Functional Fractionation of Platelets: Aggregation Kinetics and Glycoprotein Labeling of Differing Platelet Populations

Summary

Platelet heterogeneity has been studied with a technique called functional fractionation which employs gentle centrifugation to yield subpopulations (“reactive” and “less-reactive” platelets) after exposure to small doses of aggregating agent. Aggregation kinetics of the different platelet populations were investigated by quenched-flow aggregometry. The large, “reactive” platelets were more sensitive to ADP (K_a = 1.74 μM) than the smaller “less-reactive” platelets (K_a = 4.08 μM). However, their maximal rate of aggregation (V_max, % of platelets aggregating per sec) of 23.3 was significantly lower than the “less-reactive” platelets (V_max = 34.7). The “reactive” platelets had a 2.2 fold higher level of cyclic AMP.

Platelet glycoproteins were labeled using the neuraminidase-galactose oxidase – [H³]-NaBH₄ technique. When platelets were labeled after reversible aggregation, the “reactive” platelets showed a two-fold decrease in labeling efficiency (versus control platelets). However, examination of whole cells or membrane preparations from reversibly aggregated platelets revealed no significant difference in Coomassie or PAS (Schiff) staining.

These results suggest that the large, “reactive” platelets are more sensitive to ADP but are not hyperaggregable in a kinetic sense. Reversible aggregation may cause a re-orientation of membrane glycoproteins that is apparently not characterized by a major loss of glycoprotein material.

Keywords

Platelets - Functional fractionation - Aggregation kinetics - Membrane glycoproteins

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