Key Points• Paired immunoglobulin-like receptor B negatively regulates platelet activation.Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin b3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet a-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin aIIbb3-mediated signaling. (Blood. 2014;124(15):2421-2430 Introduction Platelets, which are derived from megakaryocytes, circulate in mammalian blood and play essential roles in hemostasis, angiogenesis, inflammation, and metastasis, 1-3 contain a variety of receptors on their surface. The immunoglobulin superfamily (IgSF) is a large group of cell surface proteins that are involved in the adhesion, binding, or recognition of cells. 4 Several IgSF members expressed on the platelet surface regulate platelet adhesion, activation, and aggregation. Among those receptors, platelet collagen receptor glycoprotein VI (GPVI) has short cytoplasmic domains lacking signaling motifs, but it transmits activating signals by linking to immunoreceptor tyrosine-based activation motif (ITAM) of the Fc receptor g chain (FcRg chain). 5 The GPVI/FcRg chain complex can propagate potent signaling causing aIIbb3 activation and platelet aggregation and thereby play an important role in hemostasis and thrombosis formation. 6 In contrast to GPVI, platelet endothelial cell adhesion molecule-1, a platelet surface IgSF member with 6 extracellular Ig domains and a cytoplasmic immunoreceptor tyrosinebased inhibitory motif (ITIM), mildly inhibits human or mouse platelet activation by collagen, adenosine 59-diphosphate (ADP), or thrombin. 7 Similarly, antibody-mediated cross-linking of G6B, a platelet surface IgS...

Background: Reperfusion therapy is known to improve prognosis and limit myocardial damage after myocardial infarction (MI). The administration of antiplatelet drugs prior to percutaneous coronary intervention also proves beneficial to patients with acute MI (AMI). However, a good number of AMI patients do not receive reperfusion therapy, and it is not clear if they would benefit from antiplatelet pre-treatment. Methods: Experimental C57BL/6 mice were randomly allocated to five groups: the sham group, control, post-treatment, pre-treatment, and pre-and post-treatment groups. Aspirin (15 mg/kg), clopidogrel (11 mg/kg), ticagrelor (27 mg/kg), and 2 prasugrel (1.5 mg/kg) were intragastrically administered in the treatment groups. On day 7 post MI, cardiac function and cardiac fibrosis were evaluated using echocardiography and Masson's Trichrome staining respectively. Histopathological examinations were performed on tissue sections to grade inflammatory cell infiltration. Platelet inhibition was monitored by measuring thrombin-induced platelet aggregation. Results: Left ventricular ejection fraction and fractional shortening improved significantly (p < 0.01) in the pre-treatment groups when compared to the post-treatment and control groups. A significant (p < 0.01) decrease in cardiac fibrosis was observed in the pre-treatment group, compared with the post-treatment and control groups. Inflammatory cell infiltration significantly decreased in the pre-treatment group compared with the control group (p < 0.05). Thrombin-induced platelet aggregation was significantly inhibited by antiplatelet drugs, but increased with the exposure to H2O2. Conclusions: In the absence of reperfusion therapy, pre-treatment with antiplatelet drugs successfully improved cardiac function, reduced cardiac fibrosis and inflammatory cell infiltration, and inhibited oxidative stress-induced platelet aggregation after MI in the mouse model.

BackgroundLeft atrial appendage (LAA) closure (LAAC) can safely and effectively prevent stroke events caused by atrial fibrillation. However, the structure of the LAA is highly variable among individuals, and the optimal method for obtaining measurements remains unknown.HypothesisWe aimed to study the accuracy of left atrial computed tomography angiography (CTA), three‐dimensional (3D) reconstruction using CTA, two‐dimensional transesophageal echocardiography (2D‐TEE), and digital subtraction angiography (DSA) for measuring the diameter of the LAA and compare their value for selecting occluder size.MethodsWe retrospectively evaluated data for 148 patients with nonvalvular atrial fibrillation who underwent successful LAAC. CTA and 2D‐TEE of the left atrium and pulmonary vein were performed before LAAC. We performed 3D reconstruction of the left atrium and LAA using Mimics and 3‐matics software. DSA of the LAA was performed during surgery.ResultsValues measured via CTA 3D reconstruction were significantly higher than those measured using other methods. DSA‐measured values were significantly lower than those measured via CTA and CTA 3D reconstruction. Occluder size was positively correlated with LAA orifice diameter. The differences between occluder size and DSA, 2D‐TEE, CTA, CTA 3D reconstruction measurements were 4.96 ± 2.58, 4.64 ± 2.50, 4.04 ± 1.37, and 2.92 ± 1.38 mm, respectively. Intraclass correlation coefficients for these methods were −.067, .006, .241, and .519, respectively.ConclusionCTA 3D reconstruction provides the best correlation and consistency between the measured LAA orifice diameter and occluder size. Adding 2–4 mm to the maximum LAA orifice diameter based on 3D‐CTA may aid in selecting the appropriate WATCHMAN device.

The adhesion G protein-coupled receptor GPR56 mediates cell-cell and cell-extracellular matrix interactions. To examine the function of GPR56 in platelet activation and arterial thrombosis, we generated GPR56-knockout mice and evaluated GPR56 expression in human and mouse platelets. The results revealed that the levels of the GPR56 N-terminal fragment were significantly higher on the first day after myocardial infarction than on the seventh day in the plasma of patients with ST-segment-elevation myocardial infarction. Next, we investigated the effects of GPR56 on platelet function in vitro and in vivo. We observed that collagen-induced aggregation and adenosine triphosphate release were reduced in Gpr56-/- platelets. Furthermore, P-selectin expression on the Gpr56-/- platelet surface was also reduced, and the spreading area on immobilized collagen was decreased in Gpr56-/- platelets. Furthermore, collagen-induced platelet activation in human platelets was inhibited by an anti-GPR56 antibody. Gpr56-/- mice showed an extended time to the first occlusion in models with cremaster arteriole laser injury and FeCl3-induced carotid artery injury. GPR56 activated the G protein 13 signaling pathway following collagen stimulation, which promoted platelet adhesion and thrombus formation at the site of vascular injury. Thus, our study confirmed that GPR56 regulated the formation of arterial thrombosis. Inhibition of the initial response of GPR56 to collagen could significantly inhibit platelet activation and thrombus formation. Our results provide new insights for research into antiplatelet drugs.

Background: AGK (acylglycerol kinase) was first identified as a mitochondrial transmembrane protein that exhibits a lipid kinase function. Recent studies have established that AGK promotes cancer growth and metastasis, enhances glycolytic metabolism and function fitness of CD8 + T cells, or regulates megakaryocyte differentiation. However, the role of AGK in platelet activation and arterial thrombosis remains to be elaborated. Methods: We performed hematologic analysis using automated hematology analyzer and investigated platelets morphology by transmission electron microscope. We explored the role of AGK in platelet activation and arterial thrombosis utilizing transgenic mice, platelet functional experiments in vitro, and thrombosis models in vivo. We revealed the regulation effect of AGK on Talin-1 by coimmunoprecipitation, mass spectrometry, immunofluorescence, and Western blot. We tested the role of AGK on lipid synthesis of phosphatidic acid/lysophosphatidic acid and thrombin generation by specific Elisa kits. Results: In this study, we found that AGK depletion or AGK mutation had no effect on the platelet average volumes, the platelet microstructures, or the expression levels of the major platelet membrane receptors. However, AGK deficiency or AGK mutation conspicuously decreased multiple aspects of platelet activation, including agonists-induced platelet aggregation, granules secretion, JON/A binding, spreading on Fg (fibrinogen), and clot retraction. AGK deficiency or AGK mutation also obviously delayed arterial thrombus formation but had no effect on tail bleeding time and platelet procoagulant function. Mechanistic investigation revealed that AGK may promote Talin-1Ser425 phosphorylation and affect the αIIbβ3-mediated bidirectional signaling pathway. However, AGK does not affect lipid synthesis of lysophosphatidic acid/lysophosphatidic acid in platelets. Conclusions: AGK, through its kinase activity, potentiates platelet activation and arterial thrombosis by promoting Talin-1 Ser425 phosphorylation and affecting the αIIbβ3-mediated bidirectional signaling pathway.