MECHANISM OF SHAPE CHANGE IN CHILLED HUMAN PLATELETS

The so-called cold activation of platelets that precludes refrigeratio n of platelets for storage has long been recognized, but its mechanism has remained a mystery. Cooling of discoid resting platelets to tempe ratures below 15 degrees C causes shape distortions, and the chilled c ells rewarmed to above 25 degrees C are spheres rather than discs. As platelet shape change responsive to receptor activation at normal temp eratures 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 monomer s onto the fast-exchanging (barbed) ends of actin filaments accompanie s the initial physiologic platelet shape changes, and a key control po int in this growth is the removal of proteins (caps) from the filament ends. This uncapping of actin filament ends is mediated by polyphosph oinositide aggregates in vitro, suggesting that cold-induced phase cha nges in membrane lipids might uncap actin filaments and thereby accoun t for actin assembly-mediated shape alterations during cooling. Consis tent with this hypothesis, reversible inhibition of actin assembly wit h cytochalasin B prevented the distortions in shape, although cooled p latelets had increased actin nucleation sites and became spherical. An other step in normal platelet shape changes requires the severing of a ctin filaments that maintain the resting platelet. The proteins that s ever initially bind to the broken filament ends, and uncapping of thes e fragmented filaments provides numerous nucleation sites for growth o f actin filaments to fill in spreading filopodia and lamellae. Actin f ilament fragmentation requires a rise in intracellular calcium, and we showed that chilling platelets from 37 degrees C to 4 degrees C incre ases free cytosolic calcium levels from 80 nmol/L to approximately 200 nmol/L in minutes, thus providing an explanation for the spherical sh ape 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, b ut not the other shape changes of chilled and rewarmed platelets. Howe ver, a combination of micromolar cytochalasin B and millimolar intrace llular calcium chelators preserved the discoid shapes of chilled and r ewarmed platelets. After removal of cytochalasin B and addition of suf ficient extracellular calcium, these platelets responded with normal m orphologic alterations to glass and thrombin activation. (C) 1995 by T he American Society of Hematology.