Application of spray-freezing to the study of rapid platelet reactions by a quenched-flow approach

A spray-freezing approach coupled to quenched-flow techniques has been developed to study rapid (0.3-5.0 s) biochemical and morphological changes during platelet activation. Platelet-rich plasma (PRP) pumped through the quenched-flow system was atomized and quenched within 50 ms in liquid propane at -196 degrees C. Analysis of platelet size and morphology by scanning electron microscopy (SEM) indicated that platelets can be sprayed without morphological damage. The spray-frozen platelets can be subjected to freeze-fracture, freeze-substitution, or freeze-drying procedures for later analysis by electron microscopy. Quenched-flow techniques have permitted investigation of rapid increases in cytosolic calcium protein phosphorylation and morphological changes which occur within 0.5 s after exposure to an activator. The combination of quenched-flow and spray-freezing methodologies should prove useful for studying equally rapid changes in protein distribution in activated platelets by means of electron microscopy coupled with immunocytochemical approaches.