Procoagulant platelet balloons: evidence from cryopreparation and electronmicroscopy

Visualisation of the procoagulant transformation of human platelets has rec ently become possible through use of an in vitro approach combined with flu orescence and phase contrast microscopy. Here, we extended these studies to the ultrastructural level by employing both rapid freezing/freeze-substitu tion and conventional ambient-temperature chemical fixation for transmissio n and scanning electron microscopy. Procoagulant transformation was only in ducible by adhering platelets to collagen fibrils or to the collagen-relate d peptide and exposing them to physiological extracellular Ca2+ levels. Und er these conditions prominent, 2- to 4-mum-wide balloon-like structures wer e regularly observed, regardless of the specimen fixation protocol. In stro ng contrast to normal platelets in their vicinity, the balloons' subcellula r architecture proved remarkably poor: dilute cytoplasm, no cytoskeleton, o nly a few, randomly distributed organelles and/or their remnants. Cryofixed balloons displayed intact and smooth surfaces whereas conventional specime n processing caused plasma membrane perforations and shrinkage of the ballo ons. Our results clearly show that neither the balloons themselves, nor the ir simple ultrastructure reflect fixation artefacts caused by inadequate me mbrane stabilisation. The balloons are interpreted as to be transformed and /or fragmented procoagulant platelets. Thus, the generation of balloons rep resents a genuine, final stage of platelet ontogenesis, presumably occurrin g alternatively to aggregate formation.