Ferric Chloride-induced Carotid Artery Thrombosis Research Articles

The mechanism of oleic acid inhibiting platelet activation stimulated by collagen

BackgroundAbnormal platelet activation is a key factor in the occurrence and development of thrombotic diseases. However, the physiological mechanisms that underlie platelet homeostasis remain unclear. Oleic acid, one of the most abundant lipids in the human diet, has potential antithrombotic effects. This study aimed to investigate the effects of oleic acid on platelet activation and thrombosis.MethodsPlatelet aggregation, ATP release, and fibrinogen spread were evaluated to determine the role of oleic acid in platelet activation. A ferric chloride-induced carotid injury model was used to establish the effect of oleic acid on thrombus formation in vivo. Western blotting analysis and transfection experiments were performed to determine the mechanisms involved in this process.ResultsOleic acid inhibited platelet aggregation, granule release, and calcium mobilization. Furthermore, it inhibited the spread of platelets on fibrinogen. We also found that oleic acid delayed arterial thrombosis in mice, as demonstrated in a murine model of ferric chloride-induced carotid artery thrombosis. The molecular mechanism of its inhibition of platelet activity may be through the Syk-PLCγ2 and CaMKKβ/AMPKα/VASP pathways. In addition, we demonstrated that the phosphorylation of AMPK at Ser496 was an important mechanism of platelet activation.ConclusionsOur study showed that oleic acid inhibits platelet activation and reduces thrombogenesis by inhibiting the phosphorylation of multiple signaling molecules, offering new insights into the research and development of antiplatelet drugs.E5Xy1vH3DfoQAQBKnckQzxVideo

QiShenYiQi Inhibits Tissue Plasminogen Activator-Induced Brain Edema and Hemorrhage after Ischemic Stroke in Mice.

Background: Thrombolysis with tissue plasminogen activator (tPA) remains the only approved drug therapy for acute ischemic stroke. However, delayed tPA treatment is associated with an increased risk of brain hemorrhage. In this study, we assessed whether QiShenYiQi (QSYQ), a compound Chinese medicine, can attenuate tPA-induced brain edema and hemorrhage in an experimental stroke model. Methods: Male mice were subjected to ferric chloride-induced carotid artery thrombosis followed by mechanical detachment of thrombi. Then mice were treated with QSYQ at 2.5 h followed by administration of tPA (10 mg/kg) at 4.5 h. Hemorrhage, infarct size, neurological score, cerebral blood flow, Evans blue extravasation, FITC-labeled albumin leakage, tight and adherens junction proteins expression, basement membrane proteins expression, matrix metalloproteinases (MMPs) expression, leukocyte adhesion, and leukocyte infiltration were assessed 24 h after tPA administration. Results: Compared with tPA alone treatments, the combination therapy of QSYQ and tPA significantly reduced hemorrhage, infarction, brain edema, Evans blue extravasation, albumin leakage, leukocyte adhesion, MMP-9 expression, and leukocyte infiltration at 28.5 h after stroke. The combination also significantly improved the survival rate, cerebral blood flow, tight and adherens junction proteins (occludin, claudin-5, junctional adhesion molecule-1, zonula occludens-1, VE-cadherin, α-catenin, β-catenin) expression, and basement membrane proteins (collagen IV, laminin) expression. Addition of QSYQ protected the downregulated ATP 5D and upregulated p-Src and Caveolin-1 after tPA treatment. Conclusion: Our results show that QSYQ inhibits tPA-induced brain edema and hemorrhage by protecting the blood-brain barrier integrity, which was partly attributable to restoration of energy metabolism, protection of inflammation and Src/Caveolin signaling activation. The present study supports QSYQ as an effective adjunctive therapy to increase the safety of delayed tPA thrombolysis for ischemic stroke.

Amitriptyline Treatment Mitigates Sepsis-Induced Tumor Necrosis Factor Expression and Coagulopathy.

During sepsis, the early innate response and inflammatory cytokine cascade are associated with activation of the coagulation cascade. Acute hypercoagulability can contribute to lethal sequela of vascular thrombosis, tissue ischemia, and organ failure. We investigated if amitriptyline (AMIT), an antidepressant drug with a number of anti-inflammatory effects, could ameliorate sepsis in a murine model of sepsis-cecal ligation and puncture (CLP). We hypothesized that AMIT treatment would reduce inflammation and mitigate sepsis-induced coagulopathy. Coagulation was measured using thromboelastometry and ferric chloride-induced carotid artery thrombosis. Our findings demonstrate a dynamic early hypercoagulability, followed by delayed hypocoagulability in septic mice. However, septic mice treated with AMIT were unaffected by these coagulation changes and exhibited a coagulation profile similar to sham mice. TNFα was markedly elevated in septic mice, but decreased in AMIT-treated mice. Exogenous administration of recombinant TNFα in naive mice recapitulated the acute sepsis-induced hypercoagulability profile. After sepsis and endotoxemia, peritoneal macrophages were the predominant source of TNFα expression. AMIT treatment significantly decreased macrophage TNFα expression and blunted M1 polarization. Altogether, during polymicrobial sepsis, AMIT treatment suppressed macrophage TNFα expression and the M1 phenotype, mitigating an initial hypercoagulable state, and protecting septic mice from delayed hypocoagulability. We propose that AMIT treatment is a promising therapeutic approach in the treatment of sepsis-associated coagulopathy and prevention of acute thromboembolic events or delayed bleeding complications.

The Involvement of Toll-like Receptor-2 in Arterial Thrombus Formation.

There is emerging evidence for the participation of toll-like receptor-2 (TLR2) expressed on platelets and endothelial cells in the setting of arterial thrombosis. In isolated human platelets, TLR2/1 activation was demonstrated to induce platelet activation, secretion, aggregation, adhesion to collagen coatings and the formation of platelet-leukocyte conjugates, whereas murine platelets were less sensitive to TLR2/1 stimulation. Also, endothelial cells can be activated by stimulation with TLR2 agonists, resulting in increased expression of adhesion molecules, synthesis of inflammatory mediators and Weibel-Palade body exocytosis. Endothelial TLR2 signalling promotes atherosclerotic lesion development in mouse atherosclerosis models. Experiments with germ-free mouse models demonstrated that the presence of commensal microbiota increased endothelial von Willebrand factor synthesis in the liver through TLR2. In the carotid artery ligation model, the elevated von Willebrand factor plasma levels enhanced platelet deposition to the injury site. Furthermore, in the hyperlipidemic ApoE-deficient mouse model, TLR2 deficiency was shown to protect from ferric chloride-induced carotid artery thrombosis. This review article provides an overview on TLR2 signalling in platelets and the vascular endothelium and summarizes how TLR2 signalling contributes to arterial thrombosis.

Abstract 522: VWF Inhibition by an RNA Aptamer Improves Functional Outcome Compared to rTPA in Ferric Chloride-induced Carotid Artery Thrombosis in Mice

Objective: To compare the functional effects of von Willebrand Factor (VWF) inhibition by RNA aptamer (T79) vs rTPA utilizing in vivo arterial thrombosis in mice. Approach and Results: We previously demonstrated that inhibition of VWF by a targeted RNA aptamer (T79) both prevents thrombosis and thrombolyses stabilized clots in a murine model of ferric chloride (FeCl 3 )-induced vascular injury suggesting a pivotal role for VWF in the pro-thrombotic and anti-thrombotic milieu. We hypothesized that T79 treatment, which demonstrated no hemorrhagic complications and greater re-perfusion compared to rTPA, would result in improved behavioral outcome with 7 day survival after vascular injury. Baseline locomotor testing in an open field was performed on both male and female, 8-16 week old, wild-type C57BL/6J mice. Occlusive arterial thrombus formation was induced by a 3 minute exposure to 10% FeCl 3 on the right common carotid monitored by Doppler flow and time to occlusion (blood flow of 0 ml/min) was measured. Twenty minutes after thrombus stabilization either T79 (0.1 mg/kg or 0.5 mg/kg bolus), rTPA (10 mg/kg 45 min infusion) or saline vehicle (45 min infusion) was introduced via a saphenous catheter. Animals were recovered and locomotor testing was repeated 48 hours and 7 days after injury with baseline set as 100%. All groups including vehicle control resulted in a statistically significant decrease in distance traveled (meters) and speed (meters/sec) at 48 hours after injury. At 7 days, 0.1 mg/kg T79 increased 29.89% from 48 hours, 0.5 mg/kg T79 increased 22.50% from 48 hours but rTPA treatment showed no significant improvement (2.79% increase) (n=4, p<0.001). In addition, 0.1 mg/kg and 0.5 mg/kg T79 resulted in a 28.46% and 22.44% increase in speed, respectively, at 7 days compared to 48 hours whereas rTPA did not (2.94% increase) (n=4, p<0.001). Interestingly, males scored lower on most behavioral parameters at 48 hours, but recovered to the same level as females by 7 days. No significant differences in time to occlusion or baseline locomotor testing were observed in any group. Conclusion: Inhibition of VWF by T79 aptamer markedly improves behavioral outcomes compared to rTPA after survival carotid artery occlusion following ferric chloride-induced injury in mice.

Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi.

Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization. Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection. We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis. We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.

Abstract 341: The Role of Nucleotidase in Arterial Thrombosis

Objective: To determine how leukocyte nucleotidase affects arterial thrombosis. Approach and Results: Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) is expressed on circulating cells, endothelium and smooth muscle cells where it hydrolyzes extracellular ATP or ADP to AMP. We have demonstrated that transgenic mice with a global overexpression of human CD39 (hCD39-Tg) are protected against ferric chloride-induced carotid artery thrombosis. Furthermore, transplant of hCD39-Tg bone marrow into WT recipient mice increases the time to thrombosis when compared to recipient mice (wild-type or hCD39-Tg) receiving wild-type bone marrow. Based upon these data and previously published work, we hypothesized that CD39 expression on leukocytes is responsible for the prolongation of the time to thrombosis measured in hCD39-Tg mice. To test this hypothesis, we first performed ex vivo mixing experiments. Addition of hCD39-Tg monocytes to WT blood inhibits the expression of activated glycoprotein IIb/IIIa on platelets in response to ADP as measured by FACS analysis (Baseline: 1224 ± 94.9 MFI vs hCD39-Tg monocytes: 663.5 ± 61.5 activated glycoprotein IIb/IIIa MFI: n=4; p< 0.001). Subsequently, in vivo we demonstrated that monocytes with increased CD39 contribute to extending the time to thrombosis. Clodronate liposome depletion of monocytes (WT: 69% decrease; hCD39-Tg: 63% decrease) resulted in a normalization of the time to thrombosis in hCD39-Tg mice (8.0 ± 1.07 minutes, n = 10) when compared to control loaded liposomes (120.0 ± 0.0, n = 14). No changes in the time to thrombosis were detected in wild-type mice treated with clodronate (8.6 ± 1.35 minutes, n = 8) or control liposomes (7.8 ± 0.80 minutes, n=8). Conclusion: Increased expression of CD39 on monocytes can inhibit platelet activation and extend the time to thrombosis following ferric chloride-induced carotid artery injury.

Aspirin enhances protective effect of fish oil against thrombosis and injury-induced vascular remodelling.

Although aspirin (acetylsalicylic acid) is commonly used to prevent ischaemic events in patients with coronary artery disease, many patients fail to respond to aspirin treatment. Dietary fish oil (FO), containing ω3 polyunsaturated fatty acids (PUFAs), has anti-inflammatory and cardio-protective properties, such as lowering cholesterol and modulating platelet activity. The objective of the present study was to investigate the potential additional effects of aspirin and FO on platelet activity and vascular response to injury. Femoral arterial remodelling was induced by wire injury in mice. Platelet aggregation, and photochemical- and ferric chloride-induced carotid artery thrombosis were employed to evaluate platelet function. FO treatment increased membrane ω3 PUFA incorporation, lowered plasma triglyceride and cholesterol levels, and reduced systolic BP in mice. FO or aspirin alone inhibited platelet aggregation; however, when combined, they exhibited synergistic suppression of platelet activity in mice, independent of COX-1 inhibition. FO alone, but not aspirin, attenuated arterial neointimal growth in response to injury. Strikingly, a combination of FO and aspirin synergistically inhibited injury-induced neointimal hyperplasia and reduced perivascular inflammatory reactions. Moreover, co-administration of FO and aspirin decreased the expression of pro-inflammatory cytokines and adhesion molecules in inflammatory cells. Consistently, a pro-resolution lipid mediator-Resolvin E1, was significantly elevated in plasma in FO/aspirin-treated mice. Co-administration of FO and low-dose aspirin may act synergistically to protect against thrombosis and injury-induced vascular remodelling in mice. Our results support further investigation of adjuvant FO supplementation for patients with stable coronary artery disease.

Clot-targeted micellar formulation improves anticoagulation efficacy of bivalirudin.

Application of anticoagulants remains the primary strategy for prevention and treatment of thrombosis. However, high rate of bleeding complications limits their use. The peptide anticoagulant bivalirudin has been reported to exhibit a lower rate of bleeding complications than heparin, and it also has the advantage of not causing thrombocytopenia, which is a problem with heparin. Nonetheless, hemorrhage is the most common complication of bivalirudin therapy, and there is no effective antidote. Here we use a thrombus-binding peptide, CR(NMe)EKA, to accomplish selective delivery of the bivalirudin-carrying micellar nanocarrier to sites of thrombosis. Bivalirudin and CR(NMe)EKA, each with a PEG-lipid tail, spontaneously assembled into 30 nm micelles, which almost completely retained the anticoagulant activity of bivalirudin. The micellar formulations exhibited high stability both in vitro and in vivo. In a thromboplastin-induced mouse thrombosis model, the targeted micelles accumulated in lung thrombi 10-fold more than nontargeted micelles. Moreover, the micellar formulation significantly prolonged the half-life and thereby increased the bioavailability of bivalirudin. The micellar bivalirudin had significantly higher anticoagulant activity than free bivalirudin in both the lung thrombosis model and a ferric chloride-induced carotid artery thrombosis model. The specific targeting of thrombi demonstrated here makes it possible to increase the efficacy of bivalirudin as an anticoagulant. Alternatively, the dose could be reduced without loss of efficacy to lower the systemic exposure and improve safety.

The flavonols quercetin and 3′,4′-dihydroxyflavonol reduce platelet function and delay thrombus formation in a model of type 1 diabetes

Diabetes is associated with increased cardiovascular risk. We have recently shown that the naturally occurring flavonol quercetin (Que) or the synthetic flavonol 3',4'-dihydroxyflavonol (DiOHF) inhibits platelet function and delays thrombus formation in healthy mice. Therefore, the aim of this study was to investigate the effect of Que or DiOHF treatment on platelet function and ferric chloride-induced carotid artery thrombosis in a mouse model of type 1 diabetes. Diabetic mice treated with Que or DiOHF maintained blood flow at a significantly higher level than untreated diabetic mice at the end of the recording period. In addition, treatment with Que or DiOHF significantly reduced diabetes-induced platelet hyper-aggregability in response to platelet agonist stimulation. Furthermore, treatment with Que or DiOHF significantly inhibited dense, but not alpha, granule exocytosis in diabetic and control mice. Our demonstration that flavonols delay thrombus formation in diabetes suggests a potential clinical role for these compounds in anti-platelet therapy.

Acute Pulmonary Embolism (PE)—Is Echocardiography Underutilised in Regional Australia

Results: Although slope and offset of the baroreflex peripheral arc (linear relation) were similar, the neural arc response (inverted sigmoid curve) range was attenuated in SHR (71± 2% vs. 91± 3%, P< 0.01). The operating-point AP was higher in SHR than in WKY (144± 6mmHg vs. 109± 5mmHg, P< 0.01). Although NE was higher in SHR at baseline condition (403.1 pg/ml vs. 203.2 pg/ml, P< 0.01), AP response to phenylephrine was similar (linear relation), suggesting preserved peripheral response to sympathetic activity. Conclusion: The baroreflex equilibrium diagram indicates that baroreflex regulation of SNA rather than the cardiovascular response to SNA plays a critical role in the its non-targeted control (CD39 fused to a non-functional mutated single-chain antibody). Most importantly in a mouse model of ferric chloride-induced carotid artery thrombosis, targeted-CD39 was protective against vessel occlusion at a concentration at which the non-targetedCD39was ineffective (p< 0.005).At the same concentration no tail bleeding prolongation was observed for the targeted-CD39 while the ineffective non-targeted-CD39 showed a bleeding tendency (p< 0.01). Conclusion: Targeting CD39 to its desired site of action enables administration of such a low concentration as to avoid the previously observed bleeding tendencies while still being a highly effective antithrombotic drug. Thus, enriching CD39 to activated platelets at growing thrombi prevents the previously limiting bleeding side effects and advances CD39 towards potential clinical use. doi:10.1016/j.hlc.2011.05.024

Abstract 1033: Simultaneous Clot-targeted Factor Xa Inhibition And Selective Blockade Of Activated GPIIb/IIIa On Platelets Results In Delayed But Potent Antithrombotic Effects Without Bleeding Time Prolongation.

Background: We generated phage-display-derived anti-GPIIb/IIIa single-chain antibodies (e.g. scFv SCE5) that specifically bind to the activated GPIIb/IIIa only and thus specifically block activated platelets only. ScFv SCE5 demonstrates strong antithrombotic potency, comparable to the conformation-unspecific blockers tirofiban and eptifibatide. In contrast bleeding times were not prolonged with scFv SCE5. Here we now use the possibility to add effector molecules using molecular biology methods. The highly potent anticoagulant TAP (tick anticoagulant peptide), which is a direct factor Xa (fXa) inhibitor, was used as an effector molecule. Methods and Results: We genetically fused the activation-specific scFv with TAP, expressed the constructs in E.coli and purified the 39 kDa protein via its Histag binding to Nickel beads. Specific binding of the fusion molecules MA2/SCE5-TAP and strong inhibition of fibrinogen binding was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. Prolongation in occlusion time with SCE5-TAP was significantly stronger compared to SCE5 alone, recombinant TAP, non-binding mut-scFv-TAP as well as the clinical used drugs enoxaparine and eptifibatide. In contrast to the other anticoagulants tested, bleeding time was not prolonged by SCE5-TAP. Flow experiments studying platelet adhesion on collagen revealed a possible mechanism for the unique finding of a fully normal bleeding time: LIBS exposure on adhering platelets and as such the anticoagulative targeting potency of SCE5-TAP was delayed until considerable layers of platelets were deposited. Conclusions: The combination of activation-specific GPIIb/IIIa blockade and fXa inhibition in one clot-targeted molecule further improves in-vivo antithrombotic efficiency without causing any bleeding time prolongation. The delay of the observed targeting effect may allow a sealing of injuries with platelet layers but may be in time for the prevention of occlusive platelet aggregates. The described blockers represent a new type of highly selective drugs that warrant further clinical development.

Targeting Ligand-Induced Binding Sites on GPIIb/IIIa via Single-Chain Antibody Allows Effective Anticoagulation Without Bleeding Time Prolongation

Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding. Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFv(anti-LIBS)) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFv(anti-LIBS)-TAP was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. ScFv(anti-LIBS)-TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFv(anti-LIBS)-TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFv(anti-LIBS)-TAP. The novel clot-targeting approach of anticoagulants via single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa promises effective anticoagulation with reduced bleeding risk.

Differential effects of sodium nitroprusside and hydralazine in a rat model of topical FeCl 3-induced carotid artery thrombosis

Introduction: In the rat model of topical ferric chloride-induced carotid artery thrombosis, a transient blood flow velocity (VEL) increase is observed immediately following ferric chloride application. The immediacy of the response suggested vasoconstriction, as thrombotic narrowing of the vessel lumen was hypothesized to be too slow to account for the rapidity of the response. Methods: To explore this phenomenon, the effects of two mechanistically distinct vasodilators, sodium nitroprusside (SNP) and hydralazine (HYD), on velocity increase, ex vivo platelet aggregation and thrombosis, were assessed in the rat ferric chloride-induced thrombosis model. Results: Sodium nitroprusside (10, 30 and 50 μg/kg/min i.v.) and hydralazine (0.1, 0.3 and 1.0 mg/kg/min i.v.) reduced the mean arterial pressure with the higher dose regimens eliciting equivalent hypotensive effects. Both sodium nitroprusside and hydralazine blunted the initial velocity increase, but only sodium nitroprusside significantly reduced the incidence of thrombotic occlusion. No differences in ex vivo platelet aggregation responses to adenosine diphosphate (ADP), collagen (COLL) and arachidonic acid (AA) were observed between the sodium nitroprusside and hydralazine treatment groups. However, platelet aggregation response to thrombin was significantly reduced in the 50 μg/kg/min i.v. sodium nitroprusside compared to the 1.0 mg/kg/min i.v. hydralazine and vehicle groups. Conclusions: Inhibition of the initial velocity increase by two mechanistically distinct vasodilators, and the dissociation between this velocity change and antithrombotic efficacy, support the hypothesis that the early velocity increase results from a change in vascular tone rather than due to enhanced platelet activation and thrombus formation. Inhibition of thrombin-induced platelet activation may contribute to the antithrombotic actions of sodium nitroprusside in this preparation.