Mechanism of shape change in chilled human platelets

The so-called cold activation of platelets that precludes refrigeration of platelets for storage has long been recognized, but its mechanism has remained a mystery. Cooling of discoid resting platelets to temperatures below 15 degrees C causes shape distortions, and the chilled cells rewarmed to above 25 degrees C are spheres rather than discs. As platelet shape change responsive to receptor activation at normal temperatures requires the remodeling of an actin scaffolding (Hartwig JH, 1992, J Cell Biol 118:1421–1442), we examined the role of actin in the morphologic changes induced by cooling. The addition of actin monomers onto the fast-exchanging (barbed) ends of actin filaments accompanies the initial physiologic platelet shape changes, and a key control point in this growth is the removal of proteins (caps) from the filament ends. This uncapping of actin filament ends is mediated by polyphosphoinositide aggregates in vitro, suggesting that cold-induced phase changes in membrane lipids might uncap actin filaments and thereby account for actin assembly-mediated shape alterations during cooling. Consistent with this hypothesis, reversible inhibition of actin assembly with cytochalasin B prevented the distortions in shape, although cooled platelets had increased actin nucleation sites and became spherical. Another step in normal platelet shape changes requires the severing of actin filaments that maintain the resting platelet. The proteins that sever initially bind to the broken filament ends, and uncapping of these fragmented filaments provides numerous nucleation sites for growth of actin filaments to fill in spreading filopodia and lamellae. Actin filament fragmentation requires a rise in intracellular calcium, and we showed that chilling platelets from 37 degrees C to 4 degrees C increases free cytosolic calcium levels from 80 nmol/L to approximately 200 nmol/L in minutes, thus providing an explanation for the spherical shape of cooled, rewarmed platelets. Blocking the calcium transient with nanomolar concentrations of the permeant calcium chelators Quin-2 and Fura-2 prevented the increase in nucleation sites and the sphering, but not the other shape changes of chilled and rewarmed platelets. However, a combination of micromolar cytochalasin B and millimolar intracellular calcium chelators preserved the discoid shapes of chilled and rewarmed platelets. After removal of cytochalasin B and addition of sufficient extracellular calcium, these platelets responded with normal morphologic alterations to glass and thrombin activation.