PLATELET AGGREGATES AS MARKERS OF PLATELET ACTIVATION - CHARACTERIZATION OF FLOW CYTOMETRIC METHOD SUITABLE FOR CLINICAL-APPLICATIONS

The present paper describes a flow cytometric method for assay of plat elet aggregates (PAg) in blood, This method combines and simplifies pr eviously reported techniques, simultaneously enumerating PAg formed up on platelet activation, their expression of activation marker CD62P (P -selectin), and their content of bound leukocytes (LPAg). The sensitiv ity of this method to low levels of agonists (ADP, collagen) is compar ed to conventional aggregometry and some features of platelet-leukocyt e interaction are explored. The results were: (1) ADP or collagen indu ced a dose-dependent increase in PAg number and corresponding decline in free platelets. The ED50 for ADP (0.15 mu M) and for collagen (0.2 mu g/mL) was about 1/20 the ED50 found by aggregometry, indicating 20- fold greater sensitivity. (2) At higher concentrations, the fraction o f PAg with bound leukocytes (LPAg) increased to 60-70%. This rise corr elated with PAg size and CD62P expression, but not with the number of PAg formed. (3) The response of whole blood (WBD) to agonists was qual itatively different from that of platelet-rich plasma (PRP): in WBD th e population of CD62P+ PAg was much higher than in PRP and the populat ion of CD62P+ free platelets was much lower. This implies that leukocy tes rapidly recruit activated platelets. (4) Desmopressin (DDAVP) at 5 nmol/L induced a significant rise in activated (CD62P+) PAg and plate lets, even though no effect of DDAVP could be detected by conventional aggregometry; this further confirms that DDAVP acts directly on plate lets, (5) Plasma samples from TTP patients induced a rise in PAg when added to normal PRP, though little or no effect could be detected by a ggregometry. In summary, the flow cytometric method described here app ears useful for detecting low levels of platelet activation and provid es information on platelet leukocyte interaction, potentially importan t in identifying and differentiating thrombogenic states. Since it is rapid and economical, it is well suited for clinical implementation. ( C) 1998 Wiley-Liss, Inc.