Platelet plasma membrane is equally distributed between surface and osmotically-evaginable surface-connecting membrane, independent of size, subpopulation or species

Abstract

Osmotic swelling can double the external plasma membrane surface area of normal human platelets. The surface-connected open canalicular system (SCCS) has been proposed as the major source for this additional membrane. As bovine (B) platelets have been reported to lack SCCS, we compared osmotic swelling for B and human (H) cells. Addition of water to platelet-rich-plasma (10-90% v/v) caused sequential shape change and osmotic spherocyte (OS) formation, analyzed for size and surface area changes from time-dependent phase-contrast videomicroscopic images. Selected samples were fixed and stained with tannic acid prior to osmic acid fixation for visualization of open SCCS by transmission electron microscopy. Bovine platelets required 3-4x less water dilution of PRP than human platelets, with significant OS forming at 20% water addition. Continued water dilution converted 50% of platelets to OS, with maximally stable swelling and no significant lysis for bovine OS up to 60% dilution. Electron micrographs of unactivated discocytes (D) and of optimally-swollen OS showed open SCCS in human D not detectable in any of the swollen platelets, though granules, mitochondria and a small number of vesicles and vacuoles persisted. No evidence for any open SCCS was found for bovine D or OS, though the OS otherwise appeared similar to human OS. Geometric measurements of D and nonlysed OS showed a stable, maximal 2.1 +/- 0.1 fold increase in external plasma membrane surface area with osmotic swelling, identical for different-sized normal human platelets (mean volume V = 2.8-6.8 fl), for abnormally-large platelets (V = 8.7-11.6 fl), or for bovine or rabbit platelets (V = 3-4 fl). As osmotic swelling appears to primarily externalize SCCS in human platelets, the identical relative amounts of internal membrane externalized for bovine platelets is hypothesized to arise from an osmotically more labile, "closed", and structurally simpler SCCS or from a distinct membrane source than in human platelets. It appears that the surface to invaginated ("SCCS") plasma membrane is kept constant for human, bovine and rabbit platelets, independently of platelet production, size-dependent subpopulations, or of platelet ageing.