FeCl3-induced Carotid Thrombosis Research Articles

Activated Coagulation FXII: A Unique Target for In Vivo Molecular Imaging

Current clinical imaging of thromboembolic diseases often relies on indirect detection of thrombi, which may delay diagnosis and ultimately the institution of beneficial, potentially lifesaving treatment. Therefore, the development of targeting tools that facilitate the rapid, specific, and direct imaging of thrombi using molecular imaging is highly sought after. One potential molecular target is FXIIa (factor XIIa), which initiates the intrinsic coagulation pathway but also activates the kallikrein-kinin system, thereby initiating coagulation and inflammatory/immune responses. As FXII (factor XII) is dispensable for normal hemostasis, its activated form (FXIIa) represents an ideal molecular target for diagnostic and therapeutic approaches, the latter combining diagnosis/identification of thrombi and effective antithrombotic therapy. We conjugated an FXIIa-specific antibody, 3F7, to a near-infrared (NIR) fluorophore and demonstrated binding to FeCl3-induced carotid thrombosis with 3-dimensional fluorescence emission computed tomography/computed tomography and 2-dimensional fluorescence imaging. We further demonstrated ex vivo imaging of thromboplastin-induced pulmonary embolism and detection of FXIIa in human thrombi produced in vitro. We demonstrated imaging of carotid thrombosis by fluorescence emission computed tomography/computed tomography and measured a significant fold increase in signal between healthy and control vessels from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.002) ex vivo. In a model of pulmonary embolism, we measured increased NIR signal in lungs from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.0008) and healthy lungs from mice injected with 3F7-NIR (P=0.021). Overall, we demonstrate that FXIIa targeting is highly suitable for the specific detection of venous and arterial thrombi. This approach will allow direct, specific, and early imaging of thrombosis in preclinical imaging modalities and may facilitate monitoring of antithrombotic treatment in vivo.

Atherogenic lipid stress induces platelet hyperactivity through CD36-mediated hyposensitivity to prostacyclin: the role of phosphodiesterase 3A.

Prostacyclin (PGI2) controls platelet activation and thrombosis through a cyclic adenosine monophosphate (cAMP) signaling cascade. However, in patients with cardiovascular diseases this protective mechanism fails for reasons that are unclear. Using both pharmacological and genetic approaches we describe a mechanism by which oxidized low density lipoproteins (oxLDL) associated with dyslipidemia promote platelet activation through impaired PGI2 sensitivity and diminished cAMP signaling. In functional assays using human platelets, oxLDL modulated the inhibitory effects of PGI2, but not a phosphodiesterase (PDE)-insensitive cAMP analog, on platelet aggregation, granule secretion and in vitro thrombosis. Examination of the mechanism revealed that oxLDL promoted the hydrolysis of cAMP through the phosphorylation and activation of PDE3A, leading to diminished cAMP signaling. PDE3A activation by oxLDL required Src family kinases, Syk and protein kinase C. The effects of oxLDL on platelet function and cAMP signaling were blocked by pharmacological inhibition of CD36, mimicked by CD36-specific oxidized phospholipids and ablated in CD36−/− murine platelets. The injection of oxLDL into wild-type mice strongly promoted FeCl3-induced carotid thrombosis in vivo, which was prevented by pharmacological inhibition of PDE3A. Furthermore, blood from dyslipidemic mice was associated with increased oxidative lipid stress, reduced platelet sensitivity to PGI2 ex vivo and diminished PKA signaling. In contrast, platelet sensitivity to a PDE-resistant cAMP analog remained normal. Genetic deletion of CD36 protected dyslipidemic animals from PGI2 hyposensitivity and restored PKA signaling. These data suggest that CD36 can translate atherogenic lipid stress into platelet hyperactivity through modulation of inhibitory cAMP signaling.

Влияние Нотромбела на развитие FeCl3-индуцированного тромбоза сонной артерии крыс

Цель работы - изучение влияния Нотромбела (ЗАО «Вертекс», Санкт-Петербург, РФ) на развитие экспериментального тромбоза. Методика. Опыты выполнены на крысах-самцах Вистар массой 250-270 г. Нотромбел вводили внутривенно и интрагастрально в течение 10 сут (курсовая доза 25, 50 и 100 мг/кг). В качестве препаратов сравнения использовали ацетилсалициловую кислоту (АСК) и Клопидогрел (курсовая доза 10,5 и 20 мг/кг соответственно). Тромбоз сонной артерии вызывали 60-секундной аппликацией 20% FeCl3. Кровоток регистрировали методом высокочастотной ультразвуковой допплерографии («Минимакс-Допплер-К» Санкт-Петербург, РФ, с рабочей частотой датчика 20 МГц). Прекращение кровотока рассматривали как показатель тромбоокклюзии сонной артерии. Результаты. У контрольных крыс через 20-30 мин после аппликации FeCl3 кровоток в сонной артерии не определялся, что свидетельствовало о развитии тромбоза. Нотромбел как при интрагастральном, так и при внутривенном введении тормозил развитие FeCl3-индуцированного тромбоза сонной артерии. Наиболее эффективной независимо от способа введения оказалась курсовая доза препарата 100 мг/кг, при которой ни в одном из опытов тромбоз не развивался. Антитромботический эффект Нотромбела сопоставим с эффектами АСК и Клопидогрела. В механизме антитромботического влияния Нотромбела имеют значение его прямое ингибирующее влияние на функциональную активность тромбоцитов, а также антиоксидантная активность. Заключение. Нотромбел можно рассматривать как перспективный антитромботический препарат. Aim. To study the effect of Notrombel (ZAO Vertex, St. Petersburg, Russia), a drug of the N, N’-substituted piperazine class containing a carboximidamide group, on experimental thrombosis. Methods. Experiments were carried out on male Wistar rats weighing 250-270 g. Notrombel was administered intravenously and intragastrically for 10 days (course doses 25, 50, and 100 mg/kg). Acetylsalicylic acid (ASA) and Сlopidogrel (course doses 10.5 and 20 mg/kg, respectively) were used as comparator drugs. Carotid artery thrombosis was induced by a 60-s application of 20% FeCl3. Blood flow was recorded by high-frequency ultrasonic dopplerography (Minimax-Doppler-K, St. Petersburg, RF; sensor operating frequency, 20 MHz). Stagnation of blood flow was considered as an indicator of carotid artery occlusion. Results. In control rats 20-30 minutes after the application of FeCl3, the carotid blood flow was undeterminable, which indicated development of thrombosis. Notrombel, both intragastric and intravenous, inhibited the development of FeCl3-induced carotid thrombosis. The most effective dose was 100 mg/kg irrespective of the administration route. Thrombosis has not developed in any experiment. The antithrombotic effect of Notrombel was comparable to that of ASA and Clopidogrel. The Notrombel inhibition of platelet activity and its antioxidant properties contribute to the mechanism of Notrombel antithrombotic effect. Conclusion: Notrombel can be considered as a promising antithrombotic drug.

Hepatocyte-Derived Tissue Plasminogen Activator Regulates Systemic Fibrinolysis

Tissue-type plasminogen activator (tPA) is a secreted serine protease that initiates the dissolution of a fibrin clot in a process called fibrinolysis. When a fibrin clot forms on the wall of an injured vessel, tPA binds to the fibrin and converts plasminogen to plasmin, which proteolytically degrades the fibrin clot. tPA is released locally by vascular endothelial cells in response to injury, preventing excessive fibrin deposition and thrombosis. However, freshly isolated blood from healthy subjects undergoes spontaneous fibrinolysis, suggesting the presence of basal tPA, i.e., tPA activity present before injury occurs. A major gap in fibrinolysis research is the roles, regulation, and sources of basal plasma tPA. A key question that raises the possibility that a non-endothelial source of tPA may be important, is how medium-sized and large vessels respond to injury with respect to fibrinolysis. Endothelial cells of these vessels express less tPA than small vessels, and the tPA that is secreted by these cells gets rapidly diluted owing to the rapid flow and a much lower surface-to-volume ratio of large vessels. Thus, a non-endothelial source of tPA may be necessary for limiting clot extension and thrombosis after an injury to medium-sized and large arteries.The fibrinolytic activity of plasma is determined mostly by the relative concentrations of plasma tPA and its inhibitor, plasminogen activator inhibitor-1. Longitudinal studies have revealed an association between lower plasminogen activator and fibrinolytic activity in plasma with future recurrent myocardial infarction. Moreover, basal plasma fibrinolytic activity was shown to have a diurnal variation with a nadir in the morning, which is the time of day of highest risk for coronary artery disease. More recently, studies have shown that low plasma tPA activity per se predicts cardiovascular disease in humans. Collectively, these studies suggest that basal plasma fibrinolytic activity, determined in part of tPA concentration, is a functionally important mechanism to prevent pathological fibrin clot formation and thrombosis.Although hepatocytes have been shown to express tPA protein and mRNA, the fibrinolytic function of hepatocyte tPA, either in the liver or systemically, and its mode of regulation remain unknown. Therefore, we conducted a series of tests to explore the relative level of tPA expression by hepatocytes and its role in wildtype mice. Using flow cytometry, we found that hepatocytes express high levels of tPA relative to non-parenchymal cells of liver. Comparing with well-known sources of tPA, the levels of tPA mRNA, immunoreactive tPA protein, and tPA activity in liver were comparable to those in arterial tissue. By silencing tPA specifically in hepatocytes, we were able to lower plasma tPA protein by ~50% and lower plasma tPA activity by ~30%. Despite this only partially silencing efficiency, there was a 43% decrease in bleeding time (P=0.0003) and a 25% decrease in the time to occlusive carotid arterial thrombosis induced by photochemical injury (P=0.001).tPA is released rapidly from endothelial storage granules in response to endothelial injury-induced thrombosis, which we predicted would not be affected by silencing hepatocyte tPA. Indeed, the data show that although tPA release after FeCl3-induced carotid thrombosis varied among individual mice in both cohorts, the average release values were similar between control and hepatocyte-tPA-silenced mice. These data suggest that the release of tPA from the endothelium after injury is independent of hepatocyte tPA and that hepatocyte tPA works in concert with tPA released from the endothelium to affect post-injury fibrinolysis. As a final test of the role of hepatocyte tPA, we restored tPA only in the hepatocyte of holo-tPA-KO mice, resulting a substantial increase in plasma tPA protein and a significant decrease in clot lysis time.In summary, we show that hepatocytes are a significant source of plasma tPA protein and tPA-mediated fibrinolytic activity under basal conditions and that hepatocyte-derived tPA complements locally released vessel wall-derived tPA to limit clotting after arterial injury. We imagine that these insights and ones that will follow from future studies in this area will suggest new therapeutic strategies for diseases of hemostatic imbalance. DisclosuresNo relevant conflicts of interest to declare.

Direct Imaging of Cerebral Thromboemboli Using Computed Tomography and Fibrin-targeted Gold Nanoparticles.

Computed tomography (CT) is the current standard for time-critical decision-making in stroke patients, informing decisions on thrombolytic therapy with tissue plasminogen activator (tPA), which has a narrow therapeutic index. We aimed to develop a CT-based method to directly visualize cerebrovascular thrombi and guide thrombolytic therapy. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP. This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke. Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome. Furthermore, recurrent thrombosis could be diagnosed by mCT without further nanoparticle administration for up to 3 weeks. fib-GC-AuNP-based direct cerebral thrombus imaging greatly enhance the value and information obtainable by regular CT, has multiple uses in basic / translational vascular research, and will likely allow personalized thrombolytic therapy in clinic by a) optimizing tPA-dosing to match thrombus burden, b) enabling the rational triage of patients to more radical therapies such as endovascular clot-retrieval, and c) potentially serving as a theranostic platform for targeted delivery of concurrent thrombolysis.

Abstract 127: Genetic Ablation of Alternatively-spliced Extra Domain A of Fibronectin Protects Hypercholesterolemic Mice from Acute Stroke Injury

Background and Hypothesis: Fibronectin containing the alternatively-spliced extra domain A (EDA + -FN) is absent in the arteries or circulation of healthy humans and mice; but is specifically expressed in the endothelium of atherosclerotic arteries, and elevated in circulation during pathological settings, including stroke. We tested the hypothesis that deletion of EDA in FN protects hypercholesterolemic apolipoprotein E-deficient (ApoE -/- ) mice from acute stroke injury. Methods: We generated EDA hi/hi /ApoE -/- mice (constitutively overexpress EDA + -FN in plasma) and EDA -/- /ApoE -/- mice (EDA-deleted allele of FN). ApoE -/- mice (express EDA + -FN in plasma (~3-5 μg/mL) similar to levels found in human pathological settings) were used as control. Susceptibility to thrombosis was assessed using FeCl 3 -induced carotid thrombosis model. Stroke outcome was evaluated in male mice following 60 min of ischemia/ 23 h of reperfusion injury by measuring infarct size, neurological deficit and markers of inflammation within the infarct and surrounding regions (immunohistochemistry). Results: Using intravital microscopy, we found that the time to stable occlusion of the carotid artery was accelerated in EDA hi/hi /ApoE -/- mice (P<0.05 vs ApoE -/- mice), whereas EDA -/- /ApoE -/- mice showed opposite effect. EDA -/- /ApoE -/- mice had smaller infarcts and improved neurological outcome (P<0.01 vs. ApoE -/- mice), concordant with decreased vascular inflammation (neutrophils, macrophages, and NF-κB p65 expression). Conversely, EDA hi/hi /ApoE -/- mice had larger infarcts and more severe neurological deficits, concordant with increased vascular inflammation (P<0.01 vs. ApoE -/- mice). Because EDA + -FN activates toll-like receptor 4 (TLR4) in vitro , we investigated the role of TLR4 in the EDA + -FN-mediated aggravated stroke injury. Genetic ablation of TLR4 in EDA hi/hi /ApoE -/- mice completely reversed aggravated stroke injury (P<0.05 vs. EDA hi/hi /ApoE -/- mice), whereas TLR4 deficiency in EDA -/- /ApoE -/- had no effect. Conclusions: EDA + -FN is pro-thrombotic and pro-inflammatory, and promotes acute stroke injury via a TLR4 -dependent mechanism. We suggest that monitoring plasma EDA + -FN levels may have prognostic value for patients at high risk for stroke.

Inhibition of the ADP/P2Y12 Pathway Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice

Clopidogrel, a potent anti-thrombotic drug widely used for the secondary prevention of myocardial infarction and ischemic stroke, inhibits the ADP/P2Y12 pathway by inducing irreversible alteration of the platelet receptor P2Y12, ultimately uncoupling ADP from adenylyl cyclase inhibition. Clinical trials have demonstrated additional benefits of dual antiplatelet therapy, such as combined treatment with clopidogrel and aspirin, in the prevention of ischemic cardiovascular disease. We previously established that protease-activated receptor (PAR) signaling is necessary for platelet activation by thrombin and plays an important role in mouse models of hemostasis and thrombosis. Indeed a PAR antagonist is currently being used as an anti-thrombotic agent in clinical trials.In order to evaluate the possible interaction between the thrombin/PAR and ADP/P2Y12 pathways, we orally administered clopidogrel (30mg/kg) or vehicle to PAR-4 null, heterozygous and wild type littermates, and evaluated arterial thrombus formation and bleeding during surgical challenge. In a FeCl3-induced carotid thrombosis model, PAR-4 deficient mice treated with clopidogrel were completely protected from thrombosis compared to their wild type treated littermates. Interestingly, clopidogrel treated PAR-4 heterozygotes showed an intermediate level of protection compared to treated wild type and null mice that was similar to vehicle-treated PAR-4 deficient mice. Additionally, we scored bleeding during the surgical procedure while blinded to genotype and treatment. The bleeding score was increased in clopidogrel treated mice compared to vehicle treated mice and was exacerbated with increasing haplotype deficiency of Par-4.To better define the relationship between these two activation pathways, we generated mice lacking both PAR-4 and P2Y12. All genetic combinations were born at the expected Mendelian distribution. Females lacking both receptors carried offspring to term, but signs of bleeding were observed during parturition. Preliminary data from PAR-4/P2Y12 double null mice, in both the tail bleeding assay and in a FeCl3-induced carotid thrombosis model, is in agreement with the results obtained in clopidogrel treated PAR-4 deficient mice. This study provides useful information to evaluate the efficacy and safety of dual antiplatelet therapy inhibiting the thrombin/PAR and ADP/P2Y12 pathways and explores the relationship between these two critical pro-thrombotic pathways in an in vivo setting.

GP-VI Disruption Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice

Following vascular injury, platelets are recruited and activated by adhesive proteins exposed on the subendothelium (including collagen), released agonists and locally generated thrombin. In mice, protease activated receptor 4 (PAR-4) mediates the platelet signaling response to thrombin. Previous studies showed that platelets from PAR-4 null mice do not respond to thrombin and while platelet thrombi do form at the site of injury in PAR-4 deficient animals, they do not propagate and extend into the lumen like thrombi in wild-type mice. The activation pathway responsible for this juxtamural platelet accumulation remains unknown. The collagen exposed on the subendothelium activates and recruites platelets at the site of injury by interacting with membrane glycoprotein (GP) VI. Therefore, the collagen-GP-VI pathway is a putative candidate for the platelet thrombus formation near the vessel wall. Using hirudin as a thrombin inhibitor in mice with disrupted GP-VI expression, it has been previously shown that GP-VI signaling and thrombin activation pathways may cooperate during thrombus growth. To more precisely evaluate the interplay between the thrombin/PAR-4 and collagen/GP-VI signaling pathways, we crossed mice lacking PAR-4 and GP-VI, and subsequently compared bleeding time and arterial thrombus formation in PAR-4:GP-VI double or single deficient animals. All genetic combinations were born at the expected mendelian distribution. Double deficient females carried offspring to term and no bleeding was observed during parturition. Pups lacking both receptors demonstrated transient perinatal abdominal bleeding which resolved rapidly. However, we observed prolonged tail bleeding times in double deficient animals compared to their single deficient littermates. Furthermore in a FeCl3-induced carotid thrombosis model, mice lacking both receptors were completely protected compared to their littermates. This study suggests that there is an interaction between the thrombin and collagen activating pathways in the setting of hemostasis and thrombosis. [Display omitted] [Display omitted]