Abstract
While platelets are the essential mediators of hemostasis, they are being increasingly recognized for their potential of contributing to host defenses. Here, using immunofluorescent microscopy, western blot, and ELISA, we found that human β-defensin 3 (hBD-3), an important antimicrobial peptide produced by epithelial cells, can be detected in human platelets and megakaryocytes. Flow cytometry and immuno-electron microscopy revealed hBD-3 on the surface of thrombin activated platelets. Moreover, hBD-3 was also found in platelet derived extracellular vesicles (p-EVs), isolated from platelet poor plasma and from platelet supernatants following thrombin stimulation. Incubation of platelets with hBD-3 peptide resulted in modest platelet activation and pre-incubation of platelets with synthetic hBD-3 prior to exposure to thrombin appeared to increase hBD-3 content in platelet lysates as well as in p-EVs, suggesting that hBD-3 can be initially taken up by platelets, perhaps via their open canalicular system. Interestingly, in vitro exposure of primary human endothelial cells to either hBD-3 peptide or purified p-EVs, caused significant endothelial dysfunction as documented by diminished levels of phosphorylated endothelial nitric oxide synthase (eNOS), Krüppel like factor-2 (KLF-2), and elevated relative expression of von Willebrand Factor (vWF). Pre-incubation of platelets with hBD-3 appeared to augment endothelial dysfunction caused by p-EVs. Overall, the current study provides evidence that hBD-3 enriched EVs can be released by activated platelets and may play a role in positive feedback of platelet activation as well as in endothelial dysfunction. Theoretically, these effects could contribute to both cellular recruitment to the endothelium creating a pro-thrombotic vascular microenvironment which serve as a bridge between innate immunity and hemostasis.
Highlights
Platelets are specialized, non-nucleated small (2–3 μm) fragments of bone marrow resident megakaryocytes
These findings identify the presence of hBD-3 in platelets, primarily in compartments outside of platelet α-granules, and suggest that hBD-3 may originate in platelet precursor cells; i.e., megakaryocytes
Platelets are engaged in a number of non-hemostatic processes including primary anti-microbial defense (Tang et al, 2002; Shiga et al, 2017) as they express antimicrobial peptides (AMPs), more precisely the beta defensins (Kraemer et al, 2011; Ware et al, 2013)
Summary
Non-nucleated small (2–3 μm) fragments of bone marrow resident megakaryocytes. Platelets are activated and recruited at sites of damaged blood vessel walls or inflamed atherosclerotic plaques adjacent to the endothelial lining Other than their primary function in thrombosis, platelets participate in a number of non-hemostatic processes including, primary anti-microbial defense (Tang et al, 2002; Shiga et al, 2017), and modulation of immune responses by direct cross interaction with endothelial cells and cells of the immune system (Yeaman, 2014). During local and systemic inflammatory conditions, platelets are activated locally and secrete contents from their α-granule, dense granule, and lysosome components which contain adhesion molecules, coagulation factors, growth factors, protease inhibitors, inflammatory mediators, and antimicrobial peptides (AMPs) (Yeaman, 2014; Ali et al, 2015) These non-thrombotic functions are indicative of an integral role of platelet derived AMPs in host defense (Aggour and Gamil, 2017). Reportedly carry significant in vitro antibacterial properties against several pathogens including Staphylococcus species (Drago et al, 2014)
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