Background-Peripheral homing of progenitor cells in areas of diseased organs is critical for tissue regeneration. The chemokine stromal cell-derived factor-1 (SDF-1) regulates homing of CD34 ϩ stem cells. We evaluated the role of platelet-derived SDF-1 in adhesion and differentiation of human CD34 ϩ cells into endothelial progenitor cells. Methods and Results-Adherent platelets express substantial amounts of SDF-1 and recruit CD34 ϩ cells in vitro and in vivo. A monoclonal antibody to SDF-1 or to its counterreceptor, CXCR4, inhibits stem cell adhesion on adherent platelets under high arterial shear in vitro and after carotid ligation in mice, as determined by intravital fluorescence microscopy. Platelets that adhere to human arterial endothelial cells enhance the adhesion of CD34 ϩ cells on endothelium under flow conditions, a process that is inhibited by anti-SDF-1. During intestinal ischemia/reperfusion in mice, anti-SDF-1 and anti-CXCR4, but not isotype control antibodies, abolish the recruitment of CD34 ϩ cells in microcirculation. Moreover, platelet-derived SDF-1 binding to CXCR4 receptor promotes platelet-induced differentiation of CD34 ϩ cells into endothelial progenitor cells, as verified by colony-forming assays in vitro. Conclusions-These

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.

Platelets are classified as terminally differentiated cells that are incapable of cellular division. However, we observe that anucleate human platelets, either maintained in suspension culture or captured in microdrops, give rise to new cell bodies packed with respiring mitochondria and ␣-granules. Platelet progeny formation also occurs in whole blood cultures. Newly formed platelets are structurally indistinguishable from normal platelets, are able to adhere and spread on extracellular matrix, and display normal signaldependent expression of surface Pselectin and annexin V. Platelet progeny formation is accompanied by increases in biomass, cellular protein levels, and protein synthesis in expanding populations. Platelet numbers also increase during ex vivo storage. These observations indicate that platelets have a previously unrecognized capacity for producing functional progeny, which involves a form of cell division that does not require a nucleus. Because this new function of platelets occurs outside of the bone marrow milieu, it raises the possibility that thrombopoiesis continues in the bloodstream. (Blood. 2010;115(18):3801-3809) IntroductionAfter they are shed from the cytoplasm of megakaryocytes, 1 platelets circulate in the bloodstream for 9 to 11 days. There is no evidence that these anucleate cytoplasts undergo cellular division, but recent studies by our group and others have identified unexpected cellular functions of platelets, 2-4 including the capacity to process pre-mRNA 2-5 and translate mRNA into protein. [6][7][8][9][10] Platelets also continue to synthesize protein for several days when they are stored ex vivo. 11 These findings indicate that, despite their terminally differentiated state, platelets are biosynthetically more sophisticated than previously thought. 12 They also suggest that platelets may be able to adjust their phenotypic composition in response to environmental cues.Here we show that platelets give rise to new cells that are structurally and functionally similar to their parent counterparts. The formation of platelet progeny is associated with increases in platelet biomass, protein synthetic events, and total intracellular protein. This heretofore undescribed proliferative capacity of platelets may have significant consequences for normal and pathologic thrombopoiesis in humans in addition to having clinical implications for transfusion medicine. Methods Platelet isolation and cultureAll studies were approved by the University of Utah Institutional Review Board committee (no. 392). Leukocyte-depleted platelets were isolated as previously described. 2,5 Washed platelets were resuspended at 100 000/L in serum-free M199 medium, placed in round-bottom polypropylene tubes (BD Biosciences), and cultured in a 37°C humidified incubator under gentle rotation (MacsMix, slow, 45°angle; Miltenyi). The same suspension culture conditions were also used for the whole blood studies shown in Figure 2A.Stored platelets were obtained from the ARUP Blood Transfusion Services at the University o...

The septic milieu triggers expression of spliced tissue factor mRNA in human platelets. J Thromb Haemost 2011; 9: 748-58.Summary. Background: Activated platelets have previouslyunrecognized mechanisms of post-transcriptional gene expression that may influence hemostasis and inflammation. A novel pathway involves splicing of pre-mRNAs in resting platelets to mature, translatable mRNAs in response to cellular activation. Objectives: We asked if bacterial products and host agonists present in the septic milieu induce tissue factor pre-mRNA splicing in platelets from healthy subjects. In parallel, we asked if spliced tissue factor (TF) mRNA is present in platelets from septic patients in a proof-of-principle analysis. Patients/methods: TF pre-mRNA and mRNA expression patterns were characterized in platelets from septic patients and in platelets isolated from healthy subjects activated with bacteria, toxins and inflammatory agonists. Procoagulant activity was also measured. Results and conclusions: Live bacteria, staphylococcal a-toxin and lipopolysaccharide (LPS) induced TF premRNA splicing in platelets isolated from healthy subjects. Toxin-stimulated platelets accelerated plasma clotting, a response that was blocked by a previously-characterized splicing inhibitor and by an anti-tissue factor antibody. Platelets from septic patients expressed spliced TF mRNA, whereas it was absent from unselected and age-matched control subjects. Tissue factor-dependent procoagulant activity was elevated in platelets from a subset of septic patients. Thus, bacterial and host factors induce splicing of TF pre-mRNA, expression of TF mRNA and tissue factor-dependent clotting activity in human platelets. TF mRNA is present in platelets from some septic patients, indicating that it may be a marker of altered platelet phenotype and function in sepsis and that splicing pathways are induced in this syndrome.

Platelets have been regarded as static cells that do not move once they adhere to a matrix. The present study explored, whether platelets are able to migrate. In contrast to the current opinion, we found that platelets were mobile, able to migrate over a surface, and transmigrate through a transwell membrane and endothelium toward a source of stromal cell-derived factor 1 (SDF-1). Platelet migration was stimulated by SDF-1, which led to the downstream activation and phosphorylation of Wiskott-Aldrich syndrome protein. SDF-1 signaling and subsequent platelet migration could be inhibited by CXCR4-receptor blocker AMD3100, pertussis toxin, inhibition of phosphoinositol 3-kinase (PI3 kinase) with LY294002 or wortmannin, and disruption of actin polymerization with cytochalasin B. The potential of platelets to migrate in an SDF-1-mediated fashion may redefine the role of platelets in the pathophysiology of vascular inflammation, subsequent atherosclerotic degeneration, and vascular regeneration.

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...

IntroductionSepsis is still a leading cause of morbidity and mortality, even in modern times, and thrombocytopenia has been closely associated with unfavorable disease outcome. Decreases in mitochondrial membrane potential (depolarization) were found in different tissues during sepsis. Previous work suggests that mitochondrial dysfunction of platelets correlates with clinical disease activity in sepsis. However, platelet mitochondrial membrane potential (Mmp) has not been investigated in a clinical follow-up design and not with regard to disease outcome.MethodsIn this study, platelet mitochondrial membrane depolarization was assessed by means of a fluorescent Mmp-Index with flow cytometry in 26 patients with sepsis compared with control patients. Platelet Mmp-Index on admission was correlated with the clinical disease scores Acute Physiology and Chronic Health Evaluation Score II (APACHE II), Sequential Organ Failure Score (SOFA), and Simplified Acute Physiology Score II (SAPS II). Finally, platelet Mmp-Index on admission and follow-up were compared in the group of sepsis survivors and nonsurvivors. Expression of the prosurvival protein Bcl-xL in platelets was quantified by immunoblotting.ResultsPlatelet mitochondrial membrane depolarization correlated significantly with the simultaneously assessed clinical disease severity by APACHE II (r = -0.867; P < 0.0001), SOFA (r = -0.857; P <0.0001), and SAPS II score (r = -0.839; P < 0.0001). Patients with severe sepsis showed a significant reduction in platelet Mmp-Index compared with sepsis without organ failure (0.18 (0.12 to 0.25) versus 0.79 (0.49 to 0.85), P < 0.0006) or with the control group (0.18 (0.12 to 0.25) versus 0.89 (0.68 to 1.00), P < 0.0001). Platelet Mmp-Index remained persistently low in sepsis nonsurvivors (0.269 (0.230 to 0.305)), whereas we observed recovery of platelet Mmp-Index in the survivor group (0.9 (0.713 to 1.017)). Furthermore, the level of prosurvival protein Bcl-xL decreased in platelets during severe sepsis.ConclusionIn this study, we demonstrated that mitochondrial membrane depolarization in platelets correlates with clinical disease severity in patients with sepsis during the disease course and may be a valuable adjunct parameter to aid in the assessment of disease severity, risk stratification, and clinical outcome.

The lack of sufficient well-defined tumor-associated antigens is still a drawback on the way to a cytotoxic T-lymphocyte-based immunotherapy of renal cell carcinoma (RCC). We are trying to define a larger number of such targets by a combined approach involving HLA ligand characterization by mass spectrometry and gene expression profiling by oligonucleotide microarrays. Here, we present the results of a large-scale analysis of 13 RCC specimens. We were able to identify more than 700 peptides, mostly from self-proteins without any evident tumor association. However, some HLA ligands derived from previously known tumor antigens in RCC. In addition, gene expression profiling of tumors and a set of healthy tissues revealed novel candidate RCC-associated antigens. For several of them, we were able to characterize HLA ligands after extraction from the tumor tissue. Apart from universal RCC antigens, some proteins seem to be appropriate candidates in individual patients only. This underlines the advantage of a personalized therapeutic approach. Further analyses will contribute additional HLA ligands to this repertoire of universal as well as patient-individual tumor antigens.