Staphylococcus aureus α-Toxin Triggers the Synthesis of B-Cell Lymphoma 3 by Human Platelets

The frequency and severity of bacteremic infections has increased over the last decade and bacterial endovascular infections (i.e., sepsis or endocarditis) are associated with high morbidity and mortality. Bacteria or secreted bacterial products modulate platelet function and, as a result, affect platelet accumulation at sites of vascular infection and inflammation. However, whether bacterial products regulate synthetic events in platelets is not known. In the present study, we determined if prolonged contact with staphylococcal α-toxin signals platelets to synthesize B-cell lymphoma (Bcl-3), a protein that regulates clot retraction in murine and human platelets. We show that α-toxin induced αIIbβ3-dependent aggregation (EC50 2.98 µg/mL ± 0.64 µg/mL) and, over time, significantly altered platelet morphology and stimulated de novo accumulation of Bcl-3 protein in platelets. Adherence to collagen or fibrinogen also increased the expression of Bcl-3 protein by platelets. α-toxin altered Bcl-3 protein expression patterns in platelets adherent to collagen, but not fibrinogen. Pretreatment of platelets with inhibitors of protein synthesis or the mammalian Target of Rapamycin (mTOR) decreased Bcl-3 protein expression in α-toxin stimulated platelets. In conclusion, Staphylococcus aureus-derived α-toxin, a pore forming exotoxin, exerts immediate (i.e., aggregation) and prolonged (i.e., protein synthesis) responses in platelets, which may contribute to increased thrombotic events associated with gram-positive sepsis or endocarditis

Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenecity of human platelets

Tissue factor (TF) is an essential cofactor for the activation of blood coagulation in vivo. We now report that quiescent human platelets express TF pre-mRNA and, in response to activation, splice this intronic-rich message into mature mRNA. Splicing of TF pre-mRNA is associated with increased TF protein expression, procoagulant activity, and accelerated formation of clots. Pre-mRNA splicing is controlled by Cdc2-like kinase (Clk)1, and interruption of Clk1 signaling prevents TF from accumulating in activated platelets. Elevated intravascular TF has been reported in a variety of prothrombotic diseases, but there is debate as to whether anucleate platelets—the key cellular effector of thrombosis—express TF. Our studies demonstrate that human platelets use Clk1-dependent splicing pathways to generate TF protein in response to cellular activation. We propose that platelet-derived TF contributes to the propagation and stabilization of a thrombus

Novel Anti-bacterial Activities of β-defensin 1 in Human Platelets: Suppression of Pathogen Growth and Signaling of Neutrophil Extracellular Trap Formation

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

A New Role of NAP1L1 in Megakaryocytes and Human Platelets

Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD34+-derived megakaryocytes (MKs). NAP1L1 is expressed in PLTs and MKs. Co-IP experiments revealed that dihydrolipolylysine-residue acetyltransferase (DLAT encoded protein PDC-E2, ODP2) dynamically interacts with NAP1L1. PDC-E2 is part of the mitochondrial pyruvate-dehydrogenase (PDH) multi-enzyme complex, playing a crucial role in maintaining cellular respiration, and promoting ATP-synthesis via the respiratory chain. Since altered mitochondrial function is a hallmark of infectious syndromes, we analyzed PDH activity in PLTs from septic patients demonstrating increased activity, paralleling NAP1L1 expression levels. MKs PDH activity decreased following an LPS-challenge. Furthermore, overexpression of NAP1L1 significantly altered the ability of MKs to form proplatelet extensions, diminishing thrombopoiesis. These results indicate that NAP1L1 performs in other than nucleosome-assembly functions in PTLs and MKs, binding a key mitochondrial protein as a potential chaperone, and gatekeeper, influencing PDH activity and thrombopoiesis