Inhibiting monocyte migration reduces arterial thrombosis and facilitates thrombolysis

Abstract

BackgroundMonocytes contribute to the initiation and propagation of venous thrombosis. Little is known about the roles monocytes play in arterial thrombosis, the cause of stroke and MI. MethodsWe investigated how chemokine receptor 2 (CCR2) knockout (-/-) affects platelet function, blood coagulation, thrombus volume, and thrombolytic susceptibility in 627 mice with FeCl3-mediated carotid arterial thrombosis: three CX3CR1-GFP mice, 326 C57BL/6 mice, and 288 CCR2-/- mice. We performed i) intravital microscopy imaging of leukocyte recruitment to carotid thrombus, ii) flow cytometry-based quantification of leukocytes in blood vs. thrombus and leukocyte-platelet aggregates in blood, iii) platelet aggregometry, iv) coagulation assays, v) micro-computed tomography (microCT)-based thrombus imaging using gold nanoparticles after tissue plasminogen activator (tPA) therapy with or without either a) CCR2 siRNA pretreatment (for 3 days) or post-treatment or b) clopidogrel pretreatment (for 7 days), and vi) histology including scanning electron microscopy. ResultsIntravital microscopy and flow cytometry showed that both neutrophils and monocytes were recruited to the acute arterial thrombus, as observed 30 minutes post-thrombosis. Platelet function tests demonstrated platelet aggregation to be lower in the whole blood of CCR2-/- mice (vs. C57BL/6 mice) but not in their leukocyte-free platelet rich plasma, suggesting this platelet dysfunction is cell-mediated. Flow cytometry experiments revealed lower numbers of monocyte - platelet aggregates (MPAs), a marker of platelet activity, in the blood of CCR2-/- mice, compared to C57BL/6 mice. Blood levels of FXIII and monocyte levels of FXIII-A were increased after carotid thrombosis in C57BL/6 mice but not CCR2-/- mice. Further, in vivo microCT and histology, respectively, showed that CCR2-/- mice had smaller and more porous thrombi with less fibrin cross-linking, compared with C57BL/6 mice. MicroCT also demonstrated that tPA-mediated thrombolysis was faster in CCR2-/- mice and CCR2 siRNA-treated mice, compared to C57BL/6 mice. In addition, clopidogrel had a greater effect on inducing thrombus formation with smaller sizes after FeCl3 application, while CCR2-/- had a greater effect on dissolving thrombus faster after tPA administration. ConclusionsCCR2 antagonism decreases platelet aggregation and reduces FXIII levels in blood and monocytes, thus driving arterial thrombosis towards the generation of a relatively small, porous, more lysable clot. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIIn venous thrombosis, monocytes cooperate with platelets and neutrophils to promote thrombus formation; however, few studies have investigated the roles of monocytes in arterial thrombosis. C_LIO_LIAn in vitro study using arterial blood samples from patients with acute coronary syndrome showed that P-selectin on activated platelets upregulated monocyte chemokine receptor 2 (CCR2) expression, which was higher in monocyte-platelet aggregates (MPAs) than in monocytes without platelets. C_LIO_LIIn a mouse study using an in vivo model of FeCl3-induced arterial thrombosis, the time to thrombotic occlusion was prolonged in CC chemokine ligand 2 (CCL2) knockout (-/-) mice compared with wild-type mice. C_LI What New Information Does This Article Contribute?O_LIThis study is the first to demonstrate that CCR2-/--mediated monocyte deficiency exerts antithrombotic and tissue plasminogen activator (tPA)-facilitating effects in acute arterial thrombosis. C_LIO_LICCR2-/- mice had a) decreased platelet aggregability, probably due in part to reduced post-thrombosis MPA numbers, and b) lower blood and monocyte levels of Factor XIII (FXIII), compared to those of C57BL/6 mice. C_LIO_LIMicro-computed tomography (microCT) thrombus imaging showed that i) CCR2-/- mice, compared to C57BL/6 mice, had smaller thrombi, with more porosity and less fibrin cross-linking on histology and ii) CCR2-/- or CCR2 siRNA facilitates tPA-mediated thrombolysis. C_LI The majority of ischemic strokes occur due to thromboembolic occlusion of cerebral arteries. Tissue plasminogen activator (tPA), which is the only FDA-approved drug for acute ischemic stroke, has only a moderate efficacy rate regarding early recanalization, which is closely associated with thrombus volume and thrombolytic resistance. Monocytes, in cooperation with platelets and neutrophils, contribute to thrombus formation in venous thrombosis. To date, only a few studies have detailed potential prothrombotic roles for monocytes in arterial thrombosis. In the present study, using a mouse model of FeCl3-mediated carotid artery thrombosis and high-resolution microCT thrombus imaging, we demonstrate that CCR2-/--mediated monocyte deficiency exerts antithrombotic and tPA-facilitating effects. Compared to C57BL/6 mice, CCR2-/- mice exhibited decreased platelet aggregability along with lower numbers of circulating MPAs (a marker of platelet activity) and reduced monocyte recruitment to arterial thrombi. Moreover, CCR2-/- mice had lower levels of both circulating FXIII and monocyte FXIII-A, which are known to strengthen and stabilize thrombi. In line with these findings, i) CCR2-/- mice had smaller, more porous thrombi with less fibrin cross-linking and ii) tPA-mediated thrombolysis was faster in CCR2-/- mice or with CCR2 siRNA.