Flow Cyometric Studys Of Platelets Activaion By Shear Stress

Objective The present study was conducted to observe high shear stress directly activate platelets in a modified cone-and-plate viscometer by using a flow cytometric technique and to investigate the mechanisms of shear stress- induced platelets activation The study include shear stress-induced platelets aggregation(SIPA), the receptors and blood plasma medium involved in the aggregation, the expression of glycoprotein Tb/TX, Jib/lila complex and two kinds of platelet activation markers P-selectin(CD62P) and activated glycoprotein Ⅱb/Ⅲa(PAC-1), transmission signal of calcium influx. Moreover, we have detected platelets aggregation induced by high shear stress in some arterial thrombogenesis diseases in order to find the value in clinical use Methods Venous blood from normal subjects who were free from drugs in two weeks After discarding first two to three milliliters, the blood were mixed with 1/10 volume of 3.8% sodium citrate, PRP and PPP were prepared by routine method (1)Whole blood of healthy volunteers was subjected to a series of shear stress, and stained by fluorescence staining with a CD61 PerCP monoclonal antibody SIPA was detected by FCM (2) Detect SIPA induced by shear stress of the disposed whole blood with the following factors 20μg/ml purified monoclonal antibodies and RGDS associated with platelets activation mixed with the whole blood, incubated 10 min at 37℃, mixed with ADP or epinephrine and incubated 1 min at 37 ℃, washing the whole blood or adding calcium ion and EGTA respectively, incubated with aspirin or ticlopidine at 37℃ for 30 mm (3)Diluted the whole blood five-fold with itself PPP, then detected the expression of glycoprotein Ⅰb/Ⅸ, Ⅱb/Ⅲa, CD62P and PAC-1 by FCM (4)Loaded the whole blood with a 20μmol/L final concentration Fluo-3/AM, and diluted five-fold in itself PPP, measured the calcium ion level in platelets by FCM (5)Detected SLPA and expression level of some platelet activation markers such as P-selectin and PAC-1 in some arterial thrombogenesis diseases including AMI, cerebra -4- infarction, transient ischemic attack (TIA ), diabetes mellitus with vesselaffection and MDSReSults l Shear stress-induced whole blood platelets aggregationincreases with the increasing of shear stress and prolonging of time Onlya small amount of platelet aggregation can be detected at relatively lowshear stress of l00S-' However, a significant increasing in SIPA occurs at2000S-l and exposure for 3 min, which close to maximum 2 High shearstress-induced platelet aggregation was decreased obviously afterinterfering the binding of vWF to its two platelet receptors with theirspecific monoclonal antibodies The decrease was slight while blockingthe binding of Fg to GP nb/IIIa The presence of extracellular calciumions enhance SIPA, while the effect of EGTA is inhibitory Low dose ADPor epinephrine can hardly induce platelet aggregation respectively, butthere is synergistic effect when they act with high shear stress Classicalanti-platelet drug such as aspirin failed to withhold SIPA, but ticlopidineshow obviously inhibitory effect on it 3 No significant changes ofGpIb/IX expressing on the surface of platelet were measured at relativelylow shear stress When the stress increase, it rise to some extent, thendecrease continually GpIIb/IIIa begin to increase at first under low shearstress effect When the stress was increasing, it rised quickly in a shorttime, and begain to decrease at 7th min PAC-l can express under eitherhigh stress or low stress, but it decrease quickly The expression of CD62Pis not powerful at first, then straightly increased u...