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

Abstract

Background Like immune cells, platelets express the repertoire of toll-like receptors (TLR), among them TLR2 and TLR4, which are important for the recognition of bacterial patterns. Receptor-mediated functional effects in platelets have been investigated, but reliable conclusions are tampered due to heterogeneous study designs with variable platelet preparation methods. This study compares TLR2- and TLR4-dependent platelet responsiveness in platelet-rich plasma (PRP) and in washed platelets (WPs). Material and Methods Fresh peripheral blood samples from healthy donors served for the preparation of PRP and WP. Basal and agonist-stimulated TLR2 and TLR4 expression levels were evaluated by flow cytometry. Light transmission aggregometry was used to investigate functional effects of TLR2 and TLR4 stimulation with Pam3CSK4 or LPS (lipopolysaccharides from Escherichia coli) as ligands. The capacity of chemokine release was determined by immunoassays. Results Pam3CSK4 and LPS (in combination with thrombin) were able to induce aggregation in WP, but not in PRP, with threshold concentrations of 15 µg/mL. Basal expression levels of TLR2 and TLR4 were higher in WP than in PRP, increasing several-fold rapidly and persistently upon platelet activation with potent agonists. Pam3CSK4 (15 µg/mL) or LPS led to the submaximal release of RANTES, PF4, PDGF, NAP-2, and sCD40L from WP. In PRP, secretory effects are less pronounced for RANTES, PDGF, or PF4, and not detectable for NAP-2 or sCD40L. Conclusion The effects mediated by TLR2 and TLR4 stimulation are dependent on platelet preparation, an important issue for experimental designs and for manufacturing of platelet concentrates in transfusion medicine.

Effects of bacterial lipopolysaccharides on platelet function: inhibition of weak platelet activation

“…S7). This corresponded with the results from 5,14,35 , while being in disagreement with 6,10,12 . Using continuous flow cytometry of platelet activity upon weak stimulation, we determined that incubation with LPS was capable of inhibition of platelet responses to weak activation potentially via cyclicnucleotide dependent signaling pathways (Figs.…”

“…Most of the reports on LPS-mediated platelet activation were based on the experiments with washed platelets 6 , 10 , whereas, in citrated PRP (calcium-free), LPS did not alter platelet aggregation 6 . However, based on our findings here, in the case of reversible platelet aggregation (Fig.…”

“…Platelet reactivity to LPS could be caused by activation of IKκ-β involved in phosphorylation of synaptosomal-associated protein 23 (SNAP23), which controls platelet alpha-granule release 8 . However, this was in disagreement with LPS capability to enhance aggregation of washed platelets, stimulated by thrombin, but without any effect on aggregation in platelet-rich plasma (PRP) 6 . Furthermore, there is a considerable amount of data that LPS effects on platelets are elusive 5 , 14 .…”

“…TLR4 was detected on the surface of myeloid cell line: granulocytes, monocytes, dendric cells 4 . TLR4 is also expressed on the surface of platelets 5 , 6 —anucleate blood cells, responsible for hemostasis 7 . In order to recognize LPS, TLR4 requires such cofactor proteins as CD14, myeloid-differentiation factor 2 (MD2), and LPS binding protein (LBP) 8 .…”

“…LPS mediated TLR4 activation is claimed to enhance platelet stimulation by ADP and CRP 6 , 10 , 11 . LPS also were shown to induce increased oxygen consumption by platelet mitochondria 5 , 12 .…”