Induced Carotid Artery Thrombosis Research Articles

Abstract Tu032: LRRC8 complex regulates platelet activation and thrombosis

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Antipolyphosphate monoclonal antibodies derived from autoimmune mice

BackgroundInorganic polyphosphates (polyPs) are linear chains of phosphates that accelerate blood clotting. Targeting polyP in vivo has been shown to reduce thrombosis. ObjectivesTo identify and characterize anti-polyP monoclonal antibodies that could be used as analytical tools and as antithrombotic agents. MethodsHybridomas were prepared from spleen cells from autoimmune NZBWF1/J female mice and screened for anti-polyP antibodies. Antibodies that bound polyP using enzyme-linked immunosorbent assay and pull-down assays were further characterized with plate binding, surface plasmon resonance, and plasma-based clotting assays. Antithrombotic potential was evaluated in a murine ferric chloride–induced carotid artery thrombosis model. ResultsOf 4 antibodies that bound polyP in our pull-down assay, 2 (PP2069 and PP2099) were available for further characterization. While analyzing these anti-polyP antibodies, we found secretory leukocyte peptidase inhibitor (SLPI) to be a common contaminant of these antibodies and that SLPI binds polyP. We removed SLPI quantitatively from our purified immunoglobulin G. Both PP2069 and PP2099 immunoglobulin G displayed high affinity for polyP but also bound to other polyanions such as DNA, heparin, and certain other glycosaminoglycans, indicating limited specificity. Both antibodies inhibited polyP-initiated plasma clotting in vitro. When tested in vivo in a mouse thrombosis model, however, neither PP2069 nor PP2099 exhibited a significant antithrombotic effect. ConclusionAutoimmune mice spontaneously produce antibodies against polyP. The 2 examples of anti-polyP monoclonal antibodies studied here not only bound to polyP with high affinity but also cross-reacted with DNA and heparin. Neither antibody protected against thrombosis in a mouse model, but they might have some utility for in vitro studies of polyP.

Abstract 138: Platelet RIPK1 Promotes Platelet Activation And Aggregation In Arteriolar Thrombosis And Ischemic Stroke

Receptor-interacting protein Ser/Thr kinase 1 (RIPK1) is a molecular switch for inflammation and cell death in nucleated cells, and its function is controlled by phosphorylation at the Ser166 residue. However, it is unknown whether RIPK1 regulates platelet function in arterial thrombosis. Intravital microscopy using megakaryocyte-specific Ripk1 conditional knockout (CKO) mice ( Ripk1 fl/fl;Pf4-cre ) revealed that platelet Ripk1 promoted platelet thrombus formation at sites of laser-induced cremaster arteriolar thrombosis. Deletion of platelet Ripk1 prolonged the time to occlusion in FeCl 3 -induced carotid arterial thrombosis without affecting tail bleeding times. Further, compared to wild-type (WT) mice, Ripk1 CKO mice exhibited improved neurological deficits and reduced infarct volume in ischemic stroke induced by transient middle cerebral artery occlusion. Flow cytometry and aggregation assays showed that deletion of platelet Ripk1 reduced P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by thrombin, collagen-related peptides (CRP), and U46619, but not ADP. Pretreatment of Ripk1-null platelets with ADP rescued the aggregation defect. Treatment of platelets with Nec-1s, a RIPK1 inhibitor that blocks RIPK1-Ser166 phosphorylation, did not affect platelet activation and aggregation. Instead, RIPK1-Ser320/321 was rapidly phosphorylated in thrombin- or CRP-stimulated human and mouse platelets. Treatment of platelets with an inhibitor of phospholipase C, p38, or MAPK-activated protein kinase 2 (MK2), but not a protein kinase C inhibitor, decreased RIPK1-Ser320/321 phosphorylation after thrombin or CRP stimulation, implying that the p38-MK2 axis phosphorylates RIPK1-Ser320/321. Using a genetic approach in human megakaryoblastic MEG-01 cells, we found that RIPK1 knockdown reduced granule secretion from stimulated MEG-01 cells, which was rescued by overexpressing WT RIPK1, but not mutant RIPK1-Ser320Ala, in the RIPK1 knockdown MEG-01 cells. These results suggest that platelet RIPK1 enhances granule secretion during platelet activation through p38-MK2-mediated phosphorylation of RIPK1-Ser320/321, thereby contributing to the pathology of thrombotic disease.

Abstract 119: LRRC8 Complex Is An ATP Release Channel Regulating Platelet Activation And Thrombosis

Platelet shape and volume changes are early mechanical events in platelet activation proposed to contribute to arterial thrombosis. Here, we identify single-nucleotide polymorphisms (SNPs) in four leucine-rich repeat containing protein subunits (LRRC8A, B, C, and D) expressed in human platelets that are independently associated with increased mean platelet volume in human genetic studies - implicating the volume-sensing LRRC8 channel complex as regulating platelet function in humans. We show LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist -stimulated P-selectin exposure, and αIIbβ3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces, agonist-induced platelet aggregation and ATP secretion. MK-specific LRRC8A conditional knockout mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, LRRC8A mediates swelling-induced ATP release to amplify agonist-stimulated calcium influx and aggregation in platelets. Treatment of platelets with small-molecule LRRC8 channel inhibitors recapitulate the defects in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as a regulator of platelet function and thrombosis - providing a proof-of-concept for a novel anti-thrombotic drug target.

OxLDL enhances procoagulant activity of endothelial cells by TMEM16F-mediated phosphatidylserine exposure.

Oxidized low-density lipoprotein(oxLDL), a key component in atherosclerosis and hyperlipidemia, is a risk factor for atherothrombosis in dyslipidemia, yet its mechanism is poorly understood. In this study, we used oxLDL-induced human aortic endothelial cells (HAECs) and high-fat diet (HFD)-fed mice as a hyperlipidemia model. Phosphatidylserine(PS) exposure, cytosolic Ca2+, reactive oxygen species (ROS), and lipid peroxidation were measured by flow cytometer. TMEM16F expression was detected by immunofluorescence, western blot, and reverse transcription polymerase chain reaction. Procoagulant activity(PCA) was measured by coagulation time, intrinsic/extrinsic factor Xase, and thrombin generation. We found that oxLDL-inducedPS exposure and the corresponding PCA of HAECs were increased significantly compared with control, which could be inhibited over 90% by lactadherin. Importantly, TMEM16F expression in oxLDL-induced HAECs was upregulated by enhanced intracellular Ca2+ concentration, ROS, and lipid peroxidation, which led to PS exposure. Meanwhile, the knockdown of TMEM16F by short hairpin RNA significantlyinhibited PS exposure in oxLDL-induced HAECs. Moreover, we observed that HFD-fed mice dramatically increased the progress of thrombus formation and accompanied upregulated TMEM16F expression by thromboelastography analysis, FeCl3-induced carotid artery thrombosis model, and western blot. Collectively, these results demonstrate that TMEM16F-mediated PS exposure may contribute to prothrombotic status under hyperlipidemic conditions, which may serve as a novel therapeutic target for the prevention of thrombosis in hyperlipidemia.

Nanoassemblies of Self-Immolative Boronate-Bridged Retinoic Acid Dimeric Prodrug as a Clot-Targeted Self-Deliverable Antithrombotic Nanomedicine.

All trans-retinoic acid (atRA) has potent anti-inflammatory and antiplatelet activity, but its clinical translation as an antithrombotic drug has been hampered by its low therapeutic efficacy. Here, we describe a facile and elegant strategy that converts atRA into systemically injectable antithrombotic nanoparticles. The strategy involves the dimerization of two atRA molecules using a self-immolative boronate linker that is cleaved specifically by hydrogen peroxide (H2O2) to release anti-inflammatory hydroxybenzyl alcohol (HBA), followed by dimerization-induced self-assembly to generate colloidally stable nanoparticles. The boronated atRA dimeric prodrug (BRDP) could form injectable nanoparticles in the presence of fucoidan that serves as an emulsifier and a targeting ligand to P-selectin overexpressed on the damaged endothelium. In response to H2O2, fucoidan-decorated BRDP (f-BRDP) nanoassemblies dissociate to release both atRA and HBA, while scavenging H2O2. In a mouse model of ferric chloride (FeCl3)-induced carotid arterial thrombosis, f-BRDP nanoassemblies target the thrombosed vessel and significantly inhibit thrombus formation. The results demonstrate that dimerization of atRA molecules via a boronate linker enables the formation of stable nanoassemblies with several benefits: high drug loading, drug self-delivery, on-demand multiple antithrombotic actions, and simple fabrication of nanoparticles. Overall, this strategy provides a promising expedient and practical route for the development of translational self-deliverable antithrombotic nanomedicine.

Increased dipeptidyl peptidase-4 accelerates chronic stress-related thrombosis in a mouse carotid artery model.

Exposure to chronic psychosocial stress is a risk factor for metabolic cardiovascular disorders. Given that dipeptidyl peptidase-4 (DPP-4) has an important role in human pathobiology, we investigated the role of DPP-4 in stress-related thrombosis in mice, focusing on oxidative stress and the von Willebrand factor (vWF)-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Male mice randomly assigned to nonstress and 2-week immobilized-stress groups underwent iron chloride3 (FeCl3)-induced carotid artery thrombosis surgery for morphological and biochemical studies at specific times. On day 14 post-stress/surgery, stress had enhanced the lengths and weights of arterial thrombi, with alterations of plasma DPP-4, plasminogen activation inhibitor-1 and ADAMTS13. The stressed mice had increased levels of vascular cell adhesion molecule-1, intracellular adhesion molecule-1, monocyte chemoattractant protein-1, gp91phox, p22phox, matrix metalloproteinase-2 (MMP-2), MMP-9, cathepsins S and K mRNAs and/or proteins, and reduced levels of endothelial nitric oxide synthase, catalase and superoxide dismutase-1 mRNAs and/or proteins. Stress also accelerated arterial endothelial cell damage. The DPP-4 inhibitor anagliptin ameliorated the stress-induced targeted molecular and morphological changes and thrombosis. In vitro, DPP-4 inhibition also mitigated the alterations in the targeted ADAMTS13 and other oxidative and inflammatory molecules in human umbilical vein endothelial cells in response to H2O2. DPP-4 inhibition appeared to improve the FeCl3-induced thrombosis in mice that received stress, possibly via the improvement of ADAMTS13 and oxidative stress, suggesting that DPP-4 could become a novel therapeutic target for chronic psychological stress-related thrombotic events in metabolic cardiovascular disorders.

An inhibitor of myosin II, blebbistatin, suppresses development of arterial thrombosis

Arterial thrombosis (AT) causes various ischemia-related diseases, which impose a serious medical burden worldwide. As an inhibitor of myosin II, blebbistatin has an important role in thrombosis development. We investigated the effect of blebbistatin on carotid artery ligation (CAL)-induced carotid AT and its potential underlying mechanism. A model of carotid AT in mice was generated by CAL. Mice were divided into three groups: CAL model, blebbistatin-treated, and sham-operation. After 7 days, blood vessels were harvested from mice in each group. The procoagulant activity of tissue factor (TF) was tested by a chromogenic assay, and thrombus severity assessed by histopathology scores. Expression of non-muscle myosin heavy chain II A (NMMHCIIA), TF, glycogen synthase kinase 3β (GSK3β), and nuclear factor-kappa B (NF-κB) was detected by immunohistochemical and immunofluorescence staining. mRNA expression was measured by quantitative polymerase chain reaction. Blebbistatin (1 mg/kg) inhibited development of carotid AT, reduced infiltration of inflammatory cells, and prevented vascular-tissue damage, relative to the model group. Furthermore, blebbistatin also reduced the procoagulant activity of TF. Immunohistochemical and immunofluorescence data demonstrated that, compared with the model group, blebbistatin intervention reduced expression of NMMHCIIA, TF, GSK3β, p65, and p-p65 in carotid-artery endothelia in the CAL-induced AT model, but it increased levels of p-GSK3β. Blebbistatin could inhibit expression of NMMHCIIA mRNA in the CAL model. Overall, our data demonstrated that blebbistatin could inhibit TF expression and AT development in arterial endothelia (at least in part) via GSK3β/NF-κB signaling.

A bacterial metabolite, trimethylamine N-oxide, disrupts the hemostasis balance in human primary endothelial cells but no coagulopathy in mice.

: The gut microbial metabolite, trimethylamine N-oxide (TMAO), was previously reported to induce platelet hypersensitivity, which leads to thrombotic risk. However, the molecular mechanism underlying the effects of TMAO on endothelial cells (EC), which is the primary vessel wall contact with the lumen, remains unclear. Here, we investigated the impact of TMAO on procoagulant activity (PCA) in EC and mice, for a possible link between microbiota and coagulation. To test the PCA of TMAO in EC, we performed one-stage clotting assays and converted into PCA. Antitissue factor (TF) antibody was used to test the TF role in PCA. Quantitative PCR was performed to measure the TF, thrombomodulin, IL-6, TF pathway inhibitor and IL-1b expressions at mRNA levels. To test the PCA and thrombotic risk by TMAO in mice, we challenged the mice with TMAO (8 mg/kg; 3 h) and measured the thrombin-anti-thrombin complex (TAT) and D-dimer levels as well as ferric chloride (FeCl3)-induced carotid artery thrombosis model. TMAO-induced TF expression in EC at mRNA and protein levels, dose-dependently. TF blocking experiment confirmed that the increased PCA by TMAO is TF-dependent. Also, mitogen-activated protein kinase pathway inhibitors abolished TMAO-induced TF expression. However, TMAO challenged mice failed to develop systemic activation of coagulation (TAT and D-dimer), as well as a FeCl3-induced carotid arterial thrombosis model. Our results indicated that TMAO triggered TF-dependent PCA via activation of nuclear factor-κB and downregulated thrombomodulin expression in human EC, but failed to develop systemic activation of coagulation in mice.

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.

Propofol Prevents Thrombosis in a Rat Common Carotid Artery Thrombosis Model

Aim: The aim of this study was investigated the beneficial effect of propofol on thrombosis in a rat carotid artery thrombosis model. Methods and results: Sprague-Dawley rats underwent iron chloride3 (FeCl3)-induced carotid artery thrombosis model surgery were treated with vehicle (M group), low- (PL group), middle- (PM group) or high-dose propofol (PH group). Rats were received sham operation served as control (C group). As compared with the M group, propofol treatment significantly inhibited thrombus weights and improved the degree of embolization. Propofol also markedly improved blood CD62P intensity. Moreover, as compared with C group rats, the levels of plasma TXA2, PGI2, t-PA, t-PA/PAI-1 ratio, and P-selectin were harmful altered in M group rats in response to FeCl3 induction; these effects were improved in the PL and/or PM and PH groups. Conclusions: These findings suggested that the beneficial anti-thrombotic effect of propofol is likely attributable, at least in part, to attenuation of the endothelia injury, platelet activation and fibrinolytic inactivation induced by FeCl3 in a rat carotid artery thrombosis model.

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.

A rabbit model of cerebral microembolic signals for translational research: preclinical validation for aspirin and clopidogrel

Essentials Microembolic signal (MES) is an independent predictor of stroke risk in patients. A rabbit model of cerebral microembolic signals was established. Therapeutic efficacy was demonstrated for aspirin and clopidogrel on microembolic signals. Potential translational value of this preclinical model of MES was demonstrated. Objectives Cerebral microembolic signals (MESs) detected by transcranial Doppler (TCD) ultrasound constitute an independent predictor of stroke risk and prognosis. The aim of this study was to develop a novel preclinical model of MESs to facilitate translational research. Methods A clinical TCD ultrasound machine was used to detect MESs in the cerebral circulation of New Zealand White rabbits. Technical feasibility was assessed for the measurement of MESs in the middle cerebral artery (MCA) by TCD. FeCl3 -induced carotid arterial thrombosis was optimized for the generation of endogenous microemboli. Ascending doses of two antithrombotic agents (aspirin and clopidogrel) were evaluated individually and in combination for their effects on both arterial thrombosis and MESs in a 30% FeCl3 -induced carotid arterial thrombosis model, along with ex vivo functional assays. Results Dose-dependent FeCl3 -induced arterial thrombosis studies showed that 30% FeCl3 resulted in the most consistent and reproducible MESs in the MCA (3.3 ± 0.7 MESs h(-1) ). Ascending-dose studies showed that the effective doses for 50% inhibition (ED50 ) of thrombus formation, based on integrated blood flow and thrombus weight, respectively, were 3.1 mg kg(-1) and 4.2 mg kg(-1) orally for aspirin, and 0.3 mg kg(-1) and 0.28 mg kg(-1) orally for clopidogrel. The ED50 values for MES incidence were 12.7 mg kg(-1) orally for aspirin, and 0.25 mg kg(-1) orally for clopidogrel. Dual treatment with aspirin (5 mg kg(-1) ) and clopidogel (0.3 mg kg(-1) ) resulted in significant reductions in cerebral MESs (P < 0.05) as compared with monotherapy with either agent. Conclusions Our study demonstrated the successful establishment of the MES model in rabbits, and it may provide translational value for MESs and ischemic stroke research.

Atractylodes lactone compounds inhibit platelet activation

Platelets play a crucial role in the development and progression of atherosclerosis-thrombosis and, therefore, antiplatelet drugs are widely used in the treatment of coronary artery disease. Moreover, advances in understanding the biological functions of natural plant products can provide new pharmacological strategies aimed at promoting cardiovascular health. Atractylenolide I (ATL-1), ATL-2, and ATL-3 are the major bioactive components of a Qi tonifying medicinal herb Rhizoma Atractylodis Macrocephalae (Atractylodes macrocephala), which is commonly used in traditional Chinese medicine (TCM). These components possess well-documented anti-inflammatory and anticancer activities, but their effects on platelet activation are still unknown. In this study, the effects of ATL on platelet function in vitro and in vivo were investigated, and the underlying mechanism was explored. We found that ATL-2 and ATL-3 but not ATL-1 diminished agonist-induced platelet aggregation and diminished adenosine triphosphate (ATP) release from dense granules. The levels of phospho-Akt (Ser473) and phospho-p38 MAPK were downregulated in the presence of ATL-2 and ATL-3. We also found that ATL-2 and ATL-3 have a similar inhibitory effect on platelet activation as acetylsalicylic acid in response to agonists. Furthermore, ATL-2 and ATL-3 diminished the spreading of human platelets on immobilized fibrinogen (Fg), delayed clot retraction in platelet-depleted plasma containing human platelets, extended first occlusion time in a mouse model of ferric chloride (FeCl3)-induced carotid arterial thrombosis, and prolonged the bleeding time. These observations suggest that ATL-2 and ATL-3 are potential candidate therapeutic drugs for the prevention of thrombosis.

Novel phenylalanine derived diamides as Factor XIa inhibitors

The synthesis, structural activity relationships (SAR), and selectivity profile of a potent series of phenylalanine diamide FXIa inhibitors will be discussed. Exploration of P1 prime and P2 prime groups led to the discovery of compounds with high FXIa affinity, good potency in our clotting assay (aPPT), and high selectivity against a panel of relevant serine proteases as exemplified by compound 21. Compound 21 demonstrated good in vivo efficacy (EC50=2.8μM) in the rabbit electrically induced carotid arterial thrombosis model (ECAT).

Genetic Ablation of Extra Domain A of Fibronectin in Hypercholesterolemic Mice Improves Stroke Outcome by Reducing Thrombo-Inflammation.

The fibronectin-splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans but markedly elevated in patients with comorbid conditions, including diabetes mellitus and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E-deficient (Apoe(-/-)) mouse. In a transient cerebral ischemia/reperfusion injury model, Apoe(-/-) mice expressing fibronectin deficient in EDA (Fn-EDA(-/-)Apoe(-/-) mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 (P<0.05 versus Apoe(-/-) mice). Concomitantly, intracerebral thrombosis [assessed by fibrin(ogen) deposition] and postischemic inflammation (phospho-nuclear factor-κB p65, phospho-IκB kinase α/β, interleukin 1β, and tumor necrosis factor-α) within lesions of Fn-EDA(-/-)Apoe(-/-) mice were markedly decreased (P<0.05 versus Apoe(-/-) mice). In an FeCl3 injury-induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA(-/-)Apoe(-/-) mice (P<0.05 versus Apoe(-/-) mice). Genetic ablation of TLR4 improved stroke outcome in Apoe(-/-) mice (P<0.05) but had no effect on stroke outcome in Fn-EDA(-/-)Apoe(-/-) mice. Bone marrow transplantation experiments revealed that nonhematopoietic cell-derived Fn-EDA exacerbates stroke through Toll-like receptor-4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe(-/-) mouse 15 minutes after reperfusion significantly improved stroke outcome. Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.

Human Apolipoprotein a-I Decreases Platelet Activation Responses and Protects Mice from Arterial Thrombosis

Introduction: High-density lipoprotein (HDL) protects against thromboembolic coronary disease via reverse cholesterol transport mediated by HDL’s primary lipoprotein subunit, apolipoprotein A-I (apoA-I), binding to scavenger receptor BI (SR-BI). Absence of SR-BI increases platelet aggregation and venous/arterial thrombosis in mice, consistent with an antithrombotic role of this signaling axis. To date, the effects of the isolated apoA-I subunit on platelet activation remain unknown. We hypothesize that the antithrombotic effect is mediated by apoA-I signaling through platelet-specific SR-BI.Methods: Platelet function assays were performed on samples from healthy human volunteers (n=3). ApoA-I levels were determined using an ELISA kit. Microfluidic analysis of platelet aggregate formation on collagen under physiologic flow conditions (650 sec-1) was carried out in whole blood samples. Light transmission platelet aggregation in response to collagen and ristocetin was conducted using both platelet rich plasma (PRP) and washed platelets (WP). Venous and arterial thromboses were induced in wild type C57Bl/6 mice in a collagen (0.3 mg/kg)/epinephrine (0.03 mg/kg)-induced pulmonary embolism model and a 6% ferric chloride (FeCl3)-induced carotid artery thrombosis model, respectively. Recombinant human apoA-I was used with a final concentration of 300 µg/mL.Results/Discussion: ApoA-I plasma levels in the subjects studied fell within previously published ranges. Microfluidic analysis of platelet aggregate formation on collagen under shear stress did not demonstrate significant difference in total surface area coverage (n=3 in quadruplicate), but apoA1-treated samples demonstrated a significantly greater proportion of small aggregates (3-10 platelets/aggregate, 40.2% vs 31.8%, p<0.0001), compared to controls which had a significantly greater proportion of large aggregates (51-100 plts/agg, 6.3 vs 2.3%, p<0.001 and >100 plts/agg, 2.3% vs 0.7%, p<0.05). Platelet aggregation studies (Fig. 1) revealed significantly decreased collagen-induced aggregation in platelets treated with apoA-I compared to vehicle-treated controls in both PRP and WP samples, suggesting direct action of apoA-I on platelets. The difference was less pronounced in ristocetin-induced aggregation, though still significant, consistent with decreased activation of VWF, which has been recently shown to bind apoA-I. Apoa-I-treated PRP samples also had significantly decreased dense granule (ATP) release compared to non-treated samples. Pre-treatment with native HDL had no significant effect, consistent with studies demonstrating that only oxidized HDL inhibits aggregation. Recombinant human SR-BI (rhSR-BI) alone had little effect in PRP but inhibited collagen-induced aggregation in WP. Addition of rhSR-BI and apoA-I together inhibited aggregation in both PRP and WP to a greater degree than either component alone. Rabbit anti-human SR-BI alone had little effect in PRP but inhibited collagen-induced aggregation in WP. FeCl3 applicationinduced initial arterial occlusion within 8.8 +/-0.8 minutes in apoA-I-treated mice treated (n=3) compared to 6.1 +/- 0.3 minutes in vehicle-treated controls (n=3, p<0.01, t-test). Systemic thrombosis resulting in pulmonary embolism was induced by injection of collagen/epinephrine. Median time of survival after injection in mice pre-treated with apoA-I (n=5) was 30 +/- 0 minutes, compared to 3.3 +/- 0.4 minutes in vehicle-treated controls (n=5, p<0.001, t-test). Experiments were stopped after 30 minutes, at which time all apoA-1-treated mice were still alive, compared to zero controls.Conclusion: Though the mechanism is not yet completely understood, the data show an inhibitory effect of isolated human apoA-I on human platelet activation and murine arterial/venous thrombosis, likely through platelet inhibition. The increased inhibition seen with rhSR-BI and apoA-I together is consistent with previous studies demonstrating SR-BI’s regulatory role in platelet function. Ongoing studies include increasing sample size for human platelet function assays and murine venous/arterial thrombosis models in wild type mice with and without infusion of apoA-I compared to SR-BI-/- and apoA-I-/- mice with and without infusion of apoA-I. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 9768: Cr(nMe)eka-Conjugated Micellar Formulation Improves Anticoagulation Efficacy of Bivalirudin

Anticoagulant therapies are the primary treatment modalities for preventing and treating thrombosis. However, high rate of bleeding complications is a serious side effect of anticoagulant therapies, which compromised their applications. Bivalirudin is an oligopeptide anticoagulant that has the advantage of not causing thrombocytopenia and exhibits lower potential for bleeding risk compared to heparin. Nevertheless, hemorrhage is still the most common complication. And there is no effective antidote for bivalirudin. We report here a strategy to deliver bivalirudin specifically to the site of clotting area, and thereby reduce the bleeding risk. The site-specific delivery of bivalirudin was achieved with micellar nanocarriers which has a thrombosis-targeting peptide CR(Nme)EKA. These peptide-conjugated micellar nanocomplexes are highly dispersed and stable in aqueous solution with an average size of 30 nm. The anticoagulant activity of bivalirudin was well remained after it was incorporated into the micelles. These micellar nanocomplexes specifically accumulated in the site of clotting in mice lung thrombosis. Moreover, the micellar formulation significantly prolonged the half-life and increased the bioavailability of bivalirudin. Most importantly, this effective formulation significantly improved the anticoagulant activity of bivalirudin, when tested in both thromboplastin-induced lung thrombosis mouse model and ferric chloride (FeCl3)-induced carotid artery thrombosis mouse model. Taken together, these results demonstrate that the micellar nanocomplexes specifically target thrombi, deliver bivalirudin directly into site of clotting and exhibit improved antithrombotic activity over free bivalirudin. This strategy may improve the safety and efficacy of bivalirudin in clinical use.

Abstract 19: CD39 Expression on Circulating Blood Components Prolongs the Time to Ferric Chloride--Induced Carotid Artery Thrombosis in Mice

Objective: To determine the effect of increased CD39 expression on circulating blood components on in vivo arterial thrombosis in mice. Approach and Results: We have previously demonstrated that increased global expression of hCD39 conveys an anti-thrombotic phenotype in mice suggesting a pivotal role for CD39 in modulating the balance between a pro-thrombotic and anti-thrombotic milieu. We hypothesized the expression of CD39 on circulating blood components can regulate occlusive thrombus formation. Bone marrow transplantations were performed (WT to WT; WT to hCD39-Tg; hCD39-Tg to hCD39-Tg; hCD39-Tg to WT) to determine the contribution of CD39 expressed on circulating blood cells and platelets on occlusive arterial thrombus formation in mice. Complete blood counts and fluorescence-activated cell sorting (FACS) analyses were performed on whole blood obtained from mice 60 days after transplantation of bone marrow. No significant differences in the circulating levels of white blood cells, red blood cells, and platelets were observed between the groups. FACS analysis confirmed that the circulating blood components were reconstituted from the transplanted bone marrow. Carotid artery thrombosis was induced by ferric chloride (FeCl 3 ) application and the time to thrombotic occlusion (blood flow of 0 ml/min) was measured. The operator was blinded to the animal genotype and treatment during all experiments. Transplantation of CD39-Tg bone marrow into WT mice resulted in a marked resistance to thrombosis (CD39-Tg to WT: 315.7.00 ± 54.6 min; WT to WT: 7.58 ± 0.89 min; P &lt;0.001). CD39-Tg mice that received WT bone marrow demonstrated a significantly lower time to vessel occlusion than CD39-Tg mice that received CD39-Tg bone marrow (WT to CD39-Tg: 17.15 ± 6 min; CD39-Tg to CD39-Tg 414.3 ± 61.95 min; P &lt;0.001). Conclusion: Increased expression of CD39 on circulating bone-marrow-derived blood components markedly prolongs the time to carotid artery occlusion following ferric-chloride-induced injury in mice.

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.