Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, California, USA.
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, California, USA.
BMC Vet Res. 2019 Jul 15;15(1):245. doi: 10.1186/s12917-019-1997-3.
Functional Toll-like receptor 4 (TLR4) has been characterized in human and murine platelets indicating that platelets play a role in inflammation and hemostasis during sepsis. It is unclear whether canine platelets could express functional TLR4 by responding to its ligand, lipopolysaccharide (LPS). We sought to determine if dogs express functional TLR4 and if LPS-induced platelet activation requires co-stimulation with ADP or thromboxane A (TxA). Canine platelets were unstimulated (resting) or activated with thrombin or ADP prior to flow cytometric or microscopic analyses for TLR4 expression. We treated resting or ADP-primed platelets with LPS in the absence or presence of acetylsalicylic acid (ASA) and inhibited TLR4 with function blocking antibody or LPS from Rhodobacter sphaeroides (LPS-RS).
We discovered that dog platelets have variable TLR4 expression, which was upregulated following thrombin or ADP activation. LPS augmented P-selectin expression and thromboxane B secretion in ADP-primed platelets via TLR4. Inhibition of cyclooxygenase by ASA attenuated LPS-mediated P-selectin expression demonstrating that TLR4 signaling in platelets is partially dependent on TxA pathway.
Expression of functional TLR4 on canine platelets may contribute to hypercoagulability in clinical septic dogs. Cyclooxygenase and TxA pathways in TLR4-mediated platelet activation may present novel therapeutic targets in dogs with sepsis.
功能性 Toll 样受体 4（TLR4）已在人和鼠类血小板中得到鉴定，表明血小板在败血症期间的炎症和止血中发挥作用。尚不清楚犬类血小板是否可以通过对其配体脂多糖（LPS）的反应来表达功能性 TLR4。我们试图确定犬类是否表达功能性 TLR4，以及 LPS 诱导的血小板激活是否需要与 ADP 或血栓烷 A（TxA）共同刺激。在流式细胞术或显微镜分析 TLR4 表达之前，未刺激（静止）或用凝血酶或 ADP 激活犬类血小板。我们在没有或存在乙酰水杨酸（ASA）的情况下用 LPS 处理静止或 ADP 预激活的血小板，并使用功能阻断抗体或来自 Rhodobacter sphaeroides（LPS-RS）的 LPS 抑制 TLR4。
我们发现犬类血小板具有可变的 TLR4 表达，在凝血酶或 ADP 激活后上调。LPS 通过 TLR4 增强 ADP 预激活血小板中的 P-选择素表达和血栓烷 B 分泌。ASA 抑制环氧化酶减弱了 LPS 介导的 P-选择素表达，表明血小板中 TLR4 信号通路部分依赖于 TxA 途径。
犬类血小板上功能性 TLR4 的表达可能导致临床败血症犬的高凝状态。TLR4 介导的血小板激活中环氧化酶和 TxA 途径可能为败血症犬提供新的治疗靶点。
