Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, the Netherlands (C.S., S.L.N.B., J.M.E.M.C., R.R.K., S.P.W., M.J.E.K., J.W.M.H., M.N.).
Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg-University of Mainz, Germany (C.S., K.J.).
Arterioscler Thromb Vasc Biol. 2023 Sep;43(9):1700-1712. doi: 10.1161/ATVBAHA.122.318767. Epub 2023 Jul 6.
Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches.
Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbβ3.
We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca] rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide.
Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.
血小板和中性粒细胞是首先聚集在动脉血栓形成部位的血细胞，这两种细胞类型都有助于血栓形成事件的病理过程。我们旨在使用微流控方法鉴定这些细胞之间的关键相互作用机制。
在动脉剪切率下，全血在胶原表面进行灌注。使用荧光标记物在显微镜下可视化血小板和白细胞（主要是中性粒细胞）的激活。通过使用抑制剂或抗体，以及使用缺乏血小板表达的 αIIbβ3 的 GT（Glanzmann 血小板无力症）患者的血液，研究了血小板黏附受体（整合素、P-选择素、CD40L）和趋化因子的作用。
我们观察到（1）激活的血小板整合素 αIIbβ3 具有未知的阻止白细胞黏附的作用，这种作用被短暂的流动干扰所克服，从而导致大量黏附；（2）血小板表达的 CD40L 控制着细胞在血栓上的爬行模式和血栓的保真度；（3）血小板释放的物质持续促进已识别的中性粒细胞的激活，这可以通过（fMLP[甲酰甲硫氨酸亮氨酸苯丙氨酸，一种有效的趋化因子和白细胞激活剂]诱导的）[Ca]上升和抗原表达来评估；（4）血小板释放的趋化因子按 CXCL7>CCL5>CXCL4 的顺序激活黏附细胞。此外，在血栓中沉默血小板后，抑制了白细胞的激活。然而，在没有用佛波酯或脂多糖刺激的情况下，血栓上的白细胞也只能有限地形成中性粒细胞细胞外陷阱。
总的来说，这些发现揭示了血小板在血栓中对中性粒细胞黏附和激活的多方面调节，其中几种血小板黏附受体起着平衡的作用，血小板释放的物质起着促进作用。中性粒细胞-血栓相互作用的这种多价性质为药物干预提供了新的前景。
