Correlated measurement of platelet release and aggregation in whole blood

Abstract

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two‐color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP‐140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP‐140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 μm. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)‐induced aggregates. After disaggregation the proportion of GMP‐140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)‐induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation. The ability to measure such aggregates both in vitro and in vivo may be very useful.