Platelets are central mediators of thrombosis and hemostasis. At the site of vascular injury, platelet accumulation (i.e. adhesion and aggregation) constitutes the first wave of hemostasis. Blood coagulation, initiated by the coagulation cascades, is the second wave of thrombin generation and enhance phosphatidylserine exposure, can markedly potentiate cell-based thrombin generation and enhance blood coagulation. Recently, deposition of plasma fibronectin and other proteins onto the injured vessel wall has been identified as a new “protein wave of hemostasis” that occurs prior to platelet accumulation (i.e. the classical first wave of hemostasis). These three waves of hemostasis, in the event of atherosclerotic plaque rupture, may turn pathogenic, and cause uncontrolled vessel occlusion and thrombotic disorders (e.g. heart attack and stroke). Current anti-platelet therapies have significantly reduced cardiovascular mortality, however, on-treatment thrombotic events, thrombocytopenia, and bleeding complications are still major concerns that continue to motivate innovation and drive therapeutic advances. Emerging evidence has brought platelet adhesion molecules back into the spotlight as targets for the development of novel anti-thrombotic agents. These potential antiplatelet targets mainly include the platelet receptors glycoprotein (GP) Ib-IX-V complex, β3 integrins (αIIb subunit and PSI domain of β3 subunit) and GPVI. Numerous efforts have been made aiming to balance the efficacy of inhibiting thrombosis without compromising hemostasis. This mini-review will update the mechanisms of thrombosis and the current state of antiplatelet therapies, and will focus on platelet adhesion molecules and the novel anti-thrombotic therapies that target them.

An elevated CRP level at hospital admission and vegetation length at diagnosis were strong predictors of in-hospital mortality in IE, independent of other prognostic parameters, specifically taking into account patient characteristics and complications during therapy.

Infective endocarditis (IE) is the cardiac disease with the highest rates of mortality. New biomarkers that are able to identify patients at risk for death are required to improve patient management and outcome. This study aims to investigate if cytokines, chemokines and growth factors measured at IE diagnosis can predict mortality. Patients with definite IE, according to the Duke's modified criteria, were included. Using highperformance Luminex assay, 27 different cytokines, chemokines and growth factors were analyzed. Machine learning techniques were used for the prediction of death and subsequently creating a decision tree, in which the cytokines, chemokines and growth factors were analyzed together with C-reactive protein (CRP). Sixty-nine patients were included, 41 (59%) male, median age 54 [interquartile range (IQR) = 41-65 years] and median time between onset of the symptoms and diagnosis was 12 days (IQR = 5-30 days). The in-hospital mortality was 26% (n = 18). Proinflammatory cytokines interkeukin (IL)-15 and C-C motif chemokine ligand (CCL4)were found to predict death, adding value to CRP levels. The decision tree predicted correctly the outcome of 91% of the patients at hospital admission. The high-risk group, defined as CRP ≥ 72 mg/dL, IL-15 ≥ 5·6 fg/ml and CCL4 ≥ 6·35 fg/ml had an 88% in-hospital mortality rate, whereas the patients classified as low-risk had a mortality rate of 8% (P = < 0·001). Cytokines IL-15 and CCL4 were predictors of mortality in IE, adding prognostic value beyond that provided by CRP levels. Assessment of cytokines has potential value for clinical risk stratification and monitoring in IE patients.

Objectives: To characterize the plasmatic profile of cell-derived microvesicles (MVs) at diagnosis and during the treatment of patients with infective endocarditis (IE). Methods: Blood samples from 57 patients with IE were obtained on 3 consecutive moments: upon admission (T0), at 2 weeks (T1), and at the end of treatment (T2), and were compared with 22 patients with other bacterial infections. MPs were measured by flow cytometry and labeled for specific cell markers of CD45 (leukocytes), CD66b (neutrophils), CD14 (monocytes), CD41a (platelets), CD51 (endothelial cells), CD3 (T lymphocyte) and CD235a (erythrocytes). Results:MVs from platelets (pltMVs), leukocytes (leukMVs), neutrophils (neutMVs), monocytes (monoMVs) and lymphocytes (lymphMVs) were significantly more elevated in the patients with IE, compared to the patients with other bacterial infections, despite comparable age, sex, blood counts and Creactive protein levels. MVs values revealed a relatively stable pattern over time in IE, except for a significant increase in leukMVs and neutMVs in T1. LeukMVs ( p = 0.011), neutMVs ( p = 0.010), monoMVs ( p = 0.016) and lymphMVs ( p = 0.020), measured at admission, were significantly higher in IE patients that died during hospitalization in comparison with those that survived. In a multivariable analyses, the levels of neutMVs remained as an independent factor associated with mortality (odds ratio 2.203; 95% confidence interval 1.217 -3.988; p = 0.009), adjustment for heart failure during the treatment. Conclusions: Plasma levels of pltMVs, leukMVs, neutMVs, monoMVs and lymphMVs were significantly more elevated in patients with IE than in patients with other bacterial infections at hospital admission. Furthermore, neutMVs at admission have been identified as an independent predictor of mortality in patients with IE. Thus, cell derived MPs may become an important tool in the differential diagnosis and mortality risk assessment early in the course of IE suspected cases.

Introduction: Fibrinogen (Fg) and von Willebrand factor (VWF) have been considered essential for platelet adhesion and aggregation. However, platelet aggregation still occurs in mice lacking Fg and/or VWF or plasma fibronectin but not β3 integrin (JCI, 2000; JTH, 2006; Blood, 2009; JCI, 2014). This suggests that other non-classical αIIbβ3 integrin ligand(s) mediate platelet aggregation. α-dystroglycan (α-DG) is a component of the dystrophin-glycoprotein complex that binds extracellular matrix proteins containing laminin-G like domains via unique heteropolysaccharide [-GlcA-β1,3-Xyl-α1,3-]n called matriglycan, which can be targeted specifically with monoclonal antibody IIH6C4. Although α-DG was identified in a recent proteomic study of platelet releasate, its membrane expression and function in platelets have never been investigated. Methods and Results: Using the anti-α-DG monoclonal antibody IIH6C4, we found expression of α-DG in mouse and human resting platelets in Western blots. α-DG expression was also identified on the non-permeabilized mouse and human resting platelets by flow cytometry, indicating that α-DG is constitutively expressed on the platelet surface. We next examined whether disruption of the integrity of the dystrophin-glycoprotein complex affects the platelet aggregation. In a dystrophin-deficient mouse model of Duchenne muscular dystrophy with reduced α-DG expression (mdx mice), we found that ADP induced platelet aggregation in platelet-rich plasma (PRP) decreased 50%, suggesting that the integrity of the dystrophin-glycoprotein complex is required for normal platelet aggregation. To test whether inhibition of platelet aggregation can be achieved by targeting α-DG, we applied the well-established polyclonal (H300) and monoclonal (IIH6C4 and VIA4) anti-α-DG antibodies. Mouse gel-filtered platelet aggregation induced by thrombin was significantly inhibited by all three antibodies. Mouse platelet aggregation in PRP was also inhibited by H300. For platelets from healthy human donors, the inhibitive effect was more profound. Using a lower concentration of H300 (1 µg/mL in human vs. 2 µg/mL in mouse), ADP induced human platelet aggregation in PRP was inhibited to less than 50% of control and quickly de-aggregated within 5 minutes, while no de-aggregation was observed in the controls. Human gel-filtered platelet aggregation was also inhibited in a dose-dependent manner by these antibodies. Our results thus revealed a vital role of α-DG in platelet aggregation. In an ex vivo perfusion chamber model, human platelet adhesion and thrombus formation on collagen were markedly decreased at an arterial shear rate of 1800/s by anti-α-DG antibodies. Interestingly, although α-DG was found to be a ligand of laminin, platelet adhesion on laminin was not significantly altered by these antibodies, suggesting that contribution of α-DG to thrombus formation is not through its classical ligand laminin but other previously unidentified mechanisms. Next, we tested the role of α-DG in thrombus formation in vivo. Using a mouse cremaster artery laser-injury intravital microscopy model, we found that the anti-α-DG antibodies significantly delayed and decreased thrombus formation. To investigate the underlying mechanism of the surprisingly profound impact of α-DG on platelet aggregation and thrombus formation, we performed the co-immunoprecipitation assay and found that α-DG interacts with both β3 integrin and fibronectin, even in the absence of Fg and VWF, suggesting that α-DG may directly or form an α-DG-fibronectin complex to bind αIIbβ3 integrin, contributing to platelet aggregation and thrombus formation. Conclusion: Our data demonstrated that α-DG and likely other components of the dystrophin-glycoprotein complex are expressed on the platelet surface, and play a vital role in platelet aggregation and thrombus formation. α-DG may contribute to platelet aggregation independent of VWF and Fg through direct or indirect interaction with αIIbβ3 integrin. It is likely that patients with muscular dystrophies, such as those with Duchenne muscular dystrophy, are protected from thrombosis. More importantly, our data established α-DG, and potentially other components of the dystrophin-glycoprotein complex, as novel targets for the treatment of thrombotic disorders. Disclosures No relevant conflicts of interest to declare.