Hernandez Maria Rosa, Alvarez-Guerra Miriam, Escolar Ginés, Chiavaroli Carlo, Hannaert Patrick, Garay Ricardo P
Department of Hemotherapy and Hemostasis, Hospital Clinic, Barcelone, Spain.
Fundam Clin Pharmacol. 2004 Aug;18(4):423-30. doi: 10.1111/j.1472-8206.2004.00256.x.
The hemostatic agent ethamsylate enhances membrane expression of P-selectin in human platelets, but whether this promotes platelet-leukocyte aggregate formation is unknown. Here we investigated this point by flow cytometry determination of human platelet-leukocyte aggregates under basal conditions and after whole-blood perfusion through a damaged rabbit aorta segment. Actions of ethamsylate on adhesive molecules of platelets and leukocytes were investigated in parallel. Under basal conditions, ethamsylate was unable to modify whole-blood platelet-leukocyte aggregation, but following whole-blood perfusion through a damaged vessel, ethamsylate produced a modest, but significant increase in platelet-leukocyte aggregates (48+/-21 and 45+/-26% above control levels at ethamsylate 20 and 40 microm respectively). In isolated leukocyte plasma membranes, 14C-ethamsylate specifically bound up to an amount of 660 pmol/mg protein. Moreover, at concentrations > or =1 microm, ethamsylate induced an important (100-200%) and significant increase in the P-selectin glycoprotein ligand 1 (PSGL-1) fluorescence signal in isolated leukocytes and was unable to significantly modify the percentage of CD11b-positive cells. However, no significant changes in aggregate formation were found when ethamsylate was incubated with isolated leukocytes and blood was reconstituted and perfused. In isolated platelet cell membranes, anti-P-selectin antibody and the anti-integrin RGD-containing pentapeptide (GRDGS) were unable to displace 14C-ethamsylate binding. In conclusion, ethamsylate specifically binds to plasma membranes of leukocytes, enhances membrane PSGL-1 expression and promotes leukocyte-platelet aggregation in whole-blood perfused through a damaged vascular segment. These results together with the previously observed enhancement of platelet P-selectin membrane expression [Thromb. Res. (2002)107:329-335] confirms and extends the view that ethamsylate acts on the first step of hemostasis, by improving platelet homo- and heterotypic adhesiveness.
止血剂酚磺乙胺可增强人血小板中P-选择素的膜表达，但这是否会促进血小板-白细胞聚集体的形成尚不清楚。在此，我们通过流式细胞术测定基础条件下以及全血灌注受损兔主动脉段后的人血小板-白细胞聚集体来研究这一问题。同时研究了酚磺乙胺对血小板和白细胞黏附分子的作用。在基础条件下，酚磺乙胺无法改变全血中血小板-白细胞的聚集，但在全血灌注受损血管后，酚磺乙胺使血小板-白细胞聚集体有适度但显著的增加（酚磺乙胺浓度为20和40 μmol时，分别比对照水平高48±21%和45±26%）。在分离的白细胞质膜中，14C-酚磺乙胺特异性结合量高达660 pmol/mg蛋白质。此外，在浓度≥1 μmol时，酚磺乙胺可使分离的白细胞中P-选择素糖蛋白配体1（PSGL-1）荧光信号显著增强（100%-200%），且无法显著改变CD11b阳性细胞的百分比。然而，当酚磺乙胺与分离的白细胞孵育后重新构建并灌注血液时，未发现聚集体形成有显著变化。在分离的血小板细胞膜中，抗P-选择素抗体和含抗整合素RGD的五肽（GRDGS）无法取代14C-酚磺乙胺的结合。总之，酚磺乙胺特异性结合白细胞质膜，增强膜PSGL-1表达，并促进全血灌注受损血管段时白细胞与血小板的聚集。这些结果与之前观察到的血小板P-选择素膜表达增强[《血栓研究》（2002年）107:329-335]一起，证实并扩展了酚磺乙胺通过改善血小板同种和异型黏附性作用于止血第一步的观点。
