Sybille Rex

Stimulation of Toll-Like Receptor 2 in Human Platelets Induces a Thromboinflammatory Response Through Activation of Phosphoinositide 3-Kinase

Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam3CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin &agr;IIb&bgr;3, generation of reactive oxygen species, and, in human whole blood, formation of platelet–neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam3CSK4-induced platelet responses. In vivo challenge with live Porphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet–neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

Immune versus thrombotic stimulation of platelets differentially regulates signalling pathways, intracellular protein-protein interactions, and α-granule release

In addition to haemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositide-3-kinase (PI3K) and causes platelet activation and platelet-dependent thrombosis. It remains unknown if platelet activation by immune versus thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and alpha-granule release, and the physiological relevance of these potential differences. We investigated these processes after immune versus thrombotic platelet stimulation. We examined selected signalling pathways and found that phosphorylation kinetics of Akt, ERK1/2 and p38 differed dramatically between agonists. Next, we investigated platelet protein-protein interactions by mass spectrometry (MS)-based proteomics specifically targeting cytosolic factor XIIIa (FXIIIa) because of its function as a cytoskeleton-crosslinking protein whose binding partners have limited characterisation. Four FXIIIa-binding proteins were identified, two of which are novel interactions: FXIIIa-focal adhesion kinase (FAK) and FXIIIa-gelsolin. The binding of FAK to FXIIIa was found to be altered differentially by immune versus thrombotic stimulation. Lastly, we studied the effect of thrombin versus Pam(3)CSK(4) stimulation on alpha-granule release and observed differential release patterns for selected granule proteins and decreased fibrin clot formation compared with thrombin. The inhibition of PI3K caused a decrease in protein release after Pam(3)CSK(4)- but not after thrombin-stimulation. In summary, stimulation of platelets by either thrombotic or immune receptors leads to markedly different signalling responses and granular protein release consistent with differential contribution to coagulation and thrombosis.

CHAPTER 13 – Inhibition of Platelet Function by the Endothelium

Platelets circulate in a quiescent state as long as the endothelium remains biochemically and physically intact. Injury of a diseased or healthy vessel wall causes platelet adhesion initiated by the exposure of subendothelial components including von Willebrand factor (VWF), collagen, and fibronectin. Platelets bind to the exposed VWF via the glycoprotein (GP) Ib-IX-V receptor complex (see Chapter 7) that not only anchors the platelets to the injured vessel site but also leads to platelet activation via signaling events that involve the GPIIb-IIIa receptor (integrin αIIbβ3) (see Chapter 8). In addition, several collagen-binding proteins expressed on the platelet surface, such as GPVI, integrin α2β1, and CD36 (see Chapter 6), regulate collagen-induced platelet adhesion, specifi cally under flow conditions (see Chapter 18). These receptors interact in a synergistic way dependent on flow conditions, the extent of damage, and relative exposure of distinct types of collagen or extracellular matrix proteins. Subsequent platelet activation causes secretion of additional agonists such as adenosine diphosphate (ADP), thromboxane A2 (TXA2), and serotonin (see Chapter 15), which then leads to further platelet aggregation by recruitment of additional platelets to the growing thrombus. The formation of thrombin induces contraction and the formation of fi brin leads to the consolidation of the platelet plug.1-3