 per 1000person/year. Experimental models for evaluation of DVT have many short-comings, such as mechanical occlusion or stenosis to cause thrombosis, rather than the clinical scenario of thrombosis causing occlusion/stenosis. The goal of this study was to develop a model of flow-based large-vein thrombosis with resistance to resolution, to model clinical DVT behavior. Adult male C57Bl/6 mice underwent thrombus induction via an electrolytic injury to the femoral vein (3V positive current for 90s), with subsequent intra-vital fluorescence quantitation of platelet and fibrin accumulation through the first 60min, and final histomorphometric volume evaluation at 1, 7, 14, and 28days. Platelet accumulation at the injury site was comparable to a milder electrolytic injury, whereas fibrin was greatly augmented by 60min in the more severe injury model. Thrombi showed persistent presence at 1 and 7days, with remodeling to a stenotic fibrosis that encroached into the lumen at 14 and 28days. The thrombotic/fibrotic volume within the femoral vein fell by 23% from 1 to 7days, but had a residual presence at 28days that was 31% the 1-day volume. This new model may provide an alternative approach to evaluating DVT persistence and therapeutic inhibition, to develop a better understanding of the clinical progression of DVT to thrombophlebitis.

PP.39.23

Objective: We showed previously that acute aldosterone (ALDO) infusion resulted in impairment of the fibrinolytic process and activation of coagulation cascade, consequently leading to augmentation of stasis-induced venous thrombosis in rats. This prothrombotic effect of ALDO was only partially mediated by the mineralocorticoid receptor (MR). Bearing in mind that ALDO potentiates the angiotensin II (Ang II) effects, in the present study we investigated the role of Ang II in the mechanism of ALDO prothrombotic action. Design and method: Male Wistar rats (300–350 g, n = 8–12) were used in this study. Aldosterone (ALDO, 30 μg/kg/h) or 0,4% ethanol (VEH; 2 ml/kg/h) was infused into the femoral vein 5 minutes before the induction of venous thrombosis and was continued for 1 h. Valsartan (VAL, 10 mg/kg), AT1 antagonist, was injected as a bolus into femoral vein 5 minutes before ALDO infusion. Venous thrombosis was performed by the vena cava ligation. The formed thrombus was removed and weighted after 24 h. Plasminogen activator inhibitor (PAI-I), tissue plasminogen activator (t-PA), thrombin activatable fibrinolysis inhibitor (TAFI) plasma levels were measured by ELISA. Results: We observed that one-hour infusion of ALDO resulted in a significant increase in venous thrombus weight (p < 0.01) and markedly elevated incidence of venous thrombosis, paralleled by significant increase in PAI-1 (p < 0.001) and TAFI (p < 0.001) as well as decrease in t-PA (p < 0.001) plasma levels in comparison to VEH. VAL administered together with ALDO reduced thrombus formation (p < 0.05) and diminished the incidences of thrombosis. We also observed that pretreatment with VAL reversed slightly the ALDO-induced decrease in t-PA level and reduced significantly TAFI (p < 0.05) and PAI-1 plasma levels (p < 0.05, vs ALDO). Conclusions: This study provides evidence that prothrombotic effect of ALDO is also mediated by the angiotensin II AT1 receptor. The mechanism of ALDO-induced AT1 activation involved impairment of the fibrinolytic process. The present study supplies our knowledge about the multiple mechanism of ALDO prothrombotic action, which is mediated via MR and AT1 receptors.

Abstract 712: Screening of Thrombolytic Agents in vivo Using a Novel Zebrafish Model of Ischemic Stroke Induced by Photochemical Thrombosis

Background: Timely thrombolytic therapy for acute ischemic stroke is important for improving neurological prognosis. Screening of thrombolytic agents on contemporary animal models of focal ischemic stroke is generally difficult as they require sophisticated surgical procedures such as induction of cerebral artery occlusion through mechanical ligation. Herein we seek to develop a novel zebrafish (Danio rerio) model of acute ischemic stroke for screening of thrombolytic drugs. Methods: All experiments were performed on a modified confocal optical microscope, which allows the induction of thrombosis at a selected blood vessel of larval zebrafish and the imaging of thrombotic and thrombolytic processes in real time. To initiate photochemical thrombosis, a 532-nm laser was focused at a blood vessel of a larva (4 dpf) that had been injected with a photosensitizer (rose bengal). To test the thrombolytic activity of tPA, we injected tPA to the blood vessel of a larval with a thrombus that partially occluded the blood flow. Results: Through photochemical means, we induced endothelial injury at selected blood vessel which subsequently triggered thrombosis. We show that an occlusion at the 1st branch of central artery drastically diminished the hemodynamics and cardiac function of larval zebrafish and impaired their capability to maintain balance during swimming whereas that at the basilar artery resulted in a high death rate. Immunofluorescent imaging shows that the photochemically induced thrombus comprised fibrins and platelets. After the injection of tPA to a larval with a partially occluded blood vessel, the fibrin mesh on the thrombus appeared sparse and limp which eventually led to the restoration of blood flow. Conclusions: Our zebrafish model of ischemic stroke is convenient to adapt and is highly reproducible. This model can potentially become an effective platform that benefits the screening of thrombolytic agents and the optimization of their dose.

C1-esterase inhibitor treatment: preclinical safety aspects on the potential prothrombotic risk.

Human plasma-derived C1-esterase inhibitor (C1-INH) is an efficacious and safe treatment for hereditary angioedema. However, thrombotic events in subjects treated with C1-INH at recommended or off-label, high doses have been reported. In this study, we addressed the potential prothrombotic risk of C1-INH treatment in high doses using a non-clinical rabbit model. Following intravenous infusion of C1-INH to rabbits at doses up to 800 IU/kg, the exposure and the pharmacodynamic efficacy of C1-INH in rabbits were confirmed by activity measurements of C1-esterase, and coagulation factors XIa and XIIa, respectively. Potential prothrombotic effects were assessed following induction of venous and arterial thrombosis using in vivo models of venous and arterial stasis, complemented by various in vitro assays of coagulation markers. Administration of C1-INH at doses up to 800 IU/kg did not potentiate thrombus formation during venous stasis. In contrast, inhibition of arterial occlusion was observed upon C1-INH administration when compared with isotonic saline treatment, indicating antithrombotic rather than prothrombotic activity of high dose C1-INH treatment in vivo. This was further confirmed in vitro by decreased thrombin generation, increased activated partial thromboplastin time, clotting time and clot formation time, and inhibition of platelet aggregation. No relevant changes in fibrinolysis or in the levels of thrombin-antithrombin complexes, and prothrombin fragment 1+2 were observed upon high dose C1-INH treatment. The data suggest that treatment of healthy rabbits with high doses of C1-INH could potentially inhibit coagulation and thrombus formation rather than induce a prothrombotic risk.

Bioassay-guided isolation and identification of anti-platelet-active compounds from the root of Ashitaba (Angelica keiskei Koidz.)

Platelet aggregation is fundamental to a wide range of physiological and pathological processes, including the induction of thrombosis and arteriosclerosis. Anti-platelet activity of a crude methanol extract and solvent fractions of Ashitaba roots (Angelica keiskei Koidz.) was evaluated using a turbidimetric method using washed rabbit platelets. We identified the anti-platelet activities of two chalcones, 4-hydroxyderricin and xanthoangelol, isolated from the ethyl acetate-soluble fraction of Ashitaba roots by using a bioassay-guided isolation method. 4-Hydroxyderricin and xanthoangelol effectively inhibited platelet aggregation induced by collagen (IC50 of 41.9 and 35.9 μM, respectively), platelet-activating factor (IC50 of 46.1 and 42.3 μM, respectively) and phorbol 12-myristate 13-acetate (IC50 of 16.5 and 45.9 μM, respectively). These compounds did not inhibit thrombin-induced platelet aggregation (IC50 of>80 μM). The results suggest that the chalcones 4-hydroxyderricin and xanthoangelol may be potent anti-thrombotic components of A. keiskei Koidz.

Abstract 78: Extracellular RNA Induces Venous Thrombosis Through TLR3

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. Vascular and circulating cells may sense ‘pathologic ligands’ accumulating in the circulation via toll-like receptor (TLR) and translate signals into a pro-thrombotic state. TLR3 recognizes self-molecules generated during tissue damage and inflammation such as extracellular RNA (eRNA). We hypothesize that eRNA and TLR3 are involved in the development of venous thrombosis after vessel injury. Injection of RNase1 decreased the size of thrombi after FeCl3-induced inferior vena cava injury (IVC) compared to mice treated with vehicle. Using a specific fluorescent probe for RNA, we found that FeCl3 induced RNA release and thus increased RNA content in the thrombus. WT or TLR3 deficient (-/-) mice received an injection of vehicle, eRNA or poly(I:C) prior to thrombosis induction. In WT mice, thrombus size was increased by eRNA or poly(I:C). Interestingly, no change in thrombus size was observed in TLR3-/- mice regardless of the treatment. Poly(I:C) treatment was associated with higher amount of neutrophils within the thrombus of WT mice compared with vehicle. In TLR3-/- mice, poly(I:C) failed to increase the recruitment of neutrophils. Staining for citrulinated H3, a marker of neutrophil extracellular traps (NETs) formation, was increased by poly(I:C) in WT mice. Interestingly, in TLR3-/- mice, no increase of NET formation was found after poly(I:C) injection as compared to vehicle control. Poly(I:C) also increased the recruitment of monocytes within thrombi in WT but not in TLR3-/- mice. Preliminary results suggest that injection of eRNA may also promote the recruitment of monocytes, neutrophils and the formation of NETs in WT but not in TLR3-/- mice. Human neutrophils were then treated with conditioned media from HUVEC treated with poly(I:C) in presence of control siRNA or TLR3 siRNA. Media from HUVEC transfected with control siRNA and treated with poly(I:C) induced the migration of neutrophils, whereas media from TLR3 siRNA and poly(I:C) treated cells did not. These results suggest that eRNA and TLR3 may participate to thrombus formation by inducing a pro-inflammatory response leading to the recruitment of monocytes and neutrophils.

Thrombus formation induced by laser in a mouse model.

Animal models are used for the development of techniques and/or models that aid the study of thrombosis pathophysiology. The aim of the present study was to modify the technique of in vivo thrombosis induction to make it more accessible. BALB/c mice were intraperitoneally anesthetized with 0.4 ml 2,2,2-tribromoethanol (266.6 mg/kg) and xylazine (13.3 mg/kg), whilst maintaining stable blood pressure and temperature. Through abdominal surgery, the mesentery was identified and isolated for the visualization of the arteries. A simple epifluorescence magnifier was used to detect the presence of thrombi. The results obtained indicate that using rose bengal at concentrations of 25 and 50 mg/kg and a laser power of 5 mW, thrombus formation occurred. In addition, formation of the thrombus occurred ~30 min following induction and the thrombus had a total area of 4,878.3 μm2, which caused total occlusion of the mesenteric artery. For visualization, platelets were labeled with calcein acetyloxymethyl ester for 1 h, which resulted in improved observation of thrombus formation in real time. Therefore, this technique may be used to perform in vivo studies simply and at low cost, and is suitable for use in a variety of studies of thrombosis.

Histopathological effects of different therapy strategies in experimental sinus thrombosis

The optimal treatment for cerebral venous thrombosis is still under debate. The histological consequences of different treatments have not been systematically studied and may be of value in this debate. Thrombosis of the superior sagittal sinus was induced in rats by topical application of ferric chloride. Animals were treated 6 h after operation with subcutaneous injection of 450 IU/kg enoxaparin twice daily (n = 10), with 10 mg recombinant tissue plasminogen activator (rt-PA)/kg (n = 12), and with 6 mg abciximab/kg (n = 10). Eleven animals were treated with saline (controls), and four animals were sham-operated without thrombosis induction. Animals were killed on day 7. Coronal brain slices were stained with hematoxylin-eosin (HE) and against glial fibrillary acidic protein (GFAP), and factor VIII. Histology was quantified in parasagittal and temporal regions of interest. Compared with controls, counts of pyknotic neurons on HE stain were significantly lower in the enoxaparin group. Counts for GFAP-expressing astrocytes were highest in the enoxaparin (p < 0.001) and rt-PA (p < 0.05)-treated groups. Angiogenesis defined as factor VIII-expressing vessels was significantly (p < 0.01) higher in the enoxaparin and significantly lower (p < 0.01) in the rt-PA group compared with controls. In this animal model, we found histological differences related to the different treatments, which cannot be explained by recanalization and its speed alone.

Homocysteine, Paraoxonase-1 and Vascular Endothelial Dysfunction: Omnibus viis Romam Pervenitur.

Increased oxidative stress, alterations of lipid metabolism and induction of thrombosis have been suggested to be pathogenic links which are present between hyperhomocysteinaemia and atherosclerosis. However, the mechanism by which homocysteine (Hcy) can promote atherogenesis is far from clear and it has been debated. In the presence of cardiovascular risk factors, endothelial dysfunction is the central commodity which converges a plenty of factors, which have been named as atherogenic. Now-a-days, there are only few studies which have presented the correlation between antioxidant enzyme HDL-associated-paraoxonase 1(PON1) and Hcy in atherosclerosis. Both PON 1 and Hcy have been implicated in human diseases which are related to endothelial dysfunction. Although paraoxonases have the ability to hydrolyze a variety of substrates, only one of them, Hcy-thiolactone, is known to occur naturally. It seems very likely that the involvement of Hcy in atherosclerotic disease is mediated through its interactions with PON1.

A Zebrafish Model Of Antithrombin III Deficiency Displays Bleeding and Thrombosis Secondary To Disseminated Intravascular Coagulation

Thrombosis is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Identification of small molecule inhibitors of thrombosis in an in vivo model would facilitate novel and improved therapeutics for patients. The zebrafish is a powerful genetic model in which the hemostatic system is nearly entirely conserved with humans. Its external development, ability to generate thousands of offspring at low cost, and optical transparency all make it a powerful tool to screen for genetic and chemical modifiers of coagulation disorders. We generated a zebrafish model of antithrombin III (AT3) deficiency by targeted mutagenesis using zinc finger nucleases. Homozygous at3 mutants displayed a lethal phenotype due to intracardiac thrombosis between 2 and 7 months of age, yet embryos and larvae appeared grossly normal with no overt evidence of pathologic clotting. Induction of thrombosis at 3-4 days post fertilization (dpf) in homozygous mutant larvae by laser-mediated endothelial ablation resulted in diminished rates of posterior cardinal vein (PCV) occlusion, a bleeding phenotype. To prove functional conservation with mammals, we expressed recombinant zebrafish At3 and demonstrated that it binds human thrombin in vitro. Furthermore, while injection of wild type zebrafish and human cDNAs rescued the laser injury phenotype, zebrafish at3 with a mutation in the putative reactive site failed to do so.We hypothesized that the discrepant larval bleeding and adult thrombotic phenotypes could be accounted for by disseminated intravascular coagulation (DIC). Consistent with this, we observed reduced fibrinogen levels in at3 homozygous mutant plasma, and were able to rescue mutant larvae by injection of human fibrinogen prior to laser injury. To identify the location of consumed fibrinogen, we tagged human fibrinogen with FITC (fluorescein isothiocyanate), followed by infusion into larvae from heterozygous intercrosses at 3 dpf. at3 homozygous mutants displayed extensive PCV fluorescence, which was absent in wild type and heterozygous siblings. Pre-incubation with warfarin completely prevented this phenotype, co-injection of tissue plasminogen activator (TPA) partially prevented fluorescence accumulation, and post-injection of TPA reduced the signal, consistent with our hypothesis of DIC.Our data have uncovered the mechanism underlying the discrepant at3 mutant phenotypes and demonstrated conservation of At3 function in zebrafish. Loss of At3 protein results in DIC in zebrafish larvae secondary to unopposed thrombin activity. Mutants survive, only to succumb to lethal thrombosis as adults. Further study as to why larvae are able to tolerate excessive clot formation, as well as small molecule screens for novel anticoagulants using this model, could potentially lead to innovative therapeutic modalities for affected patients. Disclosures:No relevant conflicts of interest to declare.