Xiao Zhihua, Goldsmith Harry L, McIntosh Fiona A, Shankaran Harish, Neelamegham Sriram
Department of Chemical and Biological Engineering, State University of New York, Buffalo, New York, 14260, USA.
Biophys J. 2006 Mar 15;90(6):2221-34. doi: 10.1529/biophysj.105.065789. Epub 2005 Dec 30.
Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP)-stimulated platelets or P-selectin-bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14 to 3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that although blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by approximately 60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though in synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 caused dissociation of previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a shear threshold for P-selectin PSGL-1 binding was also noted at shear rates <100/s when Ps-beads collided with isolated neutrophils. Results are discussed in light of biophysical computations that characterize the collision between unequal-size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion and weak shear threshold for P-selectin PSGL-1 interactions that may be physiologically relevant.
血小板与白细胞的黏附可能会导致血栓形成和炎症。我们研究了未激活的中性粒细胞与凝血酶受体激活肽（TRAP）刺激的血小板或携带P-选择素的磁珠（Ps-磁珠）在悬浮液中的异型相互作用。使用锥板粘度计施加14至3000/s的可控剪切速率。使用流式细胞术和高速视频显微镜对血小板-中性粒细胞和磁珠-中性粒细胞的黏附进行分析。我们观察到，尽管单独使用针对P-选择素或P-选择素糖蛋白配体-1（PSGL-1）的阻断抗体在140/s时可使血小板-中性粒细胞黏附抑制约60%，但这些试剂在3000/s时可完全阻断黏附。单独使用抗Mac-1在任何剪切速率下都不会改变血小板-中性粒细胞的黏附率，不过与选择素拮抗剂协同作用时可消除细胞结合。未刺激的中性粒细胞以不依赖整合素的方式 avidly 结合Ps-磁珠和活化的血小板，这表明纯粹依赖选择素的细胞黏附是可能的。支持这一点的是，针对P-选择素或PSGL-1的拮抗剂在各种实验系统中，包括在全血检测中，会导致先前形成的血小板-中性粒细胞和Ps-磁珠-中性粒细胞聚集体在剪切力作用下解离。在改变介质粘度和剪切速率的研究中，当Ps-磁珠与分离的中性粒细胞碰撞时，在剪切速率<100/s时也注意到了P-选择素-PSGL-1结合的剪切阈值。根据描述线性剪切流中不等大小颗粒碰撞的生物物理计算对结果进行了讨论。总体而言，我们的研究揭示了细胞黏附的不依赖整合素机制以及P-选择素-PSGL-1相互作用的弱剪切阈值，这可能具有生理相关性。
