Subject(s)
Blood Platelets/metabolism , Centrifugation, Density Gradient/methods , Gene Library , Megakaryocytes/metabolism , Polymerase Chain Reaction/methods , Precipitin Tests/methods , RNA, Messenger/isolation & purification , Blood Platelets/chemistry , Humans , Megakaryocytes/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Platelet shape change is an extremely rapid process mediated by both the calcium-sensitive and p160ROCK pathways. The present study examines how different features of shape change studied by scanning electron microscopy clearly correlate to changes in the pattern of light absorbance measured in an aggregometer. Platelets change shape from the initial 'disc' form by producing: membrane 'blebs', sphere formation (cell-rounding), filopodia extension, and surface membrane folding. The presentation of these features was dramatically slower in the absence of intracellular calcium mobilization. In the presence of the p160ROCK-inhibitor, Y-27632, shape change was initially normal but platelets rapidly transformed back to smooth discs with extended filopodia. The reappearance of the disc shape is reflected by an increase in the amplitude of oscillations in the aggregometer shape change tracing. The kinetics of actin/cytoskeleton association correlated with filopodia formation but not with disc to sphere transformation. Changes in the level of tubulin polymerization correlated with changes from disc to sphere morphology. These experiments are consistent with a role for a RhoA/Rho kinase-regulated pathway in the maintenance of a spherical platelet shape after agonist-dependent activation. Continued disruption of the cytoskeletal microtubule ring, appears to be a Rhokinase-dependent event involved in the transformation of discoid platelets into spheres.
