When human platelets are chilled to 4oC, they undergo shape change, an increase in net F-actin and intracellular calcium. Here, we show that cold-induced platelet activation is charcterized by the clustering of rafts into large aggregates, and suggest that it is an early event in cold-induced signaling. Rafts (detergent resistant membranes) were isolated using detergent extraction and sucrose flotation and contained 20% sphingomylein and 40% cholesterol. Treated with methyl-beta cyclodextrin depleted 95% of cholesterol from rafts, disrupting their liquid ordered phase. Fourier transform infrared analysis demonstrated an increase in the conformational disorder of detergent resistant membranes (rafts) after such treatment. In order to demonstrate a direct correlation between biochemical studies and temporal events at the plasma membrane, platelets were labeled with the lipophilic dye dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate) that preferentially partitions into ordered domains. Rafts coalesced into microscopically visible aggregates upon cold-activation. This reversible process correlated with passage of platelets through their phase transition. Raft aggregates dispersed upon rewarming of platelets. To ensure that raft aggregation was not an artifact of chilling, we determined the effect of platelet agonists at physiological temperature and observed a similar clustering of rafts into larger domains. Under all conditions, domains were disrupted by the removal of cholesterol with cyclodextrin. Unlike CD41/61 and CD55, only the thrombospondin receptor (CD 36) biochemically partitioned into the raft fraction and demonstrated a virtually identical staining pattern to that of dil. Overall, our results demonstrate that platelet raft clustering is a dynamic reversible process, not induced solely by cold treatment or detergent extractoiin but rather is a regulated event, triggered by cell activation.
|Original language||English (US)|
|Issue number||11 PART II|
|State||Published - 2000|
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