Ferric Chloride-induced Thrombosis Research Articles

Nanogold-coated stent facilitated non-invasive photothermal ablation of stent thrombosis and restoration of blood flow.

In-stent restenosis (ISR) and stent thrombosis (ST) are the most serious complications of coronary angioplasty and stenting. Although the evolution of drug-eluting stents (DES) has significantly restricted the incidence of ISR, they are associated with an enhanced risk of ST. In the present study, we explore the photothermal ablation of a thrombus using a nano-enhanced thermogenic stent (NETS) as a modality for revascularization following ST. The photothermal activity of NETS, fabricated by coating bare metal stents with gold nanorods generating a thin plasmonic film of gold, was found to be effective in rarefying clots formed within the stent lumen in various in vitro assays including those under conditions mimicking blood flow. NETS implanted in the rat common carotid artery generated heat following exposure to a NIR-laser that led to effective restoration of blood flow within the occluded vessel in a model of ferric chloride-induced thrombosis. Our results present a proof-of-concept for a novel photothermal ablation approach by employing coated stents in the non-invasive management of ST.

Modulation of Extravascular Binding of Recombinant Factor IX Impacts duration of Efficacy in Mouse Models.

There is an emerging concept that in addition to circulating coagulation factor IX (FIX), extravascular FIX contributes to hemostasis. Evaluate efficacy of extravascular FIX using animal models of tail clip bleeding and ferric chloride induced thrombosis. Mutant rFIX proteins with described enhanced (rFIXK5R) or reduced (rFIXK5A) binding to extracellular matrix were generated and characterized using in vitro aPTT, one stage clotting and modified FX assays. Using hemophilia B mice, pharmacokinetic parameters and in vivo efficacy of these proteins was compared against rFIX wild type protein (rFIXWT) in a tail clip bleeding and FeCl3-induced thrombosis model. Respective tissue disposition of FIX was evaluated using immunofluorescence. In vitro characterization demonstrated comparable clotting activity of rFIX proteins. Pharmacokinetic profile showed that rFIXK5A displayed highest plasma exposure compared to rFIXWT and rFIXK5R. Immunofluorescence evaluation of liver tissue showed that rFIXK5R was detectable up to 24h, where as rFIXWT and rFIXK5A were detectable only at 15 minutes. In the tail clip bleeding model, rFIXK5R displayed significant hemostatic protection against bleeding incidence for up to 72h post intravenous administration of 50 IU/kg, whereas the efficacy of rFIXK5A was already reduced at 24h. Similarly, in the mesenteric artery thrombus model, rFIXK5R and rFIXWT demonstrated prolonged efficacy compared to rFIXK5A. Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding (rFIXK5R) to extravascular space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, while rFIXK5A rapidly lost its efficacy despite higher plasma exposure.

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. We investigated whether CD69 was involved in brain damage following an ischemic stroke. We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

Defective collagen binding and increased bleeding in a murine model of von Willebrand disease affecting collagen IV binding

Essentials Defective binding to collagen IV has been seen in von Willebrand factor (VWF) A1 domain variants. We developed a murine model of defective VWF-collagen IV interactions with VWF variant p.R1399H. p.1399HH homozygous mice had decreased binding to collagen IV and increased bleeding times. p.1399HH homozygous mice had increased time to thrombosis and decreased platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) binding to typeIV collagen occurs via the VWF A1 domain, with p.R1399H being the most common VWF variant affecting this interaction. Objectives We generated a murine model of 1399H VWF to investigate its invivo effects. Methods Mice expressing the murine 1399H variant were generated via gene targeting in embryonic stem cells. VWF antigen and VWF collagen binding were measured with ELISA. Tail bleeding time assays were performed by clipping a 3-mm segment. Ferric chloride-induced thrombosis was measured via ultrasound in the carotid artery. Platelet aggregation in response to collagensI and IV was measured. VWF-dependent platelet adhesion to collagenIV was measured under flow. Results Breeding of heterozygous p.R1399H and homozygous p.1399HH mice was observed to follow normal Mendelian ratios. No spontaneous bleeding was observed for any of the offspring. VWF expression was normal, but VWF binding to collagenIV was decreased in both heterozygous and homozygous offspring. Blood loss following tail resection was increased for p.1399HH mice, and occlusion times following ferric chloride-induced thrombosis were prolonged. Platelet aggregation was unaffected, but platelet adhesion to collagenIV under flow was diminished for p.1399HH mice. Conclusions These results show that a decrease in the ability of 1399H VWF to bind collagenIV under static conditions corresponds to a decrease in binding under flow conditions, an increased bleeding time, and a prolonged time to thrombosis. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagenIV binding.

Optimizing outcome measurement with murine ferric chloride-induced thrombosis.

: The murine FeCl3 model is a widely used model for studying arterial thrombosis, yet provides limited information from each mouse, often only a single time point for the onset of occlusion (defined as the time to occlusion; TTO). To optimize data from the murine ferric chloride model of thrombosis. FeCl3 injury was induced in the carotid arteries of wild-type and Factor IX (FIX) knockout mice, with infusion of recombinant FIX (rFIX) to normalize FIX deficiency at various times around FeCl3 injury. The TTO was recorded as a percentage of baseline flow as occlusion continued to zero flow, with identification of reflow events. The TTO among the treatment groups of FIX-deficient mice showed no statistical differences, except with physiological saline-treated FIX-deficient mice and those receiving delayed treatment. Incidences of occlusion were 100% for wild-type mice and FIX-deficient mice receiving slow infusions of rFIX at early times around the FeCl3 application. In contrast, only 68% of FIX-deficient mice achieved occlusion with preinfusion of rFIX and none occluded with delayed rFIX infusion. A majority of occluded vessels exhibited reflow events, with significantly lower incidence for slow infusion of rFIX starting 4 min after FeCl3 application in comparison with preinjury bolus, demonstrating characterization of a differential response to timing and infusion rates of treatment. Simple use of the time to occlusion may not maximize data available from the FeCl3 arterial thrombosis model. Inclusion of documenting reflow events can extend the useful data obtained with application of this model.

Reduced nicotinamide adenine dinucleotide phosphate inhibits rat platelet aggregation and p38 phosphorylation

Previous studies found that reduced nicotinamide adenine dinucleotide phosphate (NADPH) protected neurons against ischemia/reperfusion-induced injury. In addition to ROS reduction and ATP increment, preliminary data suggested that NADPH inhibited ADP and thrombin-induced platelet aggregation. As the effect of NADPH on platelet function was not reported by other investigators, the actions of NADPH on platelet function and mechanisms of actions were investigated in the present study. In vitro studies, the effects of different concentrations of NADPH on platelet aggregation induced by ADP (10 μM), thrombin (0.05 U/mL) or AA (50 μM) were determined. The results showed that NADPH could inhibit platelet aggregation induced by ADP, thrombin or AA in a concentration dependent manner. When the inhibitory effects of NAD+, NADH, NADP+ and NADPH on platelet aggregation were compared, NADPH demonstrated the relatively best effect on platelet aggregation. In vivo studies, the effects of NADPH on platelet aggregation, tail bleeding time, coagulation response and ferric chloride-induced thrombosis were determined in mice or rats. The maximum aggregation rate of platelets of rats injected with NADPH (5 mg/kg) was lower than platelets from control rats. NADPH transiently prolonged tail bleeding time in mice at 30 min after the injection of NADPH (7.5 mg/kg), while aspirin (15 mg/kg) significantly prolonged the tail bleeding time in mice at all time points examined. NADPH (5 mg/kg), as well as aspirin (10 mg/kg), had no effect on coagulation response in rats. Using a FeCl3-induced abdominal aorta injury thrombosis model, administration of NADPH (5 mg/kg) significantly delayed the onset of vessel occlusion, while aspirin (10 mg/kg) almost completely prevented the vessel occlusion. With microscopic examination the thrombi in injured vessel sections of rats received NADPH were much smaller and less dense than that of rats received vehicle treatment. ADP induced an increase in phosphorylation of p38 and the effect was markedly inhibited by the p38 inhibitor SB203580. Similarly, NADPH also inhibited ADP-induced phosphorylation of p38. Similar to NADPH, SB203580 robustly inhibited ADP- and thrombin-induced platelet aggregation. In addition, NADPH also reduced ADP-induced increases in ROS in platelets. The current results demonstrated that NADPH inhibited platelet aggregation, oxidative stress and p38 phosphorylation, suggesting that NADPH might be a novel compound for management of high risk of cardiovascular disease.

RLj-RGD3, a novel recombinant toxin protein from Lampetra japonica, prevents coronary thrombosis-induced acute myocardial infarction by inhibiting platelet functions in rats

Recombinant Lampetra japonica RGD-peptide (rLj-RGD3), a soluble protein containing three RGD sequences, was acquired from the oral salivary glands of Lampetra japonica using recombinant DNA technology. The aim of this study was to investigate the protective effects of rLj-RGD3 against acute myocardial infarction (AMI) induced by coronary artery thrombosis, as well as the underlying mechanisms. A rat model of AMI caused by ferric chloride-induced thrombosis on the surface of the left anterior descending (LAD) coronary artery was successfully established. Rats were given various doses of rLj-RGD3 (12 μg/kg, 24 μg/kg and 48 μg/kg) via sublingual intravenous delivery 10 min before AMI. ST segment elevation was recorded by electrocardiogram (ECG) until the end of the model. Left ventricular Evans blue content and histopathological changes were examined. Blood samples were collected to determine 5-hydroxytryptamine (5-HT), β-thromboglobulin (β-TG), platelet factor 4 (PF4) and cAMP levels. The effects of rLj-RGD3 on platelet aggregation, adhesion and intracellular calcium concentrations were also measured. rLj-RGD3 significantly reduced ST segment elevation, prevented thrombus formation in the coronary artery and decreased Evans blue content in the left ventricular myocardium. Meanwhile, rLj-RGD3 exerted an inhibitory effect on adenosine diphosphate (ADP)-induced platelet aggregation and blocked platelet adhesion to collagen. Treatment with rLj-RGD3 prevented 5-HT, β-TG and PF4 release and significantly elevated intracellular cAMP levels in a dose-dependent manner but decreased the level of cytosolic-free Ca2+, an aggregation-inducing molecule. These results show that rLj-RGD3 can effectively reduce coronary thrombosis in AMI rats by strongly inhibiting platelet function, indicating that the recombinant RGD toxin protein rLj-RGD3 may serve as a potent clinical therapeutic agent for AMI.

Acoustophoretic purification of platelets: feasibility and impact on platelet activation and function

Purity, limited platelet activation, and preservation of platelet function are important stakes of preparation of platelet concentrates (PC) for clinical use. In fact, contaminating red blood cells and leukocytes, as well as activated and/or poorly functional platelets in PC, represents a risk of poor efficiency and adverse side effects during platelet transfusion. Therefore, optimization of preparation and storage of PC is still an active field of research. Shear-induced platelet activation is an unwanted side effect of the hard-spin (up to 5000g) step of centrifugation-based methods currently used in blood banks to prepare PC from whole blood samples. Here, we evaluated the effectiveness of an acoustic-based fractionation device for the isolation of human platelets from whole blood bags. The purity, activation status, and functionality of platelets isolated by acoustopheresis were compared with those of platelets isolated using a reference protocol known to produce limited platelet activation and consisting of two consecutive soft-spin centrifugations (120g and 1200g). Platelet concentration and purity were determined using an automated hematology analyzer. Platelet activation status and platelet reactivity to collagen and thrombin were assessed in flow cytometry by measurement of surface expression of P-selectin and activated integrin αIIbβ3. The ability of isolated platelets to incorporate into a thrombus when transfused to NOD/SCID mice was investigated by intravital microscopy using the ferric chloride-induced thrombosis model. Blood fractionation by acoustophoresis led to the elimination of more than 80% of red blood cells and leukocytes from the platelet fraction, whose mean purity was of 92.8 ± 12.8%. The activation status and reactivity to collagen and thrombin of acoustophoresis-isolated platelets were similar to those of platelets isolated by soft-spin centrifugation. Finally, acoustophoresis-isolated platelets were tethered, adhered to the vessel wall, and incorporated into a growing thrombus following ferric chloride-induced vascular injury. Together, our results indicate that acoustophoresis is a suitable method for the isolation of human platelets with minimal platelet activation and preservation of platelet function.

PO-16 - ASK1 regulates tumor lung metastasis and platelet functions

Apoptosis signal-regulating kinase 1 (ASK1) is a MAP3K in the JNK and p38 MAPK pathways and responds to various stresses. Accumulating evidence indicates that ASK1 plays important roles in tumorigenesis by regulating apoptosis and inflammation. However, little is known about ASK1's roles in tumor metastasis. To investigate ASK1's roles in tumor metastasis. We performed experimental lung metastasis model by intravenous injection of Lewis lung carcinoma cells constitutively expressing luciferase (3LL-Luc2 cells). As for the analysis of platelet functions, tail bleeding assay and ferric chloride-induced thrombosis model were utilized. We measured the transition of luciferase activity of the lung lysates up to 7 days as an indicator of lung metastasis. ASK1-/- mice showed markedly lower luciferase activity as early as 3 hours after injection compared to WT mice; hence ASK1 appears to be involved in the early stage of tumor lung metastasis, which is prior to the extravasation of tumor cells. Platelets aggregate and adhere to tumor cells in the early stage and are known to support hematogenous metastasis. ASK1-/- mice were normal in hematological parameters including platelet number, while analysis by western blot revealed that platelets of ASK1-/- mice exhibited markedly reduced phosphorylation of JNK and p38, both of which have been reported to regulate platelet functions such as platelet aggregation. We found that platelets of ASK1-/- mice were less responsive to specific aggregation agonists and that ASK1-/- mice showed bleeding tendency and defect in thrombosis. These phenotypes were also observed in megakaryocyte and platelet-specific ASK1 deficient mice. It is suggested that impaired platelet functions caused by ASK1 deficiency in platelets may attenuate tumor lung metastasis.

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

New Insights Into The Mechanisms Of Ferric Chloride-Induced Thrombosis: a Reductionist Microfluidic Approach

The ferric chloride (FeCl3) murine model of thrombosis is used extensively in hematology, in which application of ferric chloride (200 mM to 1M) to the adventitial side of mouse arteries results in occlusive thrombosis. This effect has historically been attributed to the denudation of endothelial cells by way of free iron-induced oxidative stress. Recently, a well-designed SEM analysis revealed that endothelial cells actually remain intact, and that erythrocytes adhere to the endothelium prior to platelets (Barr, et al, Blood, 2013). The same study found that washed red blood cells exposed to FeCl3 adhere to endothelial cells in vitro. These novel findings inspired us to comprehensively investigate the mechanisms of FeCl3-induced thrombosis via an in vitro reductionist approach: interrogating the effect of FeCl3 on individual blood components in the absence and presence of endothelial cells.To this end, a microfluidic channel was designed to approximately recreate the in vivo blood/FeCl3 interface (Fig 1). By using a microfluidic platform, we were able to tightly control ferric chloride influx; determine the shear rate in the “artery” ; visualize the effects of ferric chloride on isolated blood components in a controlled environment; and quantitatively track clot characteristics such as composition, size, and time to occlusion. We found that a wide range of ferric chloride concentrations causes aggregation of whole blood in our system, even in the absence of endothelial cells. In addition, FeCl3 causes “clotting” of plasma proteins and blood cells in a dose-dependent manner (Fig 2A). Once aggregation is initiated, however, FeCl3 concentration does not affect the time required for stable clot formation. Interestingly, at low concentrations, the presence of blood cells are the slowest to form clots while cell/plasma mixtures (platelet-rich plasma and whole blood) are the fastest, perhaps due to plasma proteins forming net-like structures that enhances cell binding and aggregation. (Fig 2B) [Display omitted] [Display omitted] This universal aggregation effect of FeCl3 led us to posit a charge-based hypothesis. Mechanistically, as Fe3+ ions bind negatively charged surfaces, such as cell membranes and plasma proteins, the intrinsic charge equilibrium of blood may shift, resulting in protein and cell aggregation. Indeed, erythrocyte suspensions exposed to Al3+, which is chemically similar to Fe3+, resulted in cell aggregation. In contrast, erythrocyte suspensions exposed to Cr3+, which forms substitutionally inert water complexes in solution and thus would not be expected to bind protein and cell surfaces, did not result in aggregation (Fig 2C). This suggests that inner-sphere complexation of Fe(III) to plasma proteins/endothelial or blood membranes may have a mechanistic role in FeCl3-induced thrombosis and further explains how the sheet-like protein aggregates could act as Fe3+ linkers that effectively enhances cell binding.However, a complete understanding of FeCl3-induced thrombosis requires the inclusion of endothelial cells. To that end, we used our previously developed technique to “endothelialized” microfluidic channels (Tsai, et al, JCI, 2012) (Fig 3A). We found that the effect of FeCl3 on endothelial cells is likewise concentration dependent: they remain viable at low FeCl3 concentrations (<50 mM) but have compromised cell membranes at higher concentrations (Fig 3B). However, the dead endothelial cells remain intact, corroborating the results of Barr et al. As shown in Fig 3C, FeCl3-induced clotting of platelet-rich plasma nevertheless occurs when endothelial cells are alive. This would suggest that at varying apparent FeCl3 concentrations, the primary means of clotting will differ. If endothelial cells are dead, it is posited that a normal clotting cascade would proceed, in addition to the Fe3+ based aggregation of negatively charged blood components. Overall, our microfluidic studies reveal that FeCl3 has multiple dose-dependent effects involving aggregation of different components of blood and endothelial cells. In addition, we discovered a novel mechanism in which charge effects may have a role in FeCl3-induced thrombosis that is independent of biological ligand-receptor interactions. Further studies are needed to determine how these charge effects interact with the biological aspects of FeCl3-induced thrombosis and the implications for data interpretation when using this model system. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 8: TMEM16f Regulates PhosphatidylSerine (PS) Exposure and PS+ Microparticle Generation From Mouse Platelets

Transmembrane (TMEM)16f is a protein from the family of TMEM16 Ca++-dependent Chloride channels that is essential for optimal Ca2+-dependent phospholipid scrambling activity in platelets. Recently, it has been shown that TMEM16f-dependent exposure of phosphatidylserine (PS) on platelet surfaces is required for platelet-dependent pro-coagulant activity and hemostasis. However, relatively little is known about the effect of TMEM16f on platelet signaling, functional activity, and microparticle formation. TMEM16f-/- mice were generated in the laboratory of Andrea Vortkamp and bred in our laboratory as heterozygous crosses. When assessed by flow cytometry, platelets from TMEM16f-/- mice exposed 27.8% of the PS exposed by WT platelets in response to thrombin and convulxin, 23.1% of the PS in response to 1 uM A23187 , and 38% of the PS exposed by WT platelets in response to 10 uM A23187. Similar trends were obtained using high resolution immunofluorescent microscopy (TMEM16f-/- exposed 49.2% of the PS detected on WT platelets in response to thrombin + convulxin; 25.6% PS in response to 1 uM A23187 ). Microparticle generation from TMEM16f-/- platelets compared to WT platelets was also evaluated by both high resolution immunofluorescent microscopy and flow cytometry. The proportion of PS+αIIb+ microparticles generated per platelet was lower in platelets from TMEM16f-/- mice compared to WT when evaluated by both of these methods. Given that TMEM16f-/- platelets elicit similar calcium responses upon thrombin and convulxin exposure to those of WT platelets, these data may suggest that PS scrambling activity is required not only for PS exposure on the outer leaflet of platelet membranes, but also for the shedding of PS+ microparticles. Surprisingly, TMEM16f-/- platelets were less sensitive to AYPGKF-induced fibrinogen binding than WT platelets (n= 4, p&lt;.05). Finally, TMEM16f-/- mice form less stable occlusive thrombi following 10% ferric chloride injury of the carotid artery (0/4 stable thrombi of TMEM16f-/-) than WT mice (7/9 stable thrombi). The loss of procoagulant microparticles may contribute to the lower sensitivity of TMEM16f-/- mice to ferric chloride-induced thrombosis.

TAP-SSL5 FUNCTIONS AS AN ANTICOAGULANT AND ANTI-INFLAMMATORY PROTEIN TO INHIBIT THROMBOSIS IN RAT AND ATTENUATE ATHEROSCLEROSIS IN APOE-KNOCKOUT MOUSE

ObjectivesThis study aims to inhibit the inflammatory response and thrombosis in atherosclerotic lesions, by using the staphylococcal superantigen-like protein-5 (SSL5) to inhibit neutrophil activation, and tick anticoagulant peptide (TAP) to...

Inducible nitric oxide synthase provides protection against injury-induced thrombosis in female mice.

Nitric oxide (NO) is an important vasoactive molecule produced by three NO synthase (NOS) enzymes: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS). While eNOS contributes to blood vessel dilation that protects against the development of hypertension, iNOS has been primarily implicated as a disease-promoting isoform during atherogenesis. Despite this, iNOS may play a physiological role via the modulation of cyclooxygenase and thromboregulatory eicosanoid production. Herein, we examined the role of iNOS in a murine model of thrombosis. Blood flow was measured in carotid arteries of male and female wild-type (WT) and iNOS-deficient mice following ferric chloride-induced thrombosis. Female WT mice were more resistant to thrombotic occlusion than male counterparts but became more susceptible upon iNOS deletion. In contrast, male mice (with and without iNOS deletion) were equally susceptible to thrombosis. Deletion of iNOS was not associated with a change in the balance of thromboxane A(2) (TxA(2)) or antithrombotic prostacyclin (PGI(2)). Compared with male counterparts, female WT mice exhibited increased urinary nitrite and nitrate levels and enhanced ex vivo induction of iNOS in hearts and aortas. Our findings suggest that iNOS-derived NO in female WT mice may attenuate the effects of vascular injury. Thus, although iNOS is detrimental during atherogenesis, physiological iNOS levels may contribute to providing protection against thrombotic occlusion, a phenomenon that may be enhanced in female mice.

The Antithrombotic Activity of EP224283, a Neutralizable Dual Factor Xa Inhibitor/Glycoprotein IIbIIIa Antagonist, Exceeds That of the Coadministered Parent Compounds

EP224283 combines in a single molecule idraparinux and tirofiban, which allows obtaining a predictable and sustained antiplatelet effect through the transfer of the pharmacokinetics properties of idraparinux to the anti-αIIbβ3 antagonist. The activity can be instantaneously neutralized by injection of avidin, a specific antidote. We have tested the effects of this new profile anticoagulant in various thrombosis models. The antithrombotic effect of EP224283 was compared with those of the parent compounds used alone or in association at doses achieving low to moderate inhibition of platelet aggregation ex vivo. In a model of systemic thromboembolism independent of thrombin generation, tirofiban and EP224283 had similar effects at equimolar doses. On the other hand, EP224283 was more potent than tirofiban or idraparinux under thrombin-dependent conditions. In a ferric chloride-induced thrombosis model, EP224283 was more potent than either parent compound or their combination. Similar results were obtained after atherosclerotic plaque rupture in ApoE(-/-) mice. Thus, the dual action of EP224283 exceeds that of the parent compounds used in combination. A possible explanation is that EP224283 could concentrate antithrombin inside the thrombus by binding to αIIbβ3 through the tirofiban moiety, as shown by immunolabeling of the occluded vessel. No prolongation of the bleeding time was observed at doses achieving strong antithrombotic effects, suggesting that low to moderate αIIbβ3 inhibition combined with factor Xa inhibition minimizes the bleeding risk. The favorable antithrombotic profile of EP224283 together with its possible neutralization by avidin makes it an interesting drug candidate for the treatment and prevention of acute ischemic events.

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/β subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.

A time course study on prothrombotic parameters and their modulation by anti-platelet drugs in hyperlipidemic hamsters

The present study was undertaken to assess the chronology of major pathological events associated with high cholesterol (HC) diet and their modulation by anti-platelet drugs. Male Golden Syrian hamsters were fed HC diet up to 90 days. Plasma lipid, glucose and coagulation parameters (commercial kits), platelet activation (whole blood aggregation and static adhesion), endothelial dysfunction (aortic ring vasoreactivity), splenocyte TNF-α, IFN-γ and iNOS mRNA transcripts (RT-PCR), and ferric chloride (time to occlusion) induced thrombosis were monitored at 15, 30, 60, and 90 days after HC feeding and compared with normolipidemic hamsters. A significant increase in plasma lipid levels was observed at 15 days of HC feeding, but other parameters remain unaltered. Enhanced ADP, collagen, and thrombin-induced platelet aggregation, splenocyte TNF-α expression along with endothelial dysfunction were observed from 30 to 90 days of HC feeding. Platelet adhesion on collagen-/fibrinogen-coated surface and IFN-γ expression were augmented only after 60 days, while enhanced iNOS expression, reduction in thrombin time, and potentiation of ferric chloride-induced thrombosis was observed only at 90 days of HC feeding. Thus, pathological changes induced by HC diet depend on the duration and extent of hyperlipidemia. Moreover, hamsters treated with anti-platelet drugs aspirin (5 mg/kg) or clopidogrel (10 mg/kg) along with HC feeding exhibited reduction in platelet activation as well as subsequent changes observed in the abovementioned parameters following HC feeding. Since reduction in TNF-α was associated with reversion in endothelial dysfunction and prothrombotic state, the role of platelets is implicated in the pathological changes associated with HC feeding.

Effects of Modified Je-Ho-Tang on Ferric Chloride-induced Thrombosis in

To examine the efficacy of modified Je-Ho-Tang extract in terms of its effects on arterial thrombosis and peripheral circulation, in vivo models of both FeCl3-induced thrombosis and peripheral circulatory disturbance induced by cooling were developed. MJHT extract showed potent anti-thrombotic effects and improvement of peripheral blood flow.

Antisense Oligonucletide Mediated Depletion of Factor VII Provides Protection from Ferric Chloride Induced Thrombosis without Increased Bleeding Risk in Mice

The Tissue Factor-Factor VIIa complex is the initiating protease in the extrinsic pathway of the coagulation cascade. Knockout of Factor VII in mice results in embryonic lethality. However, only humans with severe Factor VII deficiency exhibit any coagulopathy. In order to determine the potential of Factor VII as an anticoagulant target and the utility of antisense oligonucleotides (ASOs) as Factor VII inhibitors, ASOs were designed to promote RNaseH mediated degradation of Factor VII RNA and evaluated in mice. Factor VII is predominantly produced in the liver, a tissue that is tractable for antisense mediated target depletion. Following three weeks of treatment, the Factor VII ASO was well tolerated and supported significant reduction of Factor VII RNA and protein in mouse liver (ED50 of 3.5 mg/kg for RNA and 2.8 mg/kg for protein). Greater than 95% reduction in Factor VII RNA levels was observed at a dose of 25 mg/kg, administered twice weekly. Following three weeks exposure to 100 mg/kg/wk, prothrombin times (PT) from platelet poor mouse plasma was more than doubled, compared to untreated animals, while activated partial thromboplastin times (aPTT) was only increased 16%. Antithrombotic activity of Factor VII ASO was determined following ferric chloride injury to the inferior vena cava. ED50 for inhibition of thrombosis was 24 mg/kg/wk. The effects of Factor VII depletion on hemostatic control were also determined using a mouse tail nick model. No increase in blood loss was detected, compared to untreated animals, up to a weekly exposure of 80 mg/kg, the highest dose utilized in the study. Compared to warfarin, ASO mediated depletion of Factor VII promoted equivalent protection from thrombosis following ferric chloride injury while maintaining better hemostatic control. These findings support the development of an ASO drug targeting Factor VII as an anticoagulant.