Research On Mechanism Of Activated Platelets In Inflammatory Reaction After Acute Ischemic Stroke

ObjectiveBy evaluated the level of platelets activation in patients with acute ischemic stroke and the influence of activated platelets induced by ADP on expressing of chemokine (IL-8, MCP-1 and MIP-1α) in human umbilical vein endothelial cells (HUVECs) cultured in vitro, explore the pathomechanism of activated platelets involving in inflammatory reaction in acute ischemic stroke.Methods.1 To detect activation of platelets in 60 patients with acute ischemic stroke, and mean platelet component (MPC), as a new index, were used as a marker of platelet activation measured by Bayer Advia 120 Hematology system.2 Peripheral blood from 30 healthy people were taken and platelet suspension were prepared with the method used by Ahnadi CE etc. Three concentrations of ADP (0.8μmol/l, 4.0μmol/1,20.0μmol/l) were added in and MPC were measured in different time interval. Then, level-effect tie was established. Thus, the activation of platelets of peripheral blood of acute ischemic stroke patients could be deducted.3 Frozen HUVECs (ECV304) was revived and subcultured. Mixed platelet suspension was prepared with peripheral blood from healthy people and divided into the following 4 experimental groups: (1) activated platelets group: achieved by mixing 0.8 umol/l (final concentration) ADP and platelets for 20min at room temperature and incubating with HUVECs. (2) Resting platelets group: achieved by resting platelets suspension for 20min at room temperature and incubating with HUVECs. (3) ADP control group. (4) Blank control group. Cells from each group were incubated for 12 hours at 37℃in circumstance of 5%CO₂. RNA was extracted with TRNzol. Expressions of IL-8, MCP-1 and MIP-1αmRNA in endothelial cell were detected by RT - PCR technology.Results1 The average level of MPC in patients with acute ischemic stroke was 21.46± 3.44g/dl. The difference with healthy subjects in control group was statistically significant, t=32.24, P＜0.01.2 The decreases in MPC of platelets induced by ADP had dependence with the dosage. Platelets with normal activation degree could be achieved by inducting with small dose (0.8umol/l) of ADP for 20 min. Then the level of activation of platelets in acute ischemic stroke patients could be modeled.3 When cultured with HUVECs, activated platelets could stimulate expressing of chemokines (IL-8,MCP-1 and MIP-1αmRNA) in endothelial cell. While both resting platelets and ADP with the same content could not stimulate expressing of IL-8, MCP-1 and MIP-1αmRNA.ConclusionPlatelets in peripheral blood of acute ischemic stroke patients should be at activated state. The activated platelets achieved in vitro could involve in inflammatory reaction through stimulating chemokines (IL-8, MCP-1 and MIP-1α)in endothelial cell of vessels. This kind of platlet-endothlium reaction may be an important pathomechanism of inflammatory reaction in acute ischemic stroke.