Time To Occlusion Research Articles

Identification of Nfel1a and Nfel3 as novel regulators for zebrafish thrombopoiesis

In mammalian hematopoiesis, megakaryocytes mature and become polyploid in the bone marrow before releasing platelets into circulation. In contrast, fish produce thrombocytes in kidney marrow, where young thrombocytes undergo maturation in circulation, akin to platelets. Despite morphological differences, our single-cell sequencing revealed significant gene expression similarities between fish thrombocytes and mammalian megakaryocytes, including Nfe2, which is crucial in platelet production. In addition to nfe2 expression, we found four nfe2-related genes, nfe2I1a, nfe2I1b, nfe2I2a, and nfe2I3, were expressed in mature thrombocytes. However, only nfe2, nfe2l2a, and nfe2l3 are expressed in young thrombocytes. Thus, we hypothesized that Nfe2-related factors may also be involved in thrombocyte production. To address this, we knocked down the four novel nfe2-related genes by the piggyback method and found both young and mature thrombocytes reduced when nfe2, nfe2l1a, and nfe2l3 were knocked down. They also exhibited greater gill bleeding and prolonged time to occlusion (TTO) compared to controls. In summary, our study shows Nfe2I1a and Nfe2l3 as novel transcription factors that are positive regulators influencing adult zebrafish thrombopoiesis.

Vagus Nerve Stimulation Accelerates Thrombosis via α7 Nicotinic Acetylcholine Receptors and Improves Clot Formation in Hemophilia A Mice

INTRODUCTION: Hemophilia A, factor VIII deficiency, decreases thrombin and fibrin formation, resulting in abnormal bleeding. Vagus nerve stimulation (VNS) increases thrombin generation via α7 nicotinic acetylcholine receptors (α7nAChR). We reasoned that VNS would improve local clot stability in a FeCl3-induced thrombosis model in wild-type (WT) and factor VIII–deficient (F8-KO), but not in α7nAChR-deficient (α7-KO) mice. METHODS: Mice underwent left cervical VNS (1 V, 30 Hz, 2 ms, 5 minute), and then FeCl3 solution was applied to the right carotid artery. For WT and α7-KO experiments, a 5% solution was applied for 1 minute. For F8-KO experiments, a 10% solution was applied for 3 minutes. Time to occlusion (TTO) was defined as absence of arterial waveform for greater than 1 minute. RESULTS: In WT mice, VNS significantly decreases TTO (VNS vs sham: 7.1 ± 1.3 vs 15.8 ± 3.4 minutes, n = 5, p < 0.05), but fails to decrease TTO in α7-KO mice (VNS vs sham: 19.3 ± 3.7 vs 17.9 ± 4.3 minutes, n = 5, p = NS). F8-KO mice have significantly longer TTO vs WT mice (WT vs F8-KO sham: 6.55 ± 0.63 minutes vs 25 ± 0 minutes, n = 6, p <0.0001). VNS significantly decreases TTO in F8-KO mice (F8-KO VNS vs F8-KO sham: 9.26 ± 1.38 minutes vs 25 ± 0 minutes, n = 6, p < 0.0001) (Figure).Figure.: F8-KO, factor VIII–deficient; VNS, vagus nerve stimulation.CONCLUSION: VNS improves local clot stability. VNS requires α7nAChR, but bypasses factor VIII deficiency, suggesting that vagal control of thrombin generation is independent of this critical clotting factor.

Diindolylmethane ameliorates platelet aggregation and thrombosis: In silico, in vitro, and in vivo studies

Diindolylmethane (DIM), a major metabolite of indole-3-carbinol (I3C), plays a vital role in the pharmacological actions of I3C. The role of DIM in the inhibition of platelet aggregation and thrombus generation is yet to be revealed. However, how DIM and I3C modulate the interaction of platelets with the glycoproteinVI (GPVI) and purinergic receptor Y12 (P2Y12) receptors is unknown. In silico studies revealed that the indole group of DIM and indole and the hydroxyl group of I3C are responsible for modulating platelet interaction with GPVI and P2Y12 receptors. In silico studies further predicted that DIM more superiorly modulates platelet interaction with GPVI and P2Y12 receptors than I3C. In vitro studies identified that DIM significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), collagen, thrombin, and arachidonic acid, increasing the thrombin-induced clot retraction size and clot retraction weight. Moreover, in vivo results of ferric chloride (FeCl3) induced carotid artery thrombus generation indicate that DIM significantly reduced the reactive oxygen species (ROS), hydrogen peroxide (H2O2), thromboxane 2 (TXB2), cyclooxygenase 1 (COX-1), prostaglandin E2 (PGE2), thrombus weight, increased the cyclic adenosine monophosphate (cAMP), and extended the time to occlusion (TTO). Furthermore, DIM did not show thrombolytic activity. Therefore, DIM acts as an antiplatelet aggregation and antithrombotic agent. Moreover, DIM is responsible for the antiplatelet aggregation and antithrombotic activity of I3C. Therefore, DIM could be used to treat thrombotic diseases.

Preferential Inhibition of Fibrin Mediated Thrombosis in Rodent Models By JNJ-9375, a Novel exosite1 Thrombin Inhibitor

We have previously reported an improved therapeutic index (TI) for JNJ-9375 vs apixaban in rat and cynomolgus monkey models of venous thrombosis and bleeding. We have also reported that JNJ-9375 preferentially inhibited the fibrin component of the thrombus without significant impact on the platelet component under low and high shear conditions in a human translational model of thrombosis (Badimon chamber model). We hypothesized that the novel mode of action of JNJ-9375 which by selectively inhibiting exosite 1 substrates from binding to thrombin (such as fibrinogen and PAR-1) may allow for other thrombin interactions at the active site and exosite 2 including platelet activation by PAR-4 and GP1b.In the present studies, we have compared the antithrombotic effects of JNJ-9375, the active site thrombin inhibitor dabigatran and the factor Xa inhibitor apixaban in well characterized and widely used rat AV-shunt and carotid ferric chloride models of venous and arterial thrombosis, respectively. JNJ-9375, dabigatran and apixaban all maximally inhibited thrombus formation in a dose dependent manner in the rat AV-shunt model of venous thrombosis. Significant inhibition of thrombus weights of 50%, 44% and 57% were observed at 0.3 mg/kg, i.v., 0.1 mg/kg, i.v. and 1 mg/kg, i.v. with JNJ-9375, dabigatran and apixaban respectively. Dose dependent prolongation in coagulation parameters were also observed ex-vivo with JNJ-9375 (TT, ECT, aPTT and PT), dabigatran (TT, ECT, aPTT) and apixaban (aPTT and PT). In addition, there were dose dependent increases in drug exposure.In the rat ferric chloride model of arterial thrombosis, JNJ-9375, dabigatran and apixaban administration also resulted in dose dependent inhibition of thrombus formation as assessed by the time to occlusion (TTO) of the carotid artery and area under the curve (AUC) for carotid blood flow. However, the dose required to inhibit arterial thrombosis Vs venous thrombosis varied substantially for the different agents. With JNJ-9375 we only observed a significant inhibition of arterial thrombosis (TTO and AUC) at a dose of 10 mg/kg, i.v. (Vs 0.3 mg/kg in the AV-shunt model) which represents a 33-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). In contrast, we observed a significant inhibition of arterial thrombosis with dabigatran (AUC) and apixaban (TTO and AUC) at doses of 0.3 mg/kg, i.v. and 3 mg/kg, i.v. (Vs 0.1 mg/kg and 1 mg/kg in the AV-shunt model) respectively. These doses of dabigatran and apixaban represent a 3-fold shift in the dose required to inhibit arterial thrombosis (Vs venous thrombosis). We have also documented dose dependent prolongations in coagulation parameters and drug exposure with all three agents in the these studies. In addition, we performed ex-vivo platelet aggregation in platelet rich plasma with various platelet agonists (ADP, collagen, AA, rat and human thrombin) in those animals dosed with JNJ-9375. As anticipated and consistent with its mechanism of action, JNJ-9375 only inhibited platelet aggregation to thrombin stimulation but not with other platelet agonists confirming the specificity of JNJ-9375. We observed about 50%, >95% and >95% inhibition of thrombin-induced platelet aggregation at 1, 3 and 10 mg/kg, i.v. with JNJ-9375 respectively. However, we only observed inhibition of arterial thrombosis at 10 mg/kg.In summary, we have demonstrated dose dependent inhibition of thrombosis with JNJ-9375, dabigatran and apixaban in venous and arterial model of thrombosis in rats. However, we have observed a larger difference in the dose required for inhibition of arterial Vs venous thrombosis with JNJ-9375 (33-fold) compared to a more modest 3-fold shift observed for dabigatran and apixaban. These findings with JNJ-9375 demonstrate an anticoagulant profile that preferentially inhibits fibrin mediated clot formation with a more modest impact on platelet driven thrombosis in the present models. This differential profile may account in part for the improved TI over apixaban we had reported earlier in animal models. This profile suggests JNJ-9375 which is currently in early clinical development may also be more amenable to dosing in combination with antiplatelet therapy for indications such as acute coronary syndromes and needs to be tested experimentally. DisclosuresChintala:Janssen Research & Development: Employment. Li:Janssen R&D: Employment. Huang Devine:Janssen Research & Development: Employment. Bunce:Janssen R&D: Employment.

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

AbstractPlasma fibrinogen molecules comprise 2 copies of Aα, Bβ, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.

Surprisingly Rapid Onset of Estrogen-Induced Venous Thrombosis in an Animal Model

Venous thrombosis is a well-known complication of sex hormone therapy, with onset typically within weeks to months after initiation. Worldwide, more than 100 million pre-menopausal women use combined oral contraceptives, thus tens to hundreds of thousands develop thrombosis annually, resulting in significant morbidity and mortality. Estrogens are thought to be the major thrombotic risk, although the impact of progestins is less clear. It has been hypothesized that progestin effects may be secondary to mitigation of the estrogen effect. Although it is known that estrogens and progestins can alter expression of coagulation factors, the pathways and mechanisms that connect the two systems, as well as the proteins involved in progression to thrombosis, are poorly understood. Identification of these mediators are central to any comprehensive understanding of hormone-induced pathophysiology, could help ascertain patients at higher risk for thrombosis, and might also pinpoint future therapeutic targets. 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 study the genetics of coagulation disorders. We previously produced a transgenic line (fabp-fgb-egfp) that generates GFP tagged fibrinogen beta (Fgb), which is incorporated into induced and spontaneous fibrin-rich thrombi. Here we show surprisingly rapid onset of estrogen-induced thrombosis. We exposed 5-6 day old transgenic zebrafish larvae to a range of concentrations of estradiol over 4-24 hours. We discovered green fluorescent deposits co-localizing to the posterior cardinal vein (PCV, orthologous to the inferior vena cava), but not other vessels. This occurred within 4 hours in a dose-dependent fashion. Confocal imaging demonstrated that the fluorescence was intravascular and deposited along the endothelium. Pre-incubation with warfarin and the direct oral anticoagulants rivaroxaban and dabigatran inhibited the fluorescent signal, confirming that the deposits were fibrin thrombi. This phenotype was similar to spontaneous PCV thrombosis observed in zebrafish mutants deficient in antithrombin, which also demonstrate a consumptive coagulopathy with absence of venous occlusion in response to endothelial injury. Therefore we treated 3 dpf larvae with estradiol for 4 hours, followed by laser-mediated endothelial injury and measurement of the time to occlusion (TTO) in the PCV by a blinded observer. Although the median TTO only increased from 12.5 to 15 seconds with treatment, this was highly statistically significant (p=0.0001, Mann Whitney U test, n = 81-98 for each group), consistent with a consumptive coagulopathy. To evaluate the role of progestins, 6 dpf larvae were treated with estradiol and levonorgestrel, desogestrel, or norethisterone, alone or in combination, and quantitatively scored for the presence of thrombosis by a blinded observer. The rates of thrombosis were 72% for estradiol, ranged 15-31% for the progestins, and ranged 66-70% for the combined estrogen/progestin treatments. In summary, our results are nearly identical to loss of antithrombin in zebrafish, although the consumptive coagulopathy is not as potent. These data are consistent with human estrogen-induced venous thromboembolism, but it was completely unexpected and concerning to see the production of thrombi in such a rapid fashion. Our data also suggest that progestins do not mitigate nor potentiate this effect. Future screens using this model are ideally posed to identify the mediators of sex hormone-induced thrombosis, and the results could potentially lead to innovative preventive or therapeutic modalities. DisclosuresShavit:CSL Behring: Consultancy; Octapharma: Consultancy; Shire: Consultancy; Bayer: Consultancy, Research Funding.

G protein-coupled receptor gpr34l mutation affects thrombocyte function in zebrafish.

Haemostasis is a defence mechanism that has evolved to protect organisms from losing their circulating fluid. We have previously introduced zebrafish as a model to study the genetics of haemostasis to identify novel genes that play a role in haemostasis. Here, we identify a zebrafish mutant that showed prolonged time to occlusion (TTO) in the laser injury venous thrombosis assay. By linkage analysis and fine mapping, we found a mutation in the orphan G protein-coupled receptor 34 like gene (gpr34l) causing a change of Val to Glu in the third external loop of Gpr34l. We have shown that injection of zebrafish gpr34l RNA rescues the prolonged TTO defect. The thrombocytes from the mutant showed elevated levels of cAMP that supports the defective thrombocyte function. We also have demonstrated that knockdown of this gene by intravenous Vivo-Morpholino injections yielded a phenotype similar to the gpr34l mutation. These results suggest that the lack of functional Gpr34l leads to increased cAMP levels that result in defective thrombocyte aggregation.

Intraflagellar transport proteins are involved in thrombocyte filopodia formation and secretion

Intraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to thrombocyte aggregation (TTA) in the plate tilt assay after thrombocyte activation by the following agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in thrombocyte function.

경동맥 혈전 유발 동물모델을 이용한 한약재 추출물의 효능 검색

Objectives : The aim of this study was to examine anti-thrombotic effect of traditional herbal extracts in a rat model of ferric chloride (<TEX>$FeCl_3$</TEX>)-induced carotid arterial thrombosis. Methods : Thirty minutes prior to a 35% <TEX>$FeCl_3$</TEX> application, Sprague Dawley(SD) rats were injected with the 10 types of traditional herbal extracts (100mg/kg, intraperitoneal injection), respectively. The effect of these herbal extracts was examined for time to occlusion(TTO) using the Laser doppler flow meter and measured for thrombus weight (TW) in <TEX>$FeCl_3$</TEX>-induced thrombosis model. Results : In the TTO, Salvia miltiorrhiza (Sm, <TEX>$2.30{\pm}0.28$</TEX> min, p<0.001) and Santalum album (Sa, <TEX>$2.19{\pm}0.19$</TEX> min, p<0.001) showed significantly delayed TTO more than twice compared with Saline-treated group. Cnidium officinale (Co), Psoralea corylifolia (Pc), Scutellatia baicalensis (Sba), Panax notoginseng (Pn), Angelica tenuissima (At), Scrophularia buergeriana (Sbu), Rhus verniciflua (Rv) and Picrasma quassioides (Pq), except for Rhus verniciflua (Rv) also meaningfully impeded TTO more than one fold. In addition, Salvia miltiorrhiza, Santalum album, Cnidium officinale, Psoralea corylifolia and Scutellatia baicalensis significantly reduced TW more than 10% compared with Saline-treated group. Especially, Salvia miltiorrhiza and Santalum album showed the most excellent anti-thrombotic effect among the 10 herbal extracts tested on the restoration of altered TTO and TW. Conclusions : These results suggest that Sm and Sa extracts have outstanding anti-thrombotic effect in <TEX>$FeCl_3$</TEX>-induced thrombosis model and is potentially useful as herbal medicines for the treatment and prevention of thrombosis.

Screening of Preventive Effects of Herbal Prescriptions in Rat Model of Arterial Thrombosis Induced by Ferric Chloride

Objectives: This study examined the effects of 8 types of herbal prescriptions prescribed to alleviate dementia symptoms in a rat model of arterial thrombosis induced by ferric chloride (FeCl3). Methods: Thirty minutes before 35% FeCl3 treatment, SD rats were intraperitoneally injected with the 8 types of herbal prescriptions, respectively. We tested the effects of the herbal prescriptions on time to occlusion (TTO) in an arterial thrombosis model using a laser Doppler flow meter. In addition, thrombus weight (TW) and collagen fiber damages were evaluated in the same condition. Results: Herbal prescriptions showed the following rank-order based on their TTO: Chong-myung-tang (CMT) > modified Jangwonhan 02 (LMK02) > Toki-shakuyaku-san (TSS) Oren-gedoku-to (OGT) Yokukansan (YKS). In particular, CMT (100 mg/kg, i.p.) and LMK02 (100 mg/kg, i.p.) delayed the TTO the most (14.83 ± 0.98 and 13.67 ± 1.03 min, respectively) compared with the vehicle group (7.95 ± 0.78 min, P<0.001). In addition, CMT, LMK02, and OGT treatment (0.63 ± 0.01, 0.66 ± 0.02 and 0.67 ± 0.01 mg/mm, respectively) significantly reduced thrombus weight compared with the vehicle treatment (0.78 ± 0.03, P<0.001) and also alleviated collagen fiber damage (CMT; 28.40 ± 2.22%, LMK02; 30.79 ± 4.07%, OGT; 26.20 ± 1.48%) in the vessels injured by FeCl3. Therefore, CMT and LMK02 showed the greatest preventive activity in rat model of arterial thrombosis induced by FeCl3. Conclusions: These results provide experimental evidence for traditional use of herbal prescriptions, suggesting that CMT and LMK02 extracts could be used to prevent vascular injury and thrombosis in the early stages of dementia.

Anti-thrombotic Effects of Modified using a FeCl-induced Carotid Arterial Thrombosis Model

Objectives: The aim of this study was to examine the antithrombotic effects of the four herbal ingredients (Mume Fructus, MF; Santali Albi Lignum, SAL; Amomi Tsao-Ko Fructus, ATF; and Amomi Fructus, AF) of modified Jeho-tang (MJHT) in a ferric chloride (FeCl₃)-induced carotid arterial thrombosis model. Methods: Thirty minutes prior to a 35% FeCl₃ application, Sprague-Dawley (SD) rats were injected with saline, MF, SAL, ATF or AF (100 mg/kg, intraperitoneal injection), respectively. The effect of the MJHT ingredients was examined for time to occlusion (TTO) and thrombus weight (TW) in a FeCl₃-induced thrombosis model. Histological analysis was performed to examine the effect of the MJHT ingredients on collagen fiber damage using hematoxylin & eosin and Masson’s trichrome staining. Results: Compared with vehicle treatment, MF, SAL and ATF treatment delayed TTO (vehicle, 8.11 ± 0.60 min; MF, 16.67 ± 1.03 min; SAL, 17.50 ± 1.52 min and ATF, 13.33 ± 1.21 min; P Conclusions: Altogether, these results are the first evidence that the MJHT ingredients MF, SAL and ATF have the ability to prevent vascular damage and thrombus formation in FeCl₃-induced carotid arterial thrombosis.

α(1,3)-Fucosyltransferases FUT4 and FUT7 Control Murine Susceptibility to Thrombosis

The α(1,3)-fucosyltransferases, types IV and VII (FUT4 and FUT7, respectively), are required for the synthesis of functional selectin-type leukocyte adhesion molecule ligands. The selectins and their ligands modulate leukocyte trafficking, and P-selectin and its ligand, P-selectin glycoprotein ligand-1, can modulate hemostasis and thrombosis. Regulation of thrombosis by FUT4 and/or FUT7 activity was examined in mouse models of carotid artery thrombosis and collagen/epinephrine-induced thromboembolism. Mice lacking both FUT4 and FUT7 (Fut(-/-) mice) had a shorter time to occlusive thrombus formation in the injured carotid artery and a higher mortality due to collagen/epinephrine-induced pulmonary thromboemboli. Mice lacking P-selectin or P-selectin glycoprotein ligand-1 did not have a prothrombotic phenotype. Whole blood platelet aggregation was enhanced, and plasma fibrinogen content, clot weight, and clot strength were increased in Fut(-/-) mice, and invitro clot lysis was reduced compared with wild type. Fut4(-/-), but not Fut7(-/-), mice had increased pulmonary thromboembolism-induced mortality and decreased thromboemboli dissolution invivo. These data show that FUT4 and FUT7 activity regulates thrombosis in a P-selectin- and P-selectin glycoprotein ligand-1-independent manner and suggest that FUT4 activity is important for thrombolysis.

Ibudilast, a Phosphodiesterase Inhibitor, in Combination with Low-dose Aspirin Potently Inhibits Guinea Pig Carotid Artery Thrombosis without Extending Bleeding Time and Causing Gastric Mucosal Injury

A combination of low-dose aspirin (ASA) and a phosphodiesterase inhibitor has been clinically tried for the secondary prevention of atherothrombotic diseases. The in vivo antithrombotic property of ibudilast (CAS 50847-11-5), a phosphodiesterase 4 (PDE4) inhibitor, was evaluated in a photochemically-induced guinea pig carotid artery thrombosis model in combination with low-dose ASA. The time required to decrease the carotid artery blood flow to the reading "zero" was defined as the time to occlusion (TTO) of the artery through thrombogenesis. Each independent use of ASA (300 mg/kg, p.o.) and ibudilast (3 and 10 mg/kg, p.o.) significantly prolonged the TTO, and ASA (300 mg/kg) significantly increased bleeding time (BT) and gastric mucosal injury. A selective PDE4 inhibitor rolipram (1 and 5 mg/kg, p.o.) tended to prolong the TTO without extending BT. ASA (100 mg/kg) plus ibudilast (3 mg/kg) and ASA (100 mg/kg) plus rolipram (5 mg/kg) markedly prolonged the TTO compared with each agent alone. Interestingly, ASA (100 mg/kg) plus ibudilast (3 mg/kg) caused a longer TTO than ASA (300 mg/kg) alone, without significant extension of BT and gastric mucosal injury as observed in ASA (300 mg/kg). These results indicate that the combination of low-dose ASA and ibudilast has a more potent antithrombotic effect than ASA alone without increasing bleeding tendency and gastric mucosal injury. The potent in vivo antithrombotic effect of this combination may be brought about by an action that is associated with PDE4 inhibition of ibudilast.

Abstract 414: Combined Tissue Factor/Factor VIIa Inhibitor and Acetylsalicylic Acid Show Increased Antithrombotic Potency Without Affecting Bleeding Time

Since increased tissue factor (TF) in human atherosclerotic plaques is believed to trigger thrombotic events, TF/factor VIIa (FVIIa) inhibition might have prophylactic potential for atherothrombosis such as acute coronary syndrome (ACS). Although recent clinical observation by rivaroxaban provides a scientific rationale for the addition of anticoagulant to current antiplatelet treatment, these might have potential to increase bleeding. It is therefore important that emerging anticoagulants demonstrate additive antithrombotic effects with no concomitant increase in bleeding for ACS indication. We recently discovered ER-410660, a potent TF/FVIIa inhibitor. It suppressed venous thrombosis without affecting bleeding time and suggested that ER-410660 could act as a novel anticoagulant with a low bleeding risk. In the photochemically-induced thrombosis (PIT) model, the thrombus after vessel-wall injury has been developed by the deposition of TF, platelets, and fibrin. Therefore, we hypothesized that ER-410660 would be effective in the PIT model and could show enough margin against bleeding. The antithrombotic effects of ER-410660 and acetylsalicylic acid (ASA), both alone and in combination, was assessed using a rat PIT model. We also measured the bleeding time of ER-410660 in combination with ASA using a rat tail-transection model. Intravenous (iv) pretreatment with ER-410660 prolonged the time to occlusion (TTO) of the artery. The TTO was doubled at 0.99 mg/kg (95% CI, 0.51-1.9). Orally administered ASA also showed antithrombotic effect, and the TTO was doubled at approximately 12 mg/kg. Combined administration of 1 mg/kg ER-410660 (iv) and 10 mg/kg ASA (orally) showed an additive antithrombotic effect, with no cessation of blood flow during the 30-min observation period. Intravenous administration of ER-410660 (0.6, 2, and 6 mg/kg) to rats treated with ASA (10 mg/kg) did not significantly increased when compared with ASA alone, even at the highest dose. In conclusion, a TF/FVIIa inhibitor suppressed arterial thrombosis without affecting the bleeding time associated with ASA. Owing to these favorable characteristics, anticoagulation via TF/FVIIa inhibition may be a superior approach to the prophylaxis of atherothrombotic diseases.

Arterial Thrombosis in Mice with Factor V Leiden Mutation

The factor V Leiden (FVL) mutation has been demonstrated to be associated with the development of venous thrombosis in humans. Whether such a propensity also exists in the arterial circulation remains controversial. In an effort to minimize the variability that clouds the clinical study of arterial thrombosis, we studied FVL-associated arterial thrombosis in an experimental model of homozygous, heterozygous, and wild-type mice. Heterozygous FVL mice were crossbred to C57BL/6J mice over several generations. The genotypes of the resulting three genotype groups (wild type, heterozygous FVL, and homozygous FVL) were blinded to the investigators. Arterial injury was produced with the injection of ferric chloride into an isolated segment of carotid artery. Arterial thrombosis was assessed with an ultrasonic flow probe and the time to occlusion (TTO) was recorded. The carotid artery occluded within 60 minutes of injury in 72 of the animals studied (97.3%). The carotid artery remained patent at 60 minutes in the remaining two animals, both of whom were subsequently found to be genotypically wild type. There was a statistically significant relationship between TTO and genotype (p = .002). TTO was greatest in the wild-type mice (p < .001 vs heterozygous, < .001 vs homozygous) and least in the homozygotes (p < .001 vs heterozygotes). Increased thrombogenicity is present in mice with the FVL mutation and is more prolonged in homozygotes than heterozygotes. These findings provide some corroboration to the clinical studies that suggest an increased risk of arterial events in patients with the FVL mutation.

YS02 Screening and Characterisation of Novel Platelet Candidate Genes Derived from Genome‐Wide Association Studies for Function in Relation to Haemostasis and Thrombosis in the Model Organism Danio Rerio

Complementary approaches of microarray, proteomics, genome wide association and platelet functional studies have identified novel platelet proteins which are candidates for playing a role in thrombosis or conferring risk of myocardial infarction. The function of many of the proteins encoded by these candidate genes is unknown, and will need to be determined. To address the need for high‐throughput screening of candidate genes in a model organism, we have investigated the suitability of the zebrafish (ZF; Danio rerio), a genetically tractable vertebrate with nucleated thrombocytes. It is known that mouse knockouts of critical pro‐thrombotic genes [e.g. coagulation factor (F) VIII, integrin αIIb (ITGA2B), or glycoprotein VI] have subtle phenotypes and do not bleed spontaneously. We hypothesised that the sensitivity of the ZF model to screen for loss of platelet function could be enhanced by concurrent knock down of the FVIII gene and a pro‐thrombotic platelet gene. To test this, the genes for FVIII, ITGA2B or both were silenced by antisense oligonucleotides (morpholinos). Embryos were then assessed for body plan and bleeding at 3 days post‐fertilization (dpf). In parallel, a thrombosis model using laser injury to initiate thrombus formation was developed. This permits time to adhesion (TTA), time to occlusion (TTO) and time to thrombolysis (TTL) to be determined in morpholino‐injected and wild type ZF. As expected, individual knockdown of the FVIII or ITGA2B genes did not lead to spontaneous bleeding. Knockdown of the ITGA2B gene did, however, result in morphological malformations. Simultaneous knockdown of both genes resulted in a clear and reproducible bleeding phenotype visible in ∼20% of embryo's. Using the laser injury model, venous thrombosis could be reproducibly induced in 3 and 6 dpf wild‐type ZF larvae with partial but not complete thrombolysis occurring within 5–10 min. In conclusion, ZF are a fast and informative way to screen for platelet function modifying genes.

Effects of SanOrg123781A, a synthetic hexadecasaccharide, in a mouse model of electrically induced carotid artery injury: synergism with the antiplatelet agent clopidogrel.

SanOrg123781A is a synthetic hexadecasaccharide that displays antithrombin-dependent inhibition of factor Xa and thrombin and potent antithrombotic effects. The antithrombotic activity of SanOrg123781A has been studied in a new mouse model of arterial thrombosis, where thrombus formation was induced by the application of an electrical current to the adventitial surface of a carotid artery. In this model, antiplatelet agents such as the ADP-receptor antagonist clopidogrel (30 mg/kg, p.o. 2 h before stimulation) and the GpIIb/IIIa antagonist SR121566A [3-(N-[4-(4-[amino(imino)methyl]phenyl)-1,3-thiazol-2-yl]-N-[1-(carboxymethyl)piperidin-4-yl]amino)propionic acid, trihydrochloride] (0.3 mg/kg, i.v. 5 min before stimulation) strongly prolonged the time to occlusion (TTO) (761 and 473% increases, respectively), whereas aspirin was devoid of antithrombotic activity. Standard heparin (2 mg/kg, i.v.), the low molecular weight heparin enoxaparin (20 mg/kg, i.v.), and the synthetic, antithrombin-dependent inhibitor of factor Xa fondaparinux (10 mg/kg, i.v.) were also active in this model (742, 707, and 602% TTO increases, respectively). Interestingly, SanOrg123781A was active at much lower doses than the other oligosaccharides (554% increase in TTO at 0.3 mg/kg, i.v. 5 min before stimulation). Low doses of SanOrg123781A administered in combination with low doses of clopidogrel led to a marked increase in TTO, which was statistically more important than the additive effects of the two compounds given alone. These results indicate that SanOrg123781A exerts a potent antithrombotic activity in a mouse model of arterial thrombosis when compared with reference compounds and show that the combination of SanOrg123781A with clopidogrel leads to a marked synergistic antithrombotic effect.

Functional thrombomodulin deficiency causes enhanced thrombus growth in a murine model of carotid artery thrombosis.

Thrombomodulin (TM) bound thrombin initiates the protein C anticoagulant pathway and defects in TM result in enhanced coagulation. Recent studies suggest a role for TM in arterial vascular disease. In order to corroborate this association we studied arterial thrombus formation in mice with a functional TM defect. We used mice homozygous for a (404)Glu-to-Pro mutation in the TM gene (TM(pro/pro)) and compared these with wildtype littermates in a model of FeCl(3) induced carotid artery thrombosis. Time-to-occlusion (TTO) was assessed by arterial blood flow measurement, using a Doppler flow probe. Complete occlusion occurred in 8/10 (80%) TM(pro/pro) mice and in 3/11 (27%) littermate controls. Mean time to occlusion (TTO) [+/- SE] was 767 +/- 196 s in the F2-TM(pro/pro) mice, versus 1507 +/- 159 s in controls (p = 0.007, Mann Whitney U test). Histology and immunostaining for tissue factor did not reveal any differences in thrombus morphology or thrombogenicity between the two groups. These data confirm and extend the finding that a functional deficiency in TM results in enhanced thrombus formation in a murine model of carotid artery thrombosis and support a role for TM defects in arterial thrombotic disease.

Assessment of ex vivo Pharmacodynamic Markers during Inhibition of Thrombosis by CI-1031 (ZK-807834), a Novel Direct Factor Xa Inhibitor

CI-1031 (ZK-807834) is a novel, synthetic factor Xa (FXa) inhibitor with a Ki of 0.11 nM against human FXa. In human plasma in vitro, CI-1031 doubled PT and aPTT at 0.23 and 0.49 µM, respectively. The in vivo antithrombotic effect of CI-1031 was evaluated in a veno-venous shunt model of thrombosis in anesthetized rabbits. After thrombus formation was verified in the first shunts, rabbits received either vehicle or CI-1031 intravenously (bolus injection of 60, 240, or 480 µg/kg followed by an infusion of 2, 8, or 16 µg/kg/min for 140 min, respectively). The second shunts were inserted after 20 min of infusion of CI-1031 or vehicle. CI-1031 dose-dependently prolonged time to occlusion (TTO) in the second shunts (35 ± 21, 62 ± 24, and 120 ± 0 min for the three dose groups, respectively, vs. 10 ± 1 min for vehicle). Thrombus mass (TM) was reduced in a dose-dependent manner by CI-1031 (42 ± 7, 27 ± 6, and 18 ± 4 mg vs. 50 ± 4 mg for vehicle). Maximal TM reduction was 70% with an IC₅₀ of 0.6 µg/ml. Among all the coagulation parameters tested, PT had the best correlation with plasma CI- 1031 concentration (r = 0.97). Ex vivo plasma anti-FXa activity was also well correlated with plasma concentration of CI-1031 and with PT (r = 0.96 and 0.98, respectively). These results indicate that CI-1031, which is currently undergoing clinical evaluation, is an effective antithrombotic compound with a favorable efficacy-to-bleeding ratio. In addition, CI-1031 concentration in plasma can be monitored using PT or anti-Xa assays, thereby providing reliable methods to ensure safe and accurate dose titration of CI-1031.

Antithrombotic effect of LB-30057 (CI-1028), a new synthetic thrombin inhibitor, in a rabbit model of thrombosis: comparison with inogatran.

LB-30057 (CI-1028) is a novel, orally bioavailable, direct thrombin inhibitor with a Ki of 0.38 nM against human thrombin. The effects of LB-30057 on thrombus formation and hemostasis were evaluated in a veno-venous shunt model of thrombosis in rabbits, and compared with inogatran, another direct inhibitor of thrombin. Each compound was studied at 5 or 6 different doses with 5 or 6 rabbits in each group. After administration as a bolus i.v. injection followed by continuous infusion, both LB-30057 and inogatran dose-dependently inhibited thrombus formation, which was measured as an increase in time to occlusion (TTO) and a decrease in thrombus weight. Both compounds also improved vena caval blood flow and reduced the overall incidence of thrombotic occlusion. LB-30057 significantly prolonged TTO from 23 +/- 4 min (before dose) to 110 +/- 10 min at the highest dose (0.7 mg/kg + 47 microg/kg/min) (p < 0.001), and reduced thrombus weight from 57 +/- 2 mg to 15 +/- 5 mg (p < 0.001). Occlusive thrombus formed in only one of six rabbits that received the highest dose of LB-30057 (vs. 13/13 in the control group, p < 0.01). At the dose that produced the maximum antithrombotic effect (0.7 mg/kg + 47 microg/kg/min), LB-30057 increased aPTT and bleeding time approximately 2-and 2.5-fold above baseline, respectively. On a gravimetric basis, LB-30057 and inogatran displayed comparable in vivo antithrombotic efficacy. When compared to equally effective anti thrombotic doses of inogatran, LB-30057 caused less prolongation in aPTT, had no effect on PT, and tended to have less of effect on bleeding time. These results indicate that LB-30057 is an effective antithrombotic compound and it appears to have a better benefit/risk profile than inogatran in this experimental model.