Rat Model Of Arterial Thrombosis Research Articles

Additive effect of mesenchymal stem cells and defibrotide in an arterial rat thrombosis model.

In this study, we aimed to investigate the additive effect of mesenchymal stem cells (MSC) and defibrotide (DFT) in a rat model of femoral arterial thrombosis. Thirty Sprague Dawley rats were included. An arterial thrombosis model by ferric chloride (FeCl3) was developed in the left femoral artery. The rats were equally assigned to 5 groups: Group 1-Sham-operated (without arterial injury); Group 2-Phosphate buffered saline (PBS) injected; Group 3-MSC; Group 4-DFT; Group 5-MSC + DFT. All had two intraperitoneal injections of 0.5 ml: the 1st injection was 4 h after the procedure and the 2nd one 48 h after the 1st injection. The rats were sacrificed 7 days after the 2nd injection. Although the use of human bone marrow-derived (hBM) hBM-MSC or DFT alone enabled partial resolution of the thrombus, combining them resulted in near-complete resolution. Neovascularization was two-fold better in hBM-MSC + DFT treated rats (11.6 ± 2.4 channels) compared with the hBM-MSC (3.8 ± 2.7 channels) and DFT groups (5.5 ± 1.8 channels) (P < 0.0001 and P= 0.002, respectively). The combined use of hBM-MSC and DFT in a rat model of arterial thrombosis showed additive effect resulting in near-complete resolution of the thrombus.

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting.

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Prevention of arterial thrombosis by edoxaban, an oral factor Xa inhibitor in rats: monotherapy and in combination with antiplatelet agents

In addition to platelet aggregation, coagulation activation is considered to be involved in arterial thrombosis. In this study, we determined antithrombotic effects of edoxaban, an oral factor Xa (FXa) inhibitor, as both a monotherapy and in combination with antiplatelet agents in a rat model of arterial thrombosis. We further examined its effects on a procoagulant biomarker and bleeding. Arterial thrombosis was induced by topical application of 15% ferric chloride to rat abdominal aortas. Bleeding time was measured by a tail incision method. Edoxaban, clopidogrel, and aspirin were orally administered 30min, 4h, and 2h before thrombus or bleeding induction. As a biomarker of coagulation activation, plasma thrombin-antithrombin complex (TAT) was measured. Edoxaban dose-dependently prevented arterial thrombosis in a manner comparable to clopidogrel and aspirin. The combination of edoxaban plus clopidogrel or edoxaban plus aspirin significantly potentiated the antithrombotic effects compared with these drugs alone. The combination of edoxaban and clopidogrel was more potent than clopidogrel and aspirin. Plasma TAT concentration was elevated after thrombus induction and suppressed by edoxaban and clopidogrel, but not by aspirin, suggesting P2Y12 receptor-mediated platelet procoagulant activity. Bleeding time was prolonged by the coadministration of edoxaban and clopidogrel, but not by edoxaban and aspirin. In conclusion, the present study demonstrates that the monotherapy with edoxaban and combination therapy with edoxaban plus clopidogrel or edoxaban plus aspirin are promising options for the prevention of arterial thrombosis as effective as the standard antiplatelet agents; however, a combination of edoxaban and clopidogrel increased the risk of bleeding.

A Manganese Alternative to Gadolinium for MRI Contrast.

Contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) are routinely used to diagnose soft tissue and vascular abnormalities. However, safety concerns limit the use of iodinated and gadolinium (Gd)-based CT and MRI contrast media in renally compromised patients. With an estimated 14% of the US population suffering from chronic kidney disease (CKD), contrast media compatible with renal impairment is sorely needed. We present the new manganese(II) complex [Mn(PyC3A)(H2O)](-) as a Gd alternative. [Mn(PyC3A)(H2O)](-) is among the most stable Mn(II) complexes at pH 7.4 (log KML = 11.40). In the presence of 25 mol equiv of Zn at pH 6.0, 37 °C, [Mn(PyC3A)(H2O)](-) is 20-fold more resistant to dissociation than [Gd(DTPA)(H2O)](2-). Relaxivity of [Mn(PyC3A)(H2O)](-) in blood plasma is comparable to commercial Gd contrast agents. Biodistribution analysis confirms that [Mn(PyC3A)(H2O)](-) clears via a mixed renal/hepatobiliary pathway with >99% elimination by 24 h. [Mn(PyC3A)(H2O)](-) was modified to form a bifunctional chelator and 4 chelates were conjugated to a fibrin-specific peptide to give Mn-FBP. Mn-FBP binds the soluble fibrin fragment DD(E) with Kd = 110 nM. Per Mn relaxivity of Mn-FBP is 4-fold greater than [Mn(PyC3A)(H2O)](-) and increases 60% in the presence of fibrin, consistent with binding. Mn-FBP provided equivalent thrombus enhancement to the state of the art Gd analogue, EP-2104R, in a rat model of arterial thrombosis. Mn metabolite analysis reveals no evidence of dechelation and the probe was >99% eliminated after 24 h. [Mn(PyC3A)(H2O)](-) is a lead development candidate for an imaging probe that is compatible with renally compromised patients.

Antithrombogenic Activity of Antioxidant Compounds

Antithrombotic activities of enoxifol, a new antioxidant with antiaggregant activity demonstrated in vitro and in vivo, and antioxidant mexidol were compared on the rat model of arterial thrombosis induced by application of 50% ferric chloride. Acetylsalicylic acid (antiaggregant) served as the reference drug. All drugs exhibited dose-dependent antithrombotic activity. Enoxifol was more effective than mexidol, both drugs being more active than the reference drug (acetylsalicylic acid). Taking into account the pathogenesis of the thrombosis in this experimental model, we can hypothesize that the pronounced antithrombotic effect of enoxifol was due to its antiaggregant and antioxidant activities.

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.

Discovery of 2-(Phenoxypyridine)-3-phenylureas as Small Molecule P2Y1 Antagonists

Two distinct G protein-coupled purinergic receptors, P2Y1 and P2Y12, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1 has the potential for reduced bleeding liability. In this account, the discovery of a 2-(phenoxypyridine)-3-phenylurea chemotype that inhibited ADP-mediated platelet aggregation in human blood samples is described. Optimization of this series led to the identification of compound 16, 1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-4-(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only prolonging cuticle and mesenteric bleeding times by 3.3- and 3.1-fold, respectively, in provoked rat bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and efficacious antithrombotic profile.

Novel Antiplatelet Activity of Protocatechuic Acid through the Inhibition of High Shear Stress-Induced Platelet Aggregation

Bleeding is the most common and serious adverse effect of currently available antiplatelet drugs. Many efforts are being made to develop novel antithrombotic agents without bleeding risks. Shear stress-induced platelet aggregation (SIPA), which occurs under abnormally high shear stress, plays a crucial role in the development of arterial thrombotic diseases. Here, we demonstrate that protocatechuic acid (PCA), a bioactive phytochemical from Lonicera (honeysuckle) flowers, selectively and potently inhibits high shear (>10,000 s(-1))-induced platelet aggregation. In isolated human platelets, PCA decreased SIPA and attenuated accompanying platelet activation, including intracellular calcium mobilization, granule secretion, and adhesion receptor expression. The anti-SIPA effect of PCA was mediated through blockade of von Willebrand factor binding to activated glycoprotein Ib, a primary and initial event for the accomplishment of SIPA. Conspicuously, PCA did not inhibit platelet aggregation induced by other endogenous agonists like collagen, thrombin, or ADP that are important in both pathological thrombosis and normal hemostasis. Antithrombotic effects of PCA were confirmed in vivo in a rat arterial thrombosis model, where PCA significantly delayed the arterial occlusion induced by FeCl(3). Of particular note, PCA did not increase bleeding times in a rat tail transection model, whereas conventional antiplatelet drugs, aspirin, and clopidogrel substantially prolonged it. Collectively, these results suggest that PCA may be a novel antiplatelet agent that can prevent thrombosis without increasing bleeding risks.

Novel TEMPO-PEG-RGDs Conjugates Remediate Tissue Damage Induced by Acute Limb Ischemia/Reperfusion

We have recently developed new Tempo-PEG-RGDs conjugates and have quantitatively examined their antithrombotic and antioxidant capabilities. These compounds were therapeutically beneficial when characterized in both in vitro platelet aggregation assays and a rat model of arterial thrombosis. Moreover, these compounds demonstrated significant protection from organ damage in a rat model of ischemia/reperfusion. Our data indicate that Tempo-PEG-RGDs represent a new class of adjuvants with therapeutic efficacy in acute and transient ischemic damage.

Anti-Atherothrombogenic Properties of PEDF

Cardiovascular disease (CVD) such as myocardial infarction and stroke is a leading cause of death in developed countries. Atherothrombosis, characterized by atherosclerotic lesion disruption with superimposed thrombus formation, is thought to be the major cause of CVD. Although remarkable therapeutic advances in the treatment of atherothrombosis have been made with anti-platelet and anti-thrombotic therapy, these therapeutic options may be limited by considerable side effects. In addition, they may not protect the endothelium and thus could not stabilize culprit lesions. Therefore, to develop a novel therapeutic strategy is needed for the prevention of atherothrombosis in high-risk patients. Recently, we, along with others, have shown that pigment epithelium-derived factor (PEDF), a glycoprotein with potent neuronal differentiating activity, exerts anti-oxidative and anti-inflammatory anti-thrombogenic and vasculoprotective properties in various cell types. In addition, PEDF not only suppresses neointimal hyperplasia after balloon angioplasty, but also blocks occlusive thrombus formation in a rat arterial thrombosis model. These observations suggest that substitution of PEDF may be a novel therapeutic strategy for atherothrombosis. This article summarizes the pathophysiological role of PEDF in atherothrombosis and its potential therapeutic implication in CVD. We also discuss here the kinetics and regulation of PEDF in high-risk patients for atherothrombosis.

The combination of modified antioxidant enzymes for anti-thrombotic protection of the vascular wall: the significance of covalent connection of superoxide dismutase and catalase activities

Vascular wall protection can be achieved by preventive attachment to the vascular wall of antioxidants and elimination/neutralization of toxic products after their disproportioning. For this purpose we have prepared covalent conjugates between the vascular wall glycosaminglycan chondroitin sulfate (CHS) and the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). The following conjugates were obtained: SOD-CHS, CAT-CHS and SOD-CHS-CAT. Their anti-thrombotic activity was compared in a rat model of arterial thrombosis by measuring the time of occlusion emergence and thrombus mass. It is noteworthy that the effectiveness of single bolus injections of SOD-CHS/CAT-CHS mixture was much lower than that of the bienzymic SOD-CHS-CAT conjugate. The conjugate SOD-CHS-CAT proved to be anti-thrombotically effective in doses two orders of magnitude lower than the native biocatalysts and an order of magnitude lower than SOD-CHS and CAT-CHS derivatives. For effective anti-thrombotic protection in oxidative conditions it is important to maintain the stable connection of SOD and CAT activity on the vascular wall and the large size of these conjugates. Covalent conjugate SOD-CHS-CAT is the best prospect for pharmaceutical development.

Atheroprotective Properties of Pigment Epithelium-Derived Factor (PEDF) in Cardiometabolic Disorders

Although remarkable therapeutic advances in the treatment of cardiometabolic disorders have been made with current therapeutic options, cardiovascular disease (CVD) is still a leading cause of mortality and morbidity in the Western world. Therefore, to develop a novel therapeutic strategy is needed for the prevention of cardiovascular disease (CVD) in high-risk patients for atherosclerosis. Recently, we, along with others, have shown that pigment epithelium-derived factor (PEDF), a glycoprotein with potent neuronal differentiating activity, exerts anti-oxidative and anti-inflammatory properties in vascular wall cells, leukocytes and platelets. In addition, PEDF not only suppresses neointimal hyperplasia after balloon angioplasty, but also blocks occlusive thrombus formation in a rat arterial thrombosis model. These observations suggest that substitution of PEDF may be a novel therapeutic strategy for atherosclerosis. This article summarizes the pathophysiological role of PEDF in atherosclerosis and its potential therapeutic implication in this devastating disorder. We also discuss here the kinetics and regulation of PEDF in cardiometabolic disorders in humans.

Synthesis and Evaluation of Pyrrolin-2-one Compounds, a Series of Plasminogen Activator Inhibitor-1 Inhibitors

A novel series of furan-2-one and pyrrolin-2-one derivatives having PAI-1 (plasminogen activator inhibitor-1) inhibitory activity were synthesized and evaluated for their antithrombotic activity in a rat arterial thrombosis model. Among the synthesized compounds, 5f (T-1776Na) was found to have good selectivity for PAI-1 over other enzymes and high antithrombotic activity.

Human activated protein C variants in a rat model of arterial thrombosis

BackgroundActivated protein C (APC) inhibits coagulation by degrading activated factor V (FVa) and factor VIII (FVIIIa), protein S (PS) functioning as a cofactor to APC.MethodsBy mutagenesis of the vitamin K-dependent Gla domain of APC, we have recently created an APC variant having enhanced anticoagulant activity due to increased affinity for negatively charged phospholipid membranes. In the present study, the potential antithrombotic effects of this APC variant, and of a variant APC that is additionally mutated in the serine protease domain, have been evaluated in a blind randomized study in a rat model of arterial thrombosis. In this model, we have previously found the combination of bovine APC and PS to be highly antithrombotic. Four treatment groups each containing 10 rats were, in a blind random fashion, given intravenous bolus injections of wild-type or mutant variants of APC (0.8 mg/kg) together with human PS (0.6 mg/kg) or human PS (0.6 mg/kg) alone. A control group with 20 animals where given vehicle only.ResultsA trend to increased patency rates was noted in a group receiving one of the APC variants, but it did not reach statistical significance.ConclusionIn conclusion, administration of human APC variants having enhanced anticoagulant efficacy together with human PS in a rat model of arterial thrombosis did not give an efficient antithrombotic effect. The lack of effect may be due to species-specific differences between the human protein C system and the rat hemostatic system.

Design, synthesis and cardioprotective effect of a new class of dual-acting agents: Phenolic tetrahydro-β-carboline RGD peptidomimetic conjugates

In this study, a new class of phenolic tetrahydro-β-carboline RGD peptidomimetic conjugates was designed and synthesized. The radical scavenging activities of these newly synthesized compounds 12a– c were evaluated in PC12 cell survival assays. The NO scavenging activities of these compounds were confirmed in the acetylcholine-induced vasorelaxation assay. Compounds 12a– c were efficacious in a rat arterial thrombosis model, and were active in ADP- or PAF-induced in vitro platelet aggregation assays, which suggests these compounds also possess anti-thrombotic activity. The beneficial effects of dual-acting agent 12c were demonstrated on the ischemia-reperfusion induced cardiac infarct size and oxidative change in an in vivo rat model.

In vivo thrombus formation.

Thrombus formation, including platelet adhesion, activation, secretion and aggregation as well as tissue factor-initiated thrombin generation and fibrin formation, has been studied in the past using in vitro systems, often with isolated components. Given the complexity of hemostasis and thrombosis, many of the concepts that have been developed to explain these processes are being revisited by studying thrombus formation in live animals using intravital microscopy and genetically altered mice. Although much of the dogma that has evolved has been confirmed by in vivo studies of thrombus formation, there have also been conflicts between old concepts and new direct observations. In vivo studies of the initiation of thrombus formation, platelet accumulation and thrombin generation have provided evidence for the participation of novel proteins and identified new pathways and mechanisms.

Antithrombotic and anticoagulant effects of wild type and Gla-domain mutated human activated protein C in rats

The antithrombotic and anticoagulant effects of recombinant wild type (WT) and mutated human activated protein C (hAPC) were investigated using a rat model of arterial thrombosis. Recent in vitro studies using human plasma have shown enhanced anticoagulant effects of hAPC by mutagenesis of either loop 148 in the serine protease domain or of the Gla domain. The Gla-domain mutant QGNSEDY-hAPC (= H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y) was found to be particularly active as an anticoagulant. We now combined the two mutations to create the variant QGNSEDY-hAPC:B148 and investigated the in vivo effects of this variant as well as of QGNSEDY-hAPC and WT hAPC using a rat model of arterial thrombosis. In vitro clotting experiments using rat plasma demonstrated WT hAPC to be inefficient, whereas both mutant hAPC variants yielded distinct dose dependent anticoagulant effects. In the arterial injury model, a segment of the left common carotid artery was opened longitudinally. An endarterectomy was performed and the arteriotomy was closed, whereafter the vessel was reperfused and the patency rate determined after 31 min. Three treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of 0.8 mg/kg WT or mutant hAPC or vehicle only. The ex vivo clotting times of plasma drawn 3 min after the injections, as compared to baseline clotting times, were approximately doubled by QGNSEDY-hAPC and tripled by QGNSEDY-hAPC:B148 infusions, while WT APC had little effect. Compared to the control group, none of the hAPC preparations had significant antithrombotic effect or increased arteriotomy bleeding.

Synthesis of new class dipeptide analogues with improved permeability and antithrombotic activity

3-( S)-1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid isolated from A. Chinese G. Don was found to possess moderate anti-aggregation activity, but with poor bioavailability. To improve its pharmacological property, we designed and synthesized a series of novel dipeptide analogues by incorporating tetrahydro-β-carboline-3-carboxylic acid skeleton as an amino acid surrogate (∗Trp). It turned out these dipeptide analogues exhibited good membrane permeability based on in vitro Caco-2 cell monolayers permeability assay. As a result, the overall biological properties of these molecules were significantly improved depending on the nature of the amino acid residues introduced onto the 3-position of the tetrahydro-β-carboline moiety. It was very interesting to notice that these dipeptide analogues ( 5b, c, h, i, n, o, p, q) displayed a remarkable dual antiaggregatory activity in both of ADP- and PAF-induced platelet aggregation assay, and their aggregation response was significantly higher than that of aspirin ( p < 0.01). In addition, these dipeptide analogues were observed for the dose-dependent antithrombotic effect using in vivo rat arterial thrombosis model. The potency of antithrombotic activity of 5h, i, n, p was significantly higher than that of aspirin ( n = 12, p < 0.01) at equal dose (5 μmol/kg).

Aldosterone Promotes Thrombosis Formation After Arterial Injury in Mice

To the Editor: The renin/angiotensin/aldosterone system (RAAS) has been broadly implicated in the pathogenesis of cardiovascular disease.1 Angiotensin converting enzyme (ACE) inhibitors have been shown to be beneficial in reducing cardiovascular events.2 The specific role of aldosterone in promoting ischemic cardiovascular disease is unclear. Recent evidence suggests that subjects with primary aldosteronism are at increased risk of cardiovascular events compared with subjects with essential hypertension,3 suggesting a potential direct role of aldosterone in mediating ischemic events. Human studies have demonstrated that pharmacological blockade of aldosterone with spironolactone in congestive heart failure patients leads to reduced cardiovascular mortality, even in the presence of ACE inhibition.4 Because many cardiovascular events are attributable to thrombosis in the setting of atherosclerosis, we used an animal model to directly determine the effect of elevated aldosterone on atherosclerosis as well as arterial thrombus formation after …

Experimental Antioxidant Biotherapy for Protection of the Vascular Wall by Modified Forms of Superoxide Dismutase and Catalase

The antithrombotic activities of superoxide dismutase and catalase are determined by their effects on reactive oxygen species. Modification of these enzymes with chondroitin sulphate enhances the effect due to accumulation of the derivatives on the surface of the vascular wall cells. We have shown that the effects of covalently modified biocatalysts exceed those of native enzymes, free chondroitin sulphate and their mixtures. The superoxide dismutase-chondroitin sulphate conjugate markedly reduced the thrombus mass, while the catalase-chondroitin sulphate conjugate predominantly preserved blood flow. The magnitude and duration of the antithrombotic activity of modified enzymes in a rat arterial thrombosis model allows one to expect a considerable protective effect after their combined application. A single-bolus intravenous injection of the combination between superoxide dismutase-chondroitin sulphate and catalase-chondroitin sulphate covalent conjugates had a significantly lower antithrombotic effect compared with that of the superoxide dismutase-chondroitin sulphate-catalase bienzymic covalent conjugate. This could be explained by different surface distribution of the conjugates in the circulation after their intravenous administration. Biomedical study of this approach promises a new therapeutic strategy of simple and effective protection of the vascular wall against various injuries with the use of the covalent conjugate superoxide dismutase-chondroitin sulphate-catalase. The review analyses the trends of combined application of enzyme preparations to enhance the effect of antioxidant therapy and to develop conjunctive courses of thrombolytic treatment.