FeCl3-induced Arterial Thrombosis Model Research Articles

Antithrombotic Effect of Sorbus commixta in an Arterial Thrombosis Mouse Model

Sorbus commixta Hedlund ( S. commixta) is known to possess cardiovascular protective activity. However, its effect on thrombosis has not been reported, although thrombosis plays a primary role in diverse circulatory diseases. Thus, this study was performed to assess the antithrombotic activity of dried S. commixta fruit extract. Mice were fed with a diet containing 8.3% extract for 4 weeks, which corresponded to the consumption of approximately 1000 mg extract/kg/day. The antithrombotic effect was tested in a FeCl3-induced arterial thrombosis model. Additionally, the effects on platelet aggregation and plasma coagulation were examined in vitro using washed platelets (WP) and isolated blood plasma, respectively. Thrombotic vascular occlusion time was significantly prolonged by the 4-week administration of the extract. Treatment of WP with the extract resulted in a decrease in thrombin-induced aggregation, which was concentration-dependent over 10 to 100 μg/mL and was not attributed to the direct inhibition of thrombin. The extract did not affect blood plasma coagulation, which was assessed by measuring prothrombin time and activated partial thromboplastin time. S. commixta is capable of preventing arterial thrombosis and such an effect may be attributed to the inhibition of platelet aggregation.

Pyruvate Dehydrogenase Kinase Modulates Platelet Function and Thrombosis

Background and objective: Pyruvate dehydrogenase kinases (PDK 1-4) are mitochondrial enzymes that regulate the activity of pyruvate dehydrogenase (PDH) complex, which converts pyruvate (end product of glycolysis in cytosol) to acetyl coenzyme A that is further oxidized to produce energy (oxidative phosphorylation; OXPHOS). Like most normal cells, resting platelets rely primarily on OXPHOS to generate ATP, whereas activated platelets exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen, a phenomenon referred to as the Warburg effect in tumor cells). It seems paradoxical that why platelets prefer aerobic glycolysis instead of OXPHOS. The rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which is well suited for high-energy demand of activated platelets to form blood clot. We tested the hypothesis that manipulating aerobic glycolysis (metabolic reprogramming), by down regulating PDK activity in the mitochondria of platelets, will inhibit platelet function and thereby thrombosis.Methods: Global knock out or floxed PDK mice does not exists, therefore we used dichloroacetic acid (DCA), a potent inhibitor of PDKs, known to divert metabolism back from aerobic glycolysis to OXPHOS. DCA has been successfully used in clinical trials to treat several type of cancer. In human and murine platelets, using standardized platelet in vitro assays, we determined the mechanistic role of PDK in regulating platelet function. Rate of glycolysis and mitochondrial respiration was measured using the Seahorse XF24 extracellular flux analyzer. Using intravital microscopy, thrombus growth was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid artery thrombosis) models.Results: Using, extracellular flux analysis, we first confirmed that activated platelets have higher aerobic glycolysis. With both human and mice platelets, we found that DCA (10 and 25 mM) significantly inhibited in vitro platelet aggregation to collagen and convulxin-induced signaling, but not thrombin or ADP-induced signaling in a dose-dependent manner (P<0.05 vs. vehicle treated). DCA also inhibited static adhesion of washed platelets to collagen in dose-dependent manner. Furthermore, we found that DCA significantly inhibited tyrosine phosphorylation of Syk and PLCγ2 in the GPVI signaling pathway with both mice and human platelets (P<0.05 vs. vehicle treated). Metabolic profiling, using extracellular flux analysis, showed that DCA significantly inhibited extracellular acidification rate in convulxin-stimulated platelets in dose-dependent manner. Using microfluidics flow chamber, we found that both mouse and human whole blood treated with DCA formed small thrombi when perfused over collagen for 10 min at a arterial shear rate of 1500s-1 (P<0.05 vs. vehicle control), suggesting that inhibiting PDK with DCA limits thrombus growth. Finally, wild-type mice pre-treated with DCA (600 mg/Kg body weight) were less susceptible to thrombosis in FeCl3-induced arterial thrombosis model (P<0.05 vs. control, N=10 mice/group), without altered hemostasis (tail-bleeding assay). The precise mechanism how aerobic glycolysis modulates GPVI signaling pathway is unclear and is currently under investigation.Conclusion: Our results suggest that PDK present in mitochondria regulates platelet function and thrombus growth, most likely via GPVI signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

Antithrombotic and Antiplatelet Effects of Cordyceps militaris

Cordyceps is a genus of ascomycete fungi and is well known as one of the important medical fungi in Chinese, Korea, and other Asian countries, because of its various beneficial effects on human health. The pharmacological activities of Cordyceps extract are mainly focused on anti-cancer, anti-metastatic, and immune modulating effects. In the present study, we investigated whether the antiplatelet effect of ethanol extract of cultured Cordyceps militaris (CMEE) with FeCl3-induced arterial thrombosis model. We observed that CMEE exhibited a significant inhibitory effect against ADP and collagen-induced platelet aggregation. However, there were no significant differences in prothrombin time (PT) and activated partial thromboplastin time (aPTT). These results suggest that antithrombotic activity of CMEE is related to antiplatelet effect rather than anticoagulation effect, and CMEE may be a positive effect on improving blood circulation against vessel injury and occlusion.

ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

Abstract 55: Traf3 Negatively Regulates Platelet Activation and Thrombosis

Platelets express CD40 ligand (CD40L) and are a major source of soluble CD40L. CD40L has been shown to potentiate platelet activation and thrombus formation, involving both CD40-dependent and -independent mechanism. CD40L-mediated platelet activation requires the family of TNF receptor-associated factor 2 (TRAF2). Here we show that platelet also express TRAF3, which plays a negative role in regulating platelet activation. Thrombin- or collagen-induced platelet aggregation and secretion are increased in TRAF3 knockout mice. The expression of integrin αIIbβ3, GPVI, and PAR4 was similar between TRAF3 knockout platelets and wild type controls, suggesting that increased platelet activation in the TRAF3 knockout mice is not due to increased expression of platelet receptors. TRAF2 expression was not affect by deletion of TRAF3 eitehr. Using the FeCl 3 -induced arterial thrombosis model, we found that time to formation of thrombi in was significantly shortened in TRAF3 knockout mice. Thus, TRAF3 plays a negative role in platelet activation and in thrombus formation in vivo.

Factor XII full and partial null in rat confers robust antithrombotic efficacy with no bleeding.

This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (∼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1 mg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1 mg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1 mg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5 μmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.

Antithrombotic effects of the effective components group of Xiaoshuantongluo formula in vivo and in vitro

The present study was designed to investigate the antithrombotic effects and underlying mechanisms of the effective components group (ECG) of Xiaoshuantongluo recipe (XECG) and to further verify the rationality and feasibility of ECG-guided methodology in traditional Chinese medicine (TCM) research. The arterial thrombosis model induced by ferric chloride (FeCl3) oxidation and the venous thrombosis model induced by inferior vena cava ligation were established to evaluate the antithrombotic potential of XECG. Our results indicated that XECG significantly prolonged the time to occlusion, activated partial thromboplastin time (APTT), and prothrombin time (PT), and markedly inhibited adenosine diphosphate (ADP)-induced platelet aggregation in the 20% FeCl3-induced arterial thrombosis model. The superoxide dismutase (SOD) activity was significantly increased and the levels of malondialdehyde (MDA) and nitric oxide (NO) were dramatically decreased in the plasma of arterial thrombosis rats after XECG treatment for 12 days. Furthermore, XECG markedly reduced the weight of thrombus formed by inferior vena cava ligation. Additionally, XECG exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and protective effect on mitochondrial lipid peroxidation. In summary, XECG played an important role in the prevention of thrombosis through interacting with multiple targets, including inhibition of platelet aggregation and coagulation and repression of oxidative stress. The ECG-guided methodology was validated as a feasible tool in TCM research.

Zucker Diabetic Fatty rats exhibit hypercoagulability and accelerated thrombus formation in the Arterio-Venous shunt model of thrombosis

IntroductionDiabetes is a significant risk factor for thrombosis. The present study aimed at assessing coagulability, platelet reactivity, and thrombogenicity of the diabetic female Zucker Diabetic Fatty (ZDF) rat model and its relevance in studying antithrombotic mechanisms. Materials and MethodsThe basal coagulant state in ZDF rats was evaluated by clotting times, thromboelastography, and thrombin generation assay. A 14-day treatment with dapagliflozin in ZDF rats was pursued to investigate if glycemic control can improve coagulability. Thrombus formation in the Arterio-Venous (A-V) shunt model and the FeCl3-induced arterial thrombosis model was studied, with the antithrombotic effect of apixaban in the former model further investigated. ResultsZDF rats exhibited significantly shortened clotting times, enhanced thrombin generation, and decreased fibrinolysis at baseline. Effective glycemic control achieved with dapagliflozin did not improve any of these parameters. ZDF rats displayed accelerated thrombus formation and were amenable to apixaban treatment in the A-V shunt model albeit with less sensitivity than normal rats. ZDF rats exhibited less platelet aggregation in response to ADP, collagen and PAR-4, and attenuated thrombotic response in the FeCl3 model. ConclusionsZDF rats are at a chronic hypercoagulable and hypofibrinolytic state yet with compromised platelet reactivity. They display accelerated and attenuated thrombosis in the A-V shunt and FeCl3 model of thrombosis, respectively. Results shed new light on the pathophysiology of the ZDF rat model and illustrate its potential value in translational research on anticoagulant agents in diabetics. Caution needs to be exerted in utilizing this model in assessing antiplatelet mechanisms in diabetes-associated atherothrombosis.

In Vivo Anti-Thrombotic Potency of Engineered Activated Protein C Variants.

Activated protein C (APC) has both anticoagulant activity via inactivation of factors Va and VIIIa and cytoprotective activities on cells that include anti-apoptotic and anti-inflammatory activities, alterations of gene expression profiles and protection of endothelial barrier function. The relative importance of APC's anticoagulant activity vs. APC's direct cytoprotective effects on cells for reduction of mortality in severe sepsis patients and protective effects in animal injury models is not entirely clear. In this current study, genetically engineered APC variants with different activity spectra were tested for in vivo anti-thrombotic potency. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191-193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, ASH 2006, J Exper Med , 2007). In contrast to 5A-APC, mutation of E149 to A in APC increased anticoagulant activity in clotting assays while diminishing cytoprotective effects on cells. Murine APC variants, E149A-APC and 5A-APC (KKK192-194AAA + RR230/231AA) were used to determine in vivo anti-thrombotic potency in an acute carotid artery thrombosis model in mice, using FeCl3-induced injury. Under the conditions employed, first occlusion occurred within 3.5 min (mean: 171 sec; range 150-200 sec) in the absence of APC. Murine wild type (wt)-APC effectively delayed time to first occlusion in a dose-dependent manner (0 to 1.8 mg/kg wt-APC; mean: 561 sec; range 400-960 sec). The E149A-APC variant exhibited potent in vivo anti-thrombotic activity (1.8 mg/kg; mean: 1020 sec; range 540- >1600 sec) and was superior to wt-APC as evident by the absence of appreciable occlusion in 2/6 E149A-APC vs. 0/6 wt-APC treated animals. Thus E149A-APC was hyperactive in plasma clotting assays as well as hyperactive in an acute FeCl3-induced arterial thrombosis model. To test the hypothesis that an increased protein S cofactor activity contributed to its enhanced anticoagulant activity, E149A-APC anticoagulant activity was tested in normal and protein S deficient plasma. Compared to wt-APC, E149A-APC showed 3-fold increased anticoagulant activity in normal plasma but not in protein S deficient plasma. In studies with purified proteins, protein S concentrations required for half-maximal stimulation of factor Va inactivation by E149A-APC were 3-fold lower compared to wt-APC, whereas factor Va inactivation rates were indistinguishable in the absence of protein S. These data support our hypothesis that increased protein S cofactor activity is, at least partially, responsible for the observed hyper anticoagulant and anti-thrombotic potency in vitro and in vivo. In contrast to E149A-APC, 5A-APC was severely deficient in anti-thrombotic activity in vivo. Even at concentrations up to 8 mg/kg, 5A-APC (mean: 245 sec; range 172-300 sec) failed to delay significantly time to first occlusion compared to no APC. These data highlight important distinctions between structural requirements for APC's anticoagulant, anti-thrombotic and cytoprotective functions. Engineered APC variants with differentially altered activities (e.g. cytoprotective vs. anticoagulant) may lead to safer or better therapeutic APC variants for a variety of indications including sepsis, ischemic stroke or other pathologies.

Factor V Leiden mutation is associated with enhanced arterial thrombotic tendency in lean but not in obese mice

The homozygous factor V Leiden mutation is associated with enhanced venous thrombotic risk. Obesity is a major risk factor for development of thrombotic cardiovascular disease. It was the objective of this study to investigate whether obesity affects the thrombotic risk associated with the mutation. Male mice with homozygous factor V Leiden mutation (Arg 504 to Gln) (FVQ/Q) and corresponding wild-type (WT) mice were kept on a standard fat diet (SFD) or high fat diet (HFD) for 14 weeks, and femoral artery thrombosis was induced by FeCl3 treatment. As compared to SFD, HFD feeding for 14 weeks resulted in significantly higher body weight and fat mass associated with adipocyte hypertrophy, which were, however, similar for both genotypes. In the FeCl3-induced arterial thrombosis model, FVQ/Q mice kept on SFD had a 40% shorter occlusion time (p = 0.015) and 40% lower blood flow (p = 0.03), as compared to WT mice. However, on HFD the occlusion time and blood flow were not significantly different for both genotypes. This finding could not be explained by differential changes of coagulation factors in either genotype fed on SFD or HFD. In conclusion, on SFD, but not on HFD, the factor V Leiden mutation is associated with enhanced thrombotic tendency after FeCl3 injury of the femoral artery, suggesting that in this model obesity rescues the increased thrombotic risk associated with the factor V Leiden mutation.

Increased venous versus arterial thrombosis in the Factor V Leiden mouse

Background Deep vein thrombosis (DVT) occurs with high prevalence in association with the Factor V Leiden (R506Q) mutation, whereas most evidence suggests no correlation with clinical arterial thrombosis. Objective This study compared arterial to venous thrombosis in the mutationally analogous Factor V Leiden mouse. Methods Three separate vascular thrombosis models were evaluated in F v +/+ (wild-type), F v Q/+ (heterozygous) and F v Q/Q (homozygous) Factor V Leiden mice. Results In a FeCl 3-induced arterial thrombosis model, no statistical differences among the three genotypes were found in the time to thrombotic occlusion. In contrast, F v Q/+ and F v Q/Q mice demonstrated larger femoral vein thrombi at 30 and 60 min compared to wild-types, with F v Q/Q mice having statistically larger thrombi than both wild-type and F v Q/+ mice at 10 and 60 min and 24 h ( p < 0.05). In a model of thrombotic occlusion following arterial and venous anastomotic repair, both F v Q/+ and F v Q/Q mice had higher rates of venous thrombosis than wild-types, but only F v Q/Q homozygotes showed a statistically greater arterial occlusion rate than wild-types. Conclusion The Factor V Leiden mouse demonstrated a greater propensity for venous vs. arterial thrombosis, paralleling clinical epidemiologic findings and supporting its use for research on deep vein thrombosis.