Evangelista V, Manarini S, Coller B S, Smyth S S
G. Bizzozero Laboratory of Blood and Vascular Cell Interactions, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy.
J Thromb Haemost. 2003 May;1(5):1048-54. doi: 10.1046/j.1538-7836.2003.00214.x.
The initial interaction of human polymorphonuclear leukocytes (PMN) with activated human platelets is mediated by P-selectin and its leukocyte ligand PSGL-1; subsequently the interaction is strengthened by activation of alphaMbeta2 via protein tyrosine phosphorylation mediated by Src kinases and binding of activated alphaMbeta2 to its platelet counterreceptor(s).
Because mouse models are being used to define the role of PMN-platelet interactions in thrombosis and the response to vascular injury, we investigated the molecular determinants responsible for the interaction of murine PMNs with activated murine platelets.
Mouse platelets were labeled with the green fluorescent dye BCECF and then activated with thrombin and fixed with 1% paraformaldehyde. Mouse PMNs were labeled with the red fluorescent dye hydroethidine and then stirred with the fixed platelets. After stopping the reaction with paraformaldehyde, formation of mixed cell conjugates was analyzed by flow cytometry.
In time course experiments, 90 +/- 1.9% of PMNs formed mixed conjugates with platelets after 2 min and the mean (+/- SEM) number of platelets per positive PMN was 8.4 +/- 1.5. A monoclonal antibody to P-selectin reduced the percentage of PMNs with attached platelets to 16 +/- 2.4% (P = 0.001), and only 8 +/- 5% of PMNs interacted with platelets from P-selectin-/- mice. In contrast, monoclonal antibodies to PSGL-1, beta2-integrin, and alphaIIbbeta3 had much less or no effect on the production of mixed cell aggregates. To better identify a secondary contribution of beta2-integrins, P-selectin interactions were disrupted by briefly adding 5 mm EGTA to already-formed mixed cell aggregates. Brief EGTA treatment alone reduced the percentage of PMNs with attached platelets to 70 +/- 3.5% (P = 0.004 vs. no treatment), but did not modify the number of platelets per positive PMN (9.5 +/- 1.7). The combination of brief EGTA treatment and a monoclonal antibody to beta2-integrin lowered the percentage of PMN with attached platelets to 50 +/- 7% and reduced the number of platelets attached per positive PMN to 3.6 +/- 0.7 (P = 0.03 vs. brief EGTA treatment only). Brief EGTA treatment did not modify the effect of the other antibodies. When the incubation was stopped with EGTA the Src inhibitors PP1 and PP2 reduced PMN-platelet adhesion, while the inactive analog PP3 was ineffective.
These results confirm that P-selectin plays a prominent role in mediating the initial interactions between mouse PMN and platelets, and provide support for additional contributions from beta2-integrins and Src family kinases.
人类多形核白细胞（PMN）与活化的人类血小板的初始相互作用由P-选择素及其白细胞配体PSGL-1介导；随后，通过Src激酶介导的蛋白酪氨酸磷酸化激活αMβ2以及活化的αMβ2与其血小板反受体的结合，这种相互作用得以加强。
由于正在使用小鼠模型来确定PMN-血小板相互作用在血栓形成和对血管损伤的反应中的作用，我们研究了负责小鼠PMN与活化小鼠血小板相互作用的分子决定因素。
用绿色荧光染料BCECF标记小鼠血小板，然后用凝血酶激活并用1%多聚甲醛固定。用红色荧光染料氢乙锭标记小鼠PMN，然后与固定的血小板搅拌。在用多聚甲醛终止反应后，通过流式细胞术分析混合细胞共轭物的形成。
在时间进程实验中，90±1.9%的PMN在2分钟后与血小板形成混合共轭物，每个阳性PMN的血小板平均（±SEM）数为8.4±1.5。一种针对P-选择素的单克隆抗体将附着血小板的PMN百分比降低到16±2.4%（P = 0.001），只有8±5%的PMN与来自P-选择素基因敲除小鼠的血小板相互作用。相比之下，针对PSGL-1、β2整合素和αIIbβ3的单克隆抗体对混合细胞聚集体的产生影响小得多或没有影响。为了更好地确定β2整合素的次要作用，通过向已经形成的混合细胞聚集体中短暂添加5 mM EGTA来破坏P-选择素相互作用。单独短暂的EGTA处理将附着血小板的PMN百分比降低到70±3.5%（与未处理相比，P = 0.004），但没有改变每个阳性PMN的血小板数量（9.5±1.7）。短暂的EGTA处理和一种针对β2整合素的单克隆抗体的组合将附着血小板的PMN百分比降低到50±7%，并将每个阳性PMN附着的血小板数量减少到3.6±0.7（与仅短暂的EGTA处理相比，P = 0.03）。短暂的EGTA处理没有改变其他抗体的作用。当用EGTA终止孵育时，Src抑制剂PP1和PP2降低了PMN-血小板的粘附，而无活性的类似物PP3无效。
这些结果证实P-选择素在介导小鼠PMN与血小板之间的初始相互作用中起重要作用，并为β2整合素和Src家族激酶的额外作用提供了支持。
