Suppressed Thrombus Formation Research Articles

Synergistic coating of Laponite swollen-layer and heparin composite for enhanced antifouling and antithrombotic performance

When implantable medical devices come into contact with blood, acute thrombosis and inflammation often occur due to the adhesion and denaturation of plasma proteins, ultimately degrading device performance. To address this issue, we developed a gel-like coating with superhydrophilic properties, incorporating a heparin based outer layer designed to minimize foreign body reactions and enhance hemocompatibility. The coating was engineered using a layer-by-layer (LbL) assembly of poly-l-lysine (PLL), laponite (Lap), and heparin (Hep), utilizing electrostatic interactions. The successful formation of stable layers was confirmed by QCM-D analysis. The inner layer, composed of PLL/Lap multilayers, formed a gel-like structure that maintained superhydrophilicity and effectively prevented cell adhesion. The outermost PLL/Hep multilayers significantly suppressed thrombus formation by inhibiting plasma protein and red blood cell adsorption as well as platelet activation. The coating exhibited excellent stability, retaining its superhydrophilic properties and heparin functionality even after 7 days of immersion in DPBS. Additionally, in vitro hemocompatibility and cytocompatibility tests confirmed its non-toxicity and overall biocompatibility. These results highlight the potential application of this coating to various implantable medical devices, providing a robust solution for improving device performance and safety.

Antithrombotic Revascularization Strategy of Bioengineered Liver Using a Biomimetic Polymer.

A bioengineered liver has the potential to save patients with end-stage liver disease, and a three-dimensional decellularized scaffold is a promising approach for practical use. The main challenge in bioengineered liver transplantation is thrombogenicity during blood perfusion. We aimed to apply a novel antithrombotic polymer to revascularize liver scaffolds and evaluate the thrombogenicity and biosafety of the polymer-treated scaffolds. A biomimetic polymer, 2-metacryloyloxyethyl phosphorylcholine (MPC) was prepared for modification of the extracellular matrix in liver scaffolds. The polymer was injected into the rat liver scaffolds' portal vein and could extensively react to the vessel walls. In an ex vivo blood perfusion experiment, we demonstrated significantly less platelet deposition in the polymer-treated scaffolds than nontreated or re-endothelialized scaffolds with human umbilical vein endothelial cells. In the heterotopic transplantation model, liver volume was better maintained in the polymer-treated groups, and platelet deposition was suppressed in these groups. Additionally, the polymer-treated liver scaffolds maintained the metabolic function of the recellularized rat primary hepatocytes during perfusion culture. The MPC polymer treatment efficiently suppressed thrombus formation during blood perfusion in liver scaffolds and maintained the function of recellularized hepatocytes. Revascularizing liver scaffolds using this polymer is a promising approach for bioengineered liver transplantation.

Clot-Targeted Antithrombotic Liposomal Nanomedicine Containing High Content of H2O2-Activatable Hybrid Prodrugs.

Liposomes have been extensively explored as drug carriers, but their clinical translation has been hampered by their low drug-loading content and premature leakage of drug payloads. It was reasoned that vesicle-forming prodrugs could be incorporated into the lipid bilayer at a high molar fraction and therefore serve as a therapeutic agent as well as a structural component in liposomal nanomedicine. Boronated retinoic acid (BORA) was developed as a prodrug, which can self-assemble with common lipids to form liposomes at a high molar fraction (40%) and release all-trans retinoic acid (atRA) and hydroxybenzyl alcohol (HBA) simultaneously, in response to hydrogen peroxide (H2O2). Here, we report fucoidan-coated BORA-incorporated liposomes (f-BORALP) as clot-targeted antithrombotic liposomal nanomedicine with H2O2-triggered multiple therapeutic actions. In the mouse model of carotid arterial thrombosis, f-BORALP preferentially accumulated in the injured blood vessel and significantly suppressed thrombus formation, demonstrating their potential as targeted antithrombotic nanomedicine. This study also provides valuable insight into the development of vesicle-forming and self-immolative prodrugs to exploit the benefits of liposomal drug delivery.

Apolipoprotein L Domain Containing 1 Inhibits Tissue Factor to Impede Thrombus Formation in a Rat Model of Deep Vein Thrombosis via Activating PI3K/Akt Pathway

Deep venous thrombosis (DVT) is one of the major health problems worldwide. Apolipoprotein L domain containing 1 (APOLD1) was reported to be downregulated in DVT. The present study intended to investigate whether APOLD1 affects thrombus formation in a rat model of DVT. The rat model of DVT was established by inferior vena cava (IVC) stenosis. At 6, 12, 24 and 48 h after IVC stenosis, the gross IVC with thrombus was dissected and observed. Then, the rats were pre-injected with the lentiviral overexpression vector, APOLD1-LVs, 1 h before IVC stenosis, to evaluate the influence of APOLD1 on thrombosis in rats. The serum levels of D-dimer and TAT as well as the content of TF in IVC tissues were detected by ELISA. IVC stenosis resulted in thrombus formation in rats, increased serum levels of D-dimer and TAT, and decreased APOLD1 expression. APOLD1 overexpression inhibited in vivo thrombosis, reduced serum levels of D-dimer, and downregulated TF activity and level. APOLD1 overexpression also increased p-PI3K and p-Akt protein levels. APOLD1 suppresses thrombus formation in a rat model of DVT via downregulating TF expression by activating the PI3K/Akt pathway.

Study about animal (NZW Rabbit, Juvenile pig) test to secure safety and effectiveness of thermoplastic polyurethane stent graft & graft using electrospinning

Background. A stent is a cylindrical medical device used to normalize the flow of blood or body fluids, such as blood vessels, gastrointestinal tract, and bile duct, by inserting it into a narrowed or blocked area under image fluoroscopy without performing a surgical operation when the flow is not smooth. In particular, stents applied to blood vessels are divided into stents for coronary arteries, stents for blood vessels, stents for blood vessels in the brain, stents for iliac arteries, and stents made of absorbent materials. It is used to improve blood flow.Coronary artery disease refers to a lesion in which blood flow to the myocardium is impaired due to stenosis of the coronary arteries due to atherosclerosis. Atherosclerotic coronary artery disease has long been known as the most common cause of death in the West. Although it has not yet occurred frequently in the East, including Korea, it is reported that coronary artery disease is on the rise with the recent change in eating habits. Coronary artery disease is clinically manifested in one of three aspects: angina pectoris, myocardial infarction, and sudden death. Double myocardial infarction is usually caused by sudden thrombus in a coronary artery with atherosclerotic stenosis, resulting in complete blockage of myocardial blood flow. Myocardial infarction, which was supplied by blood vessels from blocked blood vessels, leads to necrosis. In order to improve blood flow in blood vessels blocked from blood supply due to myocardial infarction, stenting is in the spotlight along with surgical methods. Among the causes of blocking the flow of blood vessels, thrombus is seen as the main cause, so the frequency of using vascular stents with suppressed thrombus formation is increasing.Results. A coronary stent is a medical device used to improve blood flow by expanding a coronary artery narrowed by the formation of a thrombus, and is an intermediary procedure that minimizes side effects through surgery. Among the causes of blocking the flow of blood vessels, thrombus fish are seen as the main cause, so the frequency of using vascular stents with suppressed thrombus formation is increasing. S&G Biotech Research Institute is developing a self-expanding Nitinol stent graft that can ensure patency while suppressing the formation of blood clots. The graft material used here is a material that hardly decomposes in bodily fluids such as blood by using a thermoplastic polyurethane material, and is loaded with an antithrombotic agent. Our research team produced a stent graft by spinning the TPU graft onto the self-expanding Nitinol stent skeleton with an electrospinning device, and confirmed its safety and effectiveness by conducting a biological safety evaluation and animal testing. In addition to the vascular stent for the coronary artery for the treatment of coronary artery disease, the frequency of application of the aortic vascular stent graft for the treatment of aortic vascular disease is also increasing. The aortic vascular stent graft is a stent composed of a metal stent skeleton and a graft and suture made of a biocompatible polymer material and is used for aortic dissection and aneurysm treatment.Conclusion. After blood vessel extraction, the surface of the inserted stent and the inside of the blood vessel were checked, and the result was grade 0 (no thrombus observed). No other peculiarities were identified inside the blood vessels. As a result of checking the H&E results of 3 parts (distal part, middle part, and proximal part) of each blood vessel, in the stent group. Neointimal proliferation was not confirmed.Funding: This research was financially supported by the Ministry of Trade, Industry and Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT) through the International Cooperative R&D program (Project No. P0016047).

Fibroblast growth factor-21 as a novel metabolic factor for regulating thrombotic homeostasis

Fibroblast growth factor-21 (FGF-21) performs a wide range of biological functions in organisms. Here, we report for the first time that FGF-21 suppresses thrombus formation with no notable risk of bleeding. Prophylactic and therapeutic administration of FGF-21 significantly improved the degree of vascular stenosis and reduced the thrombus area, volume and burden. We determined the antithrombotic mechanism of FGF-21, demonstrating that FGF-21 exhibits an anticoagulant effect by inhibiting the expression and activity of factor VII (FVII). FGF-21 exerts an antiplatelet effect by inhibiting platelet activation. FGF-21 enhances fibrinolysis by promoting tissue plasminogen activator (tPA) expression and activation, while inhibiting plasminogen activator inhibitor 1 (PAI-1) expression and activation. We further found that FGF-21 mediated the expression and activation of tPA and PAI-1 by regulating the ERK1/2 and TGF-β/Smad2 pathways, respectively. In addition, we found that FGF-21 inhibits the expression of inflammatory factors in thrombosis by regulating the NF-κB pathway.

Hydrogen peroxide-responsive platelet membrane-coated nanoparticles for thrombus therapy.

Occlusion of blood vessels caused by thrombi is the major pathogenesis of various catastrophic cardiovascular diseases. Thrombi can be prevented or treated by antithrombotic drugs. However, free antithrombotic drugs often have relatively low therapeutic efficacy due to a number of limitations such as short half-life, unexpected bleeding complications, low thrombus targeting capability, and negligible hydrogen peroxide (H2O2)-scavenging ability. Inspired by the abundance of H2O2 and the active thrombus-targeting property of platelets, a H2O2-responsive platelet membrane-cloaked argatroban-loaded polymeric nanoparticle (PNPArg) was developed for thrombus therapy. Poly(vanillyl alcohol-co-oxalate) (PVAX), a H2O2-degradable polymer, was synthesized to form an argatroban-loaded nanocore, which was further coated with platelet membrane. The PNPArg can effectively target the blood clots due to the thrombus-homing property of the cloaked platelet membrane, and subsequently exert combined H2O2-scavenging effect via the H2O2-degradable nanocarrier polymer and antithrombotic effect via argatroban, the released payload. The PNPArg effectively scavenged H2O2 and protected cells from H2O2-induced cellular injury in RAW 264.7 cells and HUVECs. The PNPArg rapidly targeted the thrombosed vessels and remarkably suppressed thrombus formation, and the levels of H2O2 and inflammatory cytokines in the ferric chloride-induced carotid arterial thrombosis mouse model. Safety assessment indicated good biocompatibility of the PNPArg. Taken together, the biomimetic PNPArg offers multiple functionalities including thrombus-targeting, antioxidation, and H2O2-stimulated antithrombotic action, thereby making it a promising therapeutic nanomedicine for thrombosis diseases.

A novel anticoagulant affinity membrane for enhanced hemocompatibility and bilirubin removal

Affinity membrane is widely employed to promote specific adsorption of toxins and reduce the blood purification therapeutic time. However, it suffers from insufficient toxin binding and low hemocompatibility. Herein, a novel anticoagulant affinity membrane (AAM) was developed to clear bilirubin from human blood in a pore-flow-through way. Firstly, a nylon net membrane with a regularly arranged pore as the matrix was coated with poly(pyrrole-3-carboxylic acid) via chemical vapor deposition (CVD) method. Then, poly(L-arginine) (PLA) as a highly specific ligand of bilirubin, was immobilized onto the surface of the composited membrane after the modification of heparin. Owing to the 3-dimensional molecular architecture of PLA, up to 86.1 % of bilirubin was efficiently cleared. Besides, the AAM exhibited effective anticoagulant activity in the measurement of clotting time, with suppressed thrombus formation, low hemolysis ratio, minimized platelet and leukocyte adhesion, and excellent biosafety. Therefore, the AAM has enormous potential in blood purification therapy for enhancing hemocompatibility and bilirubin removal.

The Marine-Derived Triterpenoid Frondoside A Inhibits Thrombus Formation.

Background: The marine-derived triterpenoid frondoside A inhibits the phosphatidylinositol-3-kinase (PI3K) pathway in cancer cells. Because this pathway is also crucially involved in platelet activation, we studied the effect of frondoside A on thrombus formation. Methods: Frondoside A effects on platelet viability, surface adhesion molecule expression, and intracellular signaling were analyzed by flow cytometry and Western blot. The effect of frondoside A was analyzed by photochemically induced thrombus formation in the mouse dorsal skinfold chamber model and by tail vein bleeding. Results: Concentrations of up to 15 µM frondoside A did not affect the viability of platelets, but reduced their surface expression of P-selectin (CD62P) and the activation of glycoprotein (GP)IIb/IIIa after agonist stimulation. Additional mechanistic analyses revealed that this was mediated by downregulation of PI3K-dependent Akt and extracellular-stimuli-responsive kinase (ERK) phosphorylation. Frondoside A significantly prolonged the complete vessel occlusion time in the mouse dorsal skinfold chamber model of photochemically induced thrombus formation and also the tail vein bleeding time when compared to vehicle-treated controls. Conclusion: Our findings demonstrated that frondoside A inhibits agonist-induced CD62P expression and activation of GPIIb/IIIa. Moreover, frondoside A suppresses thrombus formation. Therefore, this marine-derived triterpenoid may serve as a lead compound for the development of novel antithrombotic drugs.

Accelerated endothelialization and suppressed thrombus formation of acellular vascular grafts by modifying with neointima-inducing peptide: A time-dependent analysis of graft patency in rat-abdominal transplantation model

Acellular blood vessels have clinical potential as tissue-engineered vascular grafts. However, neointima is hard to form on their luminal surface. We recently reported the integrin α4β1 ligand peptide (Arg-Glu-Asp-Val) conjugated with a repetitive Pro-Hyp-Gly sequence as luminal surface modifier. By using this peptide, excellent patency of tissue-engineered small-caliber long-bypass grafts in minipig transplantation model was achieved. Here, the time-dependent change of the graft patency is investigated by using rat abdominal transplantation model. In vitro test showed that 86% of the endothelial cells were adhered to the peptide-modified graft surface, while cells were scarcely adhered on the unmodified and random peptide-modified surfaces. After transplantation in the abdominal aorta, the patency of unmodified and random peptide-modified grafts gradually decreased during two to three weeks and reached 20–40% in four weeks. In contrast, 80% of the modified grafts were patent without any thrombus formation at four weeks. These results suggest that the luminal surface modifier was bound to acellular surface through (Pro-Hyp-Gly)7 sequence and improved the in vivo graft patency by endothelialization and thrombus formation suppression.

Pre-clinical pharmacodynamic study of a novel oral factor Xa inhibitor zifaxaban

Zifaxaban is an orally active, direct Factor Xa (FXa) inhibitor that is in development for the prevention and treatment of arterial and venous thrombosis. This study was conducted to investigate the biochemical and pharmacological activity of zifaxaban. In vitro activity was evaluated by enzyme, platelet aggregation, and clotting assays. In vivo effects were examined in venous thrombosis, arteriovenous-shunt thrombosis, carotid thrombosis, and bleeding models in rats. Zifaxaban competitively inhibits human FXa (IC50 = 11.1 nM) with > 10,000-fold greater selectivity than other serine proteases. It did not impair platelet aggregation induced by collagen, adenosine diphosphate (ADP) or arachidonic acid. It significantly prolonged clotting time, prothrombin time (PT), and activated partial thromboplastin time (APTT) in the plasma of humans, rabbits, and rats, with a relatively weak effect on thrombin time (TT). In venous thrombosis models in rats, zifaxaban strongly suppressed thrombus formation with ED50 values of 3.09 mg/kg, and its best efficacy time occurred at 2 h after administration. In arteriovenous-shunt thrombosis and carotid thrombosis models in rats, it inhibited thrombus formation in a dose-dependent manner. And in the rat tail bleeding assay, it showed a trend of less bleeding than rivaroxaban at doses that achieved the same antithrombotic effect. In conclusion, zifaxaban is a selective and direct FXa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

Abstract 299: Theranostic nanoparticles for Thrombosed Vessels: H 2 O 2 -Activatable Contrast-Enhanced Photoacoustic Imaging and Antithrombotic Therapy

A thrombus (blood clot) is formed in the injured vessels to maintain the integrity of vasculature. A thrombus, composed mainly of activated platelets and fibrin, obstructs arteries or veins, leading to various life-threatening diseases. During thrombus formation, H 2 O 2 (hydrogen peroxide) is overproduced by activated platelets and other vascular sources and serves as a key mediator of platelet activation and aggregation, facilitating inflammation and development of obstructive vascular diseases. In this regard, high level of H 2 O 2 holds great potential as diagnostic and therapeutic markers of obstructive capsular diseases. Inspired by the distinctive physicochemical characteristics of thrombi such as abundant fibrin and the significant of elevated level of H 2 O 2 , we developed H 2 O 2 -activatable novel nanomedicine (T-FBM) that could detect thrombi through the amplification of ultrasound/photoacoustic signal and serve as an antithrombotic nanomedicine. T-FBM nanoparticles were designed to target fibrin-rich thrombi and be activated by H 2 O 2 to generate CO 2 bubbles to amplify the photoacoustic signal. In the phantom studies, T-FBM nanoparticles showed significant amplification of ultrasound/photoacoustic signals in a H 2 O 2 -triggered manner. T-FBM nanoparticles also exerted H 2 O 2 -activatable antioxidant, anti-inflammatory and antiplatelet activities on endothelial cells. In mouse models of carotid arterial injury, T-FBM nanoparticles significantly enhanced the photoacoustic contrast specifically in thrombosed vessels and significantly suppressed thrombus formation. We anticipate that T-FBM nanoparticles hold great translational potential as nanotheranostics for H 2 O 2 -associated cardiovascular diseases.

Molecularly Engineered Theranostic Nanoparticles for Thrombosed Vessels: H2O2-Activatable Contrast-Enhanced Photoacoustic Imaging and Antithrombotic Therapy.

A thrombus (blood clot), composed mainly of activated platelets and fibrin, obstructs arteries or veins, leading to various life-threatening diseases. Inspired by the distinctive physicochemical characteristics of thrombi such as abundant fibrin and an elevated level of hydrogen peroxide (H2O2), we developed thrombus-specific theranostic (T-FBM) nanoparticles that could provide H2O2-triggered photoacoustic signal amplification and serve as an antithrombotic nanomedicine. T-FBM nanoparticles were designed to target fibrin-rich thrombi and be activated by H2O2 to generate CO2 bubbles to amplify the photoacoustic signal. In the phantom studies, T-FBM nanoparticles showed significant amplification of ultrasound/photoacoustic signals in a H2O2-triggered manner. T-FBM nanoparticles also exerted H2O2-activatable antioxidant, anti-inflammatory, and antiplatelet activities on endothelial cells. In mouse models of carotid arterial injury, T-FBM nanoparticles significantly enhanced the photoacoustic contrast specifically in thrombosed vessels and significantly suppressed thrombus formation. We anticipate that T-FBM nanoparticles hold great translational potential as nanotheranostics for H2O2-associated cardiovascular diseases.

Fibrin-Targeted and H2O2-Responsive Nanoparticles as a Theranostics for Thrombosed Vessels.

A thrombus (blood clot) is formed in injured vessels to maintain the integrity of vasculature. However, obstruction of blood vessels by thrombosis slows blood flow, leading to death of tissues fed by the artery and is the main culprit of various life-threatening cardiovascular diseases. Herein, we report a rationally designed nanomedicine that could specifically image obstructed vessels and inhibit thrombus formation. On the basis of the physicochemical and biological characteristics of thrombi such as an abundance of fibrin and an elevated level of hydrogen peroxide (H2O2), we developed a fibrin-targeted imaging and antithrombotic nanomedicine, termed FTIAN, as a theranostic system for obstructive thrombosis. FTIAN inhibited the generation of H2O2 and suppressed the expression of tumor necrosis factor-alpha (TNF-α) and soluble CD40 ligand (sCD40L) in activated platelets, demonstrating its intrinsic antioxidant, anti-inflammatory, and antiplatelet activity. In a mouse model of ferric chloride (FeCl3)-induced carotid thrombosis, FTIAN specifically targeted the obstructive thrombus and significantly enhanced the fluorescence/photoacoustic signal. When loaded with the antiplatelet drug tirofiban, FTIAN remarkably suppressed thrombus formation. Given its thrombus-specific imaging along with excellent therapeutic activities, FTIAN offers tremendous translational potential as a nanotheranostic agent for obstructive thrombosis.

Dual inhibition of PI3K and mTOR by VS-5584 suppresses thrombus formation

VS-5584 is a highly selective dual kinase inhibitor which suppresses phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) activity. Because these kinases are crucially involved in primary hemostasis, we herein investigated the effect of this compound on thrombus formation in vitro and in vivo. Pretreatment of washed platelets (WP) or platelet-rich plasma (PRP) with VS-5584 inhibited the agonist-induced activation of surface glycoprotein complex (GP)IIb/IIIa and the upregulation of P-selectin. This was associated with a significantly reduced formation of platelet-leukocyte aggregates (PLA). VS-5584 further attenuated platelet aggregation and adhesion after agonist stimulation. In contrast, endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cellular adhesion molecule (VCAM)-1 and secretion of von Willebrand Factor (vWF) were not affected by the dual kinase inhibitor. In vivo, VS-5584 inhibited photochemically induced thrombus formation as shown by a significantly prolonged time to complete vessel occlusion when compared to vehicle-treated controls. This was associated with an elevated tail vein bleeding time, indicating a potential hemorrhagic risk in VS-5584-treated mice. Taken together, these novel findings demonstrate that VS-5584 is a potent inhibitor of primary hemostasis targeting multiple platelet functions.

Coordinated Membrane Ballooning and Procoagulant Spreading in Human Platelets.

Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.

Short- and long-term treatment with folic acid suppresses thrombus formation in atherogenic mice in vivo

In the present study, we examined the effects of short- and long-term treatment with folic acid (FA) on thrombus formation in vivo in atherogenic mice to explore a novel agent for the prevention of atherothrombotic disease. Apolipoprotein E and low-density lipoprotein receptor double deficient (ApoE−/−LDLR−/−) mice were orally administrated a single bolus of FA (20mg/kg) or fed an atherogenic diet with or without FA (0.02, 0.5, and 1.5mg/kg) for 12 weeks. Thrombus formation and endothelial function were assessed in vivo using the He–Ne laser-induced carotid artery thrombus formation test and the flow-mediated vasodilation method. Platelet reactivity was assessed ex vivo using haemostatometry. Short-term treatment with FA markedly increased plasma folate levels and significantly suppressed laser-induced thrombus formation in apoE−/−LDLR−/− mice. Short-term treatment with FA suppressed platelet reactivity in apoE−/−LDLR−/− mice, but FA treatment did not affect endothelial function or plasma homocysteine levels. Long-term treatment with FA increased plasma folate levels dose-dependently. Thrombus formation and endothelial dysfunction were suppressed by treatment with 0.5 and 1.5mg/kg of FA, respectively, but not with 0.02mg/kg of FA, whereas platelet reactivity was not altered by treatment with any dose of FA. Long-term treatment with all doses of FA decreased the plasma homocysteine levels in apoE−/−LDLR−/− mice, although this result was not consistent with its anti-thrombotic action. In conclusion, our data showed that short- and long-term treatment with FA could suppress in vivo thrombus formation in an atherogenic setting, independent of its hypohomocysteinemic action.

Comparative Evaluation of Direct Thrombin and Factor Xa Inhibitors with Antiplatelet Agents under Flow and Static Conditions: An In Vitro Flow Chamber Model

Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s−1. Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.

Combined Dabigatran and Antiplatelet Agents Assessed by the Novel Microchip-Based Flow Chamber System

AbstractAbstract 1167Evaluation of the overall antithrombotic activity of dabigatran in combination with antiplatelet agents is difficult because plasma-based clotting for dabigatran, and platelet aggregometry in anticoagulated blood are two separate tests which do not reflect physiological interactions between soluble factors and platelets. The use of a flow chamber could be more suitable in evaluating a flow-dependent platelet activation and coagulation responses.The aim of the current study was to comparatively evaluate antithrombotic effects of dabigatran in combination with dual antiplatelet therapy (aspirin plus P2Y12 blockade) using the microchip-based flow chamber (T-TAS, Fujimori Kogyo, Japan)(1), and thrombin generation (TG) assay (Thrombinoscope, Maastricht, the Netherlands)(2). After the local ethics committee approval, blood samples were obtained from consented 5 healthy volunteers in the tubes containing 3.2% sodium citrate. Whole blood samples were mixed with dabigatran (250, 500, 1000 nM), aspirin (100 nM) plus ARC-66096 (P2Y12 inhibitor, 1000 nM) at 25¡C for 10 min. Corn trypsin inhibitor (50 μg/ml) was used to prevent contact activation. The whole blood sample was perfused in the capillary pre-coated with collagen and thromboplastin at the shear rate of 240 or 600 s−1. The process of thrombus formation was monitored by flow pressure increases inside the capillary; (i) lag time before it reaches 10 kPa (T10), (ii) occlusion time (OT) is the lag time before it reaches 80 kPa as thrombus completely occludes the capillary, and (iii) AUC30 is an area under the flow pressure curve (under 80 kPa) after 30 min of perfusion. For TG assay, platelet-rich plasma (platelet count 150 × 103/μl) was prepared from citrated whole blood. TG was triggered by adding 20 μl of CaCl2-fluorogenic substrate buffer to 80 μl of the sample mixed with tissue factor (1 pM) in each well. The lag time (min), and peak thrombin concentration (nM) were evaluated.In the flow chamber, dabigatran inhibited white thrombus formation in a concentration dependent manner at shear rates of 240 and 600 s−1(Fig. 1). At 500 nM of dabigatran, OT was prolonged by ∼2-fold from the (non-treated) control at both shear rates. The combination of aspirin and AR-C66096 only weakly suppressed thrombus formation, but it enhanced the antithrombotic efficacy of dabigatran at both shear rates (Fig. 1). In TG measurements using platelet-rich plasma, dabigatran at 500 nM prolonged the by 3.17-fold, and reduced the peak by 57.6% compared to the untreated control (Table 1). Aspirin and AR-C66096 weakly prolonged the lag time without affecting the peak height. There were relatively small changes in these parameters when antiplatelet agents were combined with dabigatran (Table 1).Our results suggest that combined antithrombotic effects of dabigatran, aspirin, and P2Y12inhibition can be demonstrated in the whole blood using the flow chamber system compared without additional plasma preparation required for TG assay. [Display omitted] The re-calcified whole blood was perfused at the shear rate of 240 s−1 or 600 s−1. Asp/AR-C=aspirin and AR-C66096Table 1Lag time (min)Peak (nM)NativeAsp/AR-CNativeAsp/AR-CControl6.8 ± 0.89.4 ± 3.292.1 ± 23.791.2 ± 29.5Dabi 250 nM18.6 ± 5.421.1 ± 4.569.3 ± 20.652.2 ± 13.6Dabi 500 nM21.6 ± 5.326.2 ± 10.253.0 ± 5.847.8 ± 9.1Dabi 1000 nM30.2 ± 5.635.1 ± 6.323.0 ± 6.922.0 ± 8.4Dabi=dabigatran, Native=no antiplatelet agents, Asp/AR-C=aspirin and AR-C66096 Disclosures:Hosokawa:Fujimori Kogyo: Employment. Ohnishi:Fujimori Kogyo: Employment. Sameshima:Fujimori Kogyo: Employment.

Enhanced induction of heme oxygenase-1 suppresses thrombus formation and affects the protein C system in sepsis

Heme oxygenase-1 (HO-1) displays anti-inflammatory and cytoprotective activities in sepsis. Here, we investigated the effects of HO-1 on thrombus formation and the protein C system in a septic C57BL/6 mouse model induced by cecal ligation and perforation (CLP). Septic mice were either preinjected with the vehicle, pretreated with hemin (an HO-1 inducer) or zinc protoporphyrin IX (ZnPP, an HO-1 inhibitor), or given a combination of hemin + ZnPP. CLP increased significantly the hepatic expression of HO-1; increased thrombosis in livers, kidneys, and lungs; shortened the prothrombin time (PT) and activated partial thromboplastin time (APTT); elevated the levels of tumor necrosis factor-1α (TNF-1α), interleukin-6 (IL-6), and thrombomodulin (TM); reduced the levels of protein C (PC) and activated protein C (aPC); and downregulated hepatic expression of PC and TM. The preadministration of hemin to septic mice increased the expression and activity of HO-1; inhibited thrombosis in the preceding 3 organs; prolonged PT and APTT; inhibited the production of TNF-α and IL-6; upregulated the expression of PC and TM in livers; elevated the plasma levels of PCand aPC; and reduced the plasma levels of TM. In contrast, ZnPP showed opposite effects to hemin and reversed the effects of hemin by inhibiting the activity of HO-1. The administration of tricarbonyl dichloro ruthenium (II) dimer (CORM-2), which is a CO-releasing molecule, had a similar effect to hemin on thrombosis and the protein C system. The data indicate that the enhanced induction of HO-1 inhibits thrombus formation and affects the protein C system in sepsis.