Toll like receptor 2/1 mediated platelet adhesion and activation on bacterial mimetic surfaces is dependent on src/Syk-signaling and purinergic receptor P2X1 and P2Y12 activation

Abstract

Platelets are considered to have important functions in inflammatory processes as key players in innate immunity. Toll like receptors (TLRs), expressed on platelets, recognize pathogen associated molecular patterns and trigger immune responses. Pathogens are able to adhere to human tissues and form biofilms which cause a continuous activation of the immune system. The authors aimed to investigate how immobilized Pam3CSK4 (a synthetic TLR2/1 agonist) and IgG, respectively, resembling a bacterial focus, affects adhesion and activation of platelets including release of two cytokines, regulated on activation normal T-cell expressed and secreted (RANTES) and macrophage migration inhibitory factor (MIF). The authors also aim to clarify the signaling downstream of TLR2/1 and FcγRII (IgG receptor) and the role of adenine nucleotides in this process. Biolayers of Pam3CSK4 and IgG, respectively, were confirmed by null-ellipsometry and contact angle measurements. Platelets were preincubated with signaling inhibitors for scr and Syk and antagonists for P2X1 or P2Y1 [adenosine triphosphate (ATP), adenosine diphosphate (ADP) receptors] prior to addition to the surfaces. The authors show that platelets adhere and spread on both Pam3CSK4- and IgG-coated surfaces and that this process is antagonized by scr and Syc inhibitors as well as P2X1 and P2Y antagonists. This suggests that Pam3CSK4 activated platelets utilize the same pathway as FcγRII. Moreover, the authors show that ATP-ligation of P2X1 is of importance for further platelet activation after TLR2/1-activation, and that P2Y12 is the prominent ADP-receptor involved in adhesion and spreading. RANTES and MIF were secreted over time from platelets adhering to the coated surfaces, but no MIF was released upon stimulation with soluble Pam3CSK4. These results clarify the importance of TLR2/1 and FcγRII in platelet adhesion and activation, and strengthen the role of platelets as an active player in sensing bacterial infections.

Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation

“…As both Pam3CSK4 and FSL-1 signal through TLR2 heterodimers, while LPS signals through TLR4, this may be due to differential platelet-TLR signal transduction. Platelet-TLR signal transduction has been most well-studied in response to Pam3CSK4, and platelet-TLR2/1 signalling has shown to induce Src- and Syk- family kinases [40, 41], which mimics the signalling pathway of common haemostatic platelet receptors [42]. It may be that platelets can exert greater effects on neutrophils following stimulation with TLR2 agonists due to the ability to recruit or mimic haemostatic receptor pathways.…”

The Role of Human Platelet Preparation for Toll-Like Receptors 2 and 4 Related Platelet Responsiveness

“…Moreover, the stimulation of TLR2 and TLR4 with bacterial ligands has been shown to result in the activation of platelet signaling cascades accompanied by the shedding of contents. 8 17 In this regard, differences in the extent of chemokine releases have been observed. Pam3CSK4 and LPS stimulation led to the release of von Willebrand factor, as α-granule marker, from WP.…”

Phagocytic Receptors Activate Syk and Src Signaling during Borrelia burgdorferi Phagocytosis

“…SFK activity has also been linked to Toll-like receptor 2 (TLR2) inflammatory cytokine signaling (30)(31)(32). We hypothesized that SFKs and Syk may be involved in the internalization of B. burgdorferi, contributing to the intracellular immune responses activated by the bacterium.…”