Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.
Poly(amidoamine) (PAMAM) dendrimers have been proposed for a variety of biomedical applications and are increasingly studied as model nanomaterials for such use. The dendritic structure features both modular synthetic control of molecular size and shape and presentation of multiple equivalent terminal groups. These properties make PAMAM dendrimers highly functionalizable, versatile single-molecule nanoparticles with a high degree of consistency and low polydispersity. Recent nanotoxicological studies showed that intravenous administration of amine-terminated PAMAM dendrimers to mice was lethal, causing a disseminated intravascular coagulation-like condition. To elucidate the mechanisms underlying this coagulopathy, in vitro assessments of platelet functions in contact with PAMAM dendrimers were undertaken. This study demonstrates that cationic G7 PAMAM dendrimers activate platelets and dramatically alter their morphology. These changes to platelet morphology and activation state substantially altered platelet function, including increased aggregation and adherence to surfaces. Surprisingly, dendrimer exposure also attenuated platelet-dependent thrombin generation, indicating that not all platelet functions remained intact. These findings provide additional insight into PAMAM dendrimer effects on blood components and underscore the necessity for further research on the effects and mechanisms of PAMAM-specific and general nanoparticle toxicity in blood.
Dysregulated inflammation is implicated in the pathobiology of aging, yet platelet-leukocyte interactions and downstream cytokine synthesis in aging remains poorly understood. Platelets and monocytes were isolated from healthy younger (age <45, = 37) and older (age ≥65, = 30) adults and incubated together under autologous and nonautologous conditions. Synthesis of inflammatory cytokines by monocytes, alone or in the presence of platelets, was examined. Next-generation RNA-sequencing allowed for unbiased profiling of the platelet transcriptome in aging. Basal IL-8 and MCP-1 synthesis by monocytes alone did not differ between older and younger adults. However, in the presence of autologous platelets, monocytes from older adults synthesized greater IL-8 (41 ± 5 versus 9 ± 2 ng/ml, < 0.0001) and MCP-1 (867 ± 150 versus 216 ± 36 ng/ml, < 0.0001) than younger adults. Platelets from older adults were sufficient for upregulating the synthesis of inflammatory cytokines by monocytes. Using RNA-sequencing of platelets followed by validation via RT-PCR and immunoblot, we discovered that granzyme A (GrmA), a serine protease not previously identified in human platelets, increases with aging (∼9-fold versus younger adults, < 0.05) and governs increased IL-8 and MCP-1 synthesis through TLR4 and caspase-1. Inhibiting GrmA reduced excessive IL-8 and MCP-1 synthesis in aging to levels similar to younger adults. In summary, human aging is associated with changes in the platelet transcriptome and proteome. GrmA is present and bioactive in human platelets, is higher in older adults, and controls the synthesis of inflammatory cytokines by monocytes. Alterations in the platelet molecular signature and signaling to monocytes may contribute to dysregulated inflammatory syndromes in older adults.
Stroke is a common and often fatal event, and, in survivors, it is accompanied by a high risk of recurrence. Ischemic stroke is associated with abnormal platelet activity and thrombus formation. In addition to their roles in the development of acute thrombi, platelets serve as a bridge for leukocytes within the vasculature. Myeloid leukocytes are critical mediators of atherosclerosis and atherothrombosis. Interactions between platelets and leukocytes foster an inflammatory and thrombotic milieu that influences lesion progression, facilitates plaque rupture, and triggers thrombus formation and embolization. Accordingly, antiplatelet agents, including aspirin, dipyridamole, and clopidogrel, are recommended therapies for most patients with a history of stroke. In addition to mitigating thrombosis, antiplatelet drugs have direct and indirect effects on inflammation, which may translate to enhanced clinical efficacy.
Bacteria can enter the bloodstream in response to infectious insults. Bacteremia elicits several immune and clinical complications, including thrombocytopenia. A primary cause of thrombocytopenia is shortened survival of platelets. We demonstrate that pathogenic bacteria induce apoptotic events in platelets that include calpain-mediated degradation of Bcl-x L , an essential regulator of platelet survival. Specifically, bloodstream bacterial isolates from patients with sepsis induce lateral condensation of actin, impair mitochondrial membrane potential, and degrade Bcl-x L protein in platelets. Bcl-x L protein degradation is enhanced when platelets are exposed to pathogenic Escherichia coli that produce the poreforming toxin ␣-hemolysin, a response that is markedly attenuated when the gene is deleted from E coli. We also found that nonpathogenic E coli gain degrading activity when they are forced to express ␣-hemolysin. Like ␣-hemolysin, purified ␣-toxin readily degrades Bcl-x L protein in platelets, as do clinical Staphylococcus aureus isolates that produce ␣-toxin. Inhibition of calpain activity, but not the proteasome, rescues Bcl-x L protein degradation in platelets coincubated with pathogenic E coli including ␣-hemolysin producing strains. This is the first evidence that pathogenic bacteria can trigger activation of the platelet intrinsic apoptosis program and our results suggest a new mechanism by which bacterial pathogens might cause thrombocytopenia in patients with bloodstream infections. IntroductionBacteremia is a leading cause of morbidity and mortality in the United States and worldwide. 1,2 Risk factors for bacteremia include indwelling catheters, 3 trauma, 4 and surgery. 5 Bloodstream infections also are found frequently in patients with malignancies, 6 endocarditis, 7 and urinary tract infections. 8 In severe cases, bacteremia elicits a vigorous immune response that results in sepsis and septic shock. 9 Bloodstream infections often are accompanied by thrombocytopenia. 10 Adverse clinical outcomes commonly are observed in thrombocytopenic patients with documented bacteremia, and the severity of thrombocytopenia is associated with an increase in the rate of mortality of patients in the intensive care unit. 10,11 It generally is presumed that the infectious milieu induces thrombocytopenia by activating platelets that subsequently deposit in microvascular thrombi or get cleared from the circulation. 12 Indeed, bacteria or bacterial products can induce platelet activation by directly or indirectly binding to surface receptors such as integrin ␣ IIb  3 , GPIb, and TLRs. 13 However, the precise mechanisms that underpin bacteremia-induced thrombocytopenia remain poorly defined.In addition to the biochemical pathways that regulate classic platelet functional responses such as aggregation and adhesion, it has been demonstrated recently that megakaryocytes and platelets possess an intrinsic apoptosis program. 14,15 Key components of the platelet apoptotic pathway include the prosurvival protein Bcl-x L and...
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