The hemostatic agent ethamsylate promotes platelet/leukocyte aggregate formation in a model of vascular injury.
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.
Hernandez
Maria Rosa
MR
Department of Hemotherapy and Hemostasis, Hospital Clinic, Barcelone, Spain.
Alvarez-Guerra
Miriam
M
Escolar
Ginés
G
Chiavaroli
Carlo
C
Hannaert
Patrick
P
Garay
Ricardo P
RP
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004979:Ethamsylate / Q000378:metabolism / Q000494:pharmacology*; D005434:Flow Cytometry; D006490:Hemostatics / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000293:injuries; D010974:Platelet Aggregation / Q000187:drug effects*; D011817:Rabbits
D006490:Hemostatics; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D004979:Ethamsylate