No abstract

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...

Summary Methicillin-resistant Staphylococcus aureus (MRSA) induces a pro-thrombotic and pro-inflammatory milieu. Although timely antibiotic administration in MRSA sepsis may improve outcomes by arresting bacterial growth, the effects of antibiotics on mitigating injurious thrombo-inflammatory cellular responses remains unexplored. Using a newly developed human whole blood model and an in vivo mouse model of MRSA infection, we examined how antibiotics inhibit MRSA induced thrombo-inflammatory pathways. Human whole blood was inoculated with MRSA. Thrombin generation and inflammatory cytokine synthesis was measured in the presence or absence of linezolid and vancomycin. C57BL/6 mice were injected with MRSA and the effect of vancomycin administration was examined. MRSA accelerated thrombin generation in a time- and concentration-dependent manner and induced the release of cytokines, including interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1. The increase in thrombin generation and inflammatory responses was mediated through the synthesis of tissue factor and cytokines, respectively, and the release of microparticles. The early administration of antibiotics restored normal thrombin generation patterns and significantly reduced the synthesis of cytokines. In contrast, when antibiotic administration was delayed, thrombin generation and cytokine synthesis were not significantly reduced. In mice infected with MRSA, early antibiotic administration reduced thrombin anti-thrombin complexes and cytokine synthesis, whereas delayed antibiotic administration did not. These data provide novel mechanistic evidence of the importance of prompt antibiotic administration in infectious syndromes.