Measurement of Amount of Invaginated Membrane in Mammalian Platelets by Osmotically-Induced Spherocyte Formation

Invaginated membrane of the surface-connected canalicular system (SCCS) of the resting platelet can be potentially externalized. We have estimated the SCCS surface area by carefully swelling platelets in hypotonic media. Platelets were osmotically stressed by either interfacing a drop of platelet-rich plasma (PRP) with distilled water on a siliconized glass slide or directly in a test tube by careful additions of 60-80% by volume water. Surface areas of the platelets were estimated from a cinematographic analysis of freely rotating platelets. Within five minutes of the addition of water, large smooth spheres (spherocytes) are formed. Soluble, cytoplasmic lactate dehydrogenase activity is not released and visible membrane damage as estimated from dark-field microscopy observations does not occur for at least one hour. The mean value of the spherocyte diameters was 3.9 ± 0.6 um (3 human donors) and did not change over 5 - 60 minutes. It is estimated that the volume of the spherocyte is ~550% greater than that of the original disc and that the surface area has increased by ~300%. Similar results were obtained for rabbit platelets.Since platelet swelling induced osmotically has been shown to yield large spheres with the disappearance of SCCS (J.G. White, personal communication), it is concluded that: 1) the SCCS of the resting platelet can be easily externalized, and 2) osmotic spherocyte formation allows a quantitative estimation of the SCCS surface area.