FLOW CYTOMETRIC STUDIES OF PLATELET RESPONSES TO SHEAR-STRESS IN WHOLE-BLOOD

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and -plate viscometer in the absence of added agonists. Blood specimens we re fixed with formaldehyde and incubated with two monoclonal antibodie s. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb) , was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC flu orescence profiles. Anti-CD62 antibody, specific for the granule membr ane protein-140 (GMP-140), was used to measure platelet activation. Re sults showed platelet aggregation increasing with increasing shear str ess with marked increase in this response for a pathophysiological str ess level of 140 dyn/cm(2) and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (''large '' refers to particles larger than 10 mu m in equivalent sphere diamet er). These platelet responses to shear stress were insensitive to aspi rin, but strongly inhibited by agents that elevate platelet cyclic ade nosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor bi nding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb /IIIa inhibited platelet aggregation. Aggregation induced by shear at 37 degrees C was less in extent than at 23 degrees C, At physiological shear stresses, whole blood was more susceptible to shear-induced pla telet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole bl ood unaltered by cell separation methods.