A t sites of vascular injury, the subendothelial extracellular matrix is exposed to the blood and triggers sudden platelet activation and the formation of a fibrin containing thrombus. This process is essential to prevent excessive posttraumatic blood loss, but if it occurs inappropriately, for example, at sites of atherosclerotic plaque rupture, it can lead to vessel occlusion and ischemic infarction. In the clinical context of myocardial infarction, timely reopening of an occluded coronary artery by pharmacological thrombolysis or coronary intervention constitutes an effective therapeutic mean to limit cardiac damage. However, restoration of blood flow causes further cardiac damage, the so-called reperfusion injury (RI). The mechanisms of myocardial RI are not fully understood; thus, it is possible that thrombus formation by platelets in reperfused microvessels could contribute to this process. Furthermore, thrombus fragmentation by thrombolysis or coronary balloon angioplasty might add to the occlusion of microvessels by distal embolization. Additionally, platelets release factors that potentially increase RI without mechanical obstruction of microvessels. Activated © 2016 American Heart Association, Inc. Objective-The objective of this study was to investigate the effects of platelet inhibition on myocardial ischemia-reperfusion (IR) injury. Approach and Results-Timely restoration of coronary blood flow after myocardial infarction is indispensable but leads to additional damage to the heart (myocardial IR injury). Microvascular dysfunction contributes to myocardial IR injury. We hypothesized that platelet activation during IR determines microvascular perfusion and thereby the infarct size in the reperfused myocardium. The 3 phases of thrombus formation were analyzed by targeting individual key platelet-surface molecules with monoclonal antibody derivatives: (1) adhesion (anti-glycoprotein [GP]-Ib), (2) activation (anti-GPVI), and (3) aggregation (anti-GPIIbIIIa) in a murine in vivo model of left coronary artery ligation (30 minutes of ischemia followed by 24 hours of reperfusion). Infarct sizes were determined by Evans Blue/2,3,5-triphenyltetrazolium chloride staining, infiltrating neutrophils by immunohistology. Anti-GPVI treatment significantly reduced infarct size versus control, whereas anti-GPIb or anti-GPIIbIIIa antibody fragments showed no significant differences. Mechanistically, anti-GPVI antibody-mediated reduction of infarct size was not because of impaired Ca 2+ signaling or platelet degranulation because mice deficient in store-operated calcium channels (stromal interaction molecule 1, ORAI1), α-granules (Nbeal2

Rationale: In patients after acute myocardial infarction (AMI), the initial extent of necrosis and inflammation determine clinical outcome. One early event in AMI is the increased cardiac expression of atrial natriuretic peptide (NP) and B-type NP, with their plasma levels correlating with severity of ischemia. It was shown that NPs, via their cGMP-forming guanylyl cyclase-A (GC-A) receptor and cGMP-dependent kinase I (cGKI), strengthen systemic endothelial barrier properties in acute inflammation. Objective: We studied whether endothelial actions of local NPs modulate myocardial injury and early inflammation after AMI. Methods and Results: Necrosis and inflammation after experimental AMI were compared between control mice and littermates with endothelial-restricted inactivation of GC-A (knockout mice with endothelial GC-A deletion) or cGKI (knockout mice with endothelial cGKI deletion). Unexpectedly, myocardial infarct size and neutrophil infiltration/activity 2 days after AMI were attenuated in knockout mice with endothelial GC-A deletion and unaltered in knockout mice with endothelial cGKI deletion. Molecular studies revealed that hypoxia and tumor necrosis factor-α, conditions accompanying AMI, reduce the endothelial expression of cGKI and enhance cGMP-stimulated phosphodiesterase 2A (PDE2A) levels. Real-time cAMP measurements in endothelial microdomains using a novel fluorescence resonance energy transfer biosensor revealed that PDE2 mediates NP/cGMP-driven decreases of submembrane cAMP levels. Finally, intravital microscopy studies of the mouse cremaster microcirculation showed that tumor necrosis factor-α–induced endothelial NP/GC-A/cGMP/PDE2 signaling impairs endothelial barrier functions. Conclusions: Hypoxia and cytokines, such as tumor necrosis factor-α, modify the endothelial postreceptor signaling pathways of NPs, with downregulation of cGKI, induction of PDE2A, and altered cGMP/cAMP cross talk. Increased expression of PDE2 can mediate hyperpermeability effects of paracrine endothelial NP/GC-A/cGMP signaling and facilitate neutrophil extravasation during the early phase after MI.