Koessler Juergen, Niklaus Marius, Weber Katja, Koessler Angela, Kuhn Sabine, Boeck Markus, Kobsar Anna
Institute of Clinical Transfusion Medicine and Haemotherapy, University of Würzburg, Würzburg, Germany.
TH Open. 2019 Apr 17;3(2):e94-e102. doi: 10.1055/s-0039-1685495. eCollection 2019 Apr.
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). 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 ) as ligands. The capacity of chemokine release was determined by immunoassays. 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. 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.
与免疫细胞一样，血小板表达一系列Toll样受体（TLR），其中TLR2和TLR4对识别细菌模式很重要。已经研究了受体介导的血小板功能效应，但由于采用不同血小板制备方法的异质性研究设计，可靠的结论受到影响。本研究比较了富血小板血浆（PRP）和洗涤血小板（WP）中TLR2和TLR4依赖性血小板反应性。 来自健康供体的新鲜外周血样本用于制备PRP和WP。通过流式细胞术评估基础和激动剂刺激的TLR2和TLR4表达水平。采用光透射聚集法研究以Pam3CSK4或LPS（来自 的脂多糖）作为配体刺激TLR2和TLR4的功能效应。通过免疫测定法测定趋化因子释放能力。 Pam3CSK4和LPS（与凝血酶联合使用）能够在WP中诱导聚集，但在PRP中不能，阈值浓度为15μg/mL。WP中TLR2和TLR4的基础表达水平高于PRP，在用强效激动剂激活血小板后迅速且持续增加数倍。Pam3CSK4（15μg/mL）或LPS导致WP中RANTES、PF4、PDGF、NAP-2和sCD40L的次最大释放。在PRP中，RANTES、PDGF或PF4的分泌效应不太明显，而NAP-2或sCD40L则无法检测到。 TLR2和TLR4刺激介导的效应取决于血小板制备，这是实验设计和输血医学中血小板浓缩物制造的一个重要问题。
