The fluidity of the plasma membrane is thought to play a role in the activation of blood platelets. We investigated the lateral diffusion of the lipophilic probe 1 ,l'-ditetradecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC14) and derivatives in the plasma membrane of the megakaryoblast MEG-01 by fluorescence recovery after photobleaching. The lateral diffusion coefficient (D) of DiIC,, in an unstimulated cell was (3.53?0.06)X10-9 cm2/s with a mobile fraction of 75%. Similar data were found with DiIC,, and DiICII, but lipophilic probes specific for the outer leaflet showed a slower diffusion with a D value of (2.99+0.31)X10-' cm2/s and a mobile fraction of 58%. Stimulation with platelet-activating agents decreased the diffusion of DiIC,, within 2 min, but left the mobile fraction unchanged. Signal processing was required for the decrease in D as ~-Phenylalanyl-~-prolyl-~-arginyl-chloromethane-treated thrombin, which binds normally to the thrombin receptor but fails to activate the cell, had no effect. The decrease in D was accompanied by an increase in cytosolic Ca'+ content, [Ca"'],, and studies using different concentrations of thrombin, the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester and the Ca2+ ionophore ionomycin revealed that lipid mobilty in the plasma membrane is regulated by Ca2+. In contrast, treatments thought to interfere with the mobility of membrane proteins had little effect. We conclude that the rigidification of the plasma membrane during cell activation is caused by an increase in [Ca'+], and is therefore a late event and might only contribute to signal transduction at steps downstream of the mobilizatiodinflux of Ca2+.Cell activation is often accompanied by changes in lipid mobility in the plasma membrane. Polarization studies with trans-parinaric acid show a transient decrease in membrane fluidity in luteal cells during luteolysis [l], whereas the lateral diffusion of fluorescein-phosphatidyl-ethanolamine shows a more persistent decrease in stimulated endothelial cells [2]. Stimulation of hepatocytes with insulin induces a 30% decrease in lipid mobility within 7 min [3].The activation of platelets is also accompanied by a decrease in membrane fluidity as demonstrated in polarization studies with 1,6-diphenyl-1,3,5-hexatriene, 1 -(Ctrimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene [4 -61 and the stachyose derivative of pyrenebutyryl hydrazide [7]. The decrease is transient and occurs in a period in which signaltransduction pathways are initiated leading to aggregation Correspondence to J.

In the present study we measured membrane fluidity as the lateral mobility of the lipid probe l,l'-ditetradecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by fluorescence recovery after photobleaching in the plasma membrane of a single megakaryocyte, the progenitor cell of platelets. Megakaryocytes after 13 days in culture (maturation stage 111) had a lateral diffusion coefficient (D) of (4.56-+0.10)X10-9 cm2/s and a mobile fraction of 6 5 2 2 % (means ? SEM, n = 140).Megakaryocytes isolated from rib had a similar D and mobile fraction. Stimulation with a-thrombin (1-10 U/ml) induced a dose-dependent decrease in D to (3.40 ?0.22)X10-9 cmz/s between 1 -5 min after stimulation ( P < 0.001). The mobile fraction did not change. A similar decrease in D was found following stimulation with ADP (20 kM) and ionomycin (100 nM). Modulation of calpain I activity with calpain I inhibitor or tetracain had no effect. Pretreatment with cytochalasin B or colchicine decreased D to (3.64 Ifr 0.29)X em% ( P < 0.013) respectively. After stimulation D decreased further in cytochalasin-treated cells (3.37 2 0.16)X lo-' cm2/s ( P < 0.020) but remained at the same level in colchicine-treated cells. Both treatments increased the mobile fraction to 73 -75% in stimulated megakaryocytes ( P < 0.03).These data indicate that the diffusion velocity of lipids in megakaryocytes is low and decreases further after stimulation. These changes are independent of calpain I. Treatments that decrease the cytoskeletal mass and thereby increase the mobility of proteins in the plasma membrane increase the number of lipids that participate in this process.cm2/s ( P < 0.003) and (3.96 ? 0.1 8)XOne of the factors that determines the sensitivity of blood platelets to activating stimuli is the fluidity of the plasma membrane. Platelets enriched in cholesterol, as seen in hyperlipoproteinemic patients or after prolonged incubation with liposomes, have a decreased membrane fluidity and respond to thrombin and ADP with increased aggregation and secretion [l-31. On the other hand, when the fluidity is increased by incubation with alkyl alcohols, benzyl alcohol, and phenolic compounds, a lower aggregation tendency is found [4].There is little insight into the mobility of lipids after platelets have been stimulated, partly because different approaches have led to conflicting results. Studies based on fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, which detects the angular movement of a lipophilic molecule around a perpendicular axis in the plane of the membrane, showed a rigidification of the membrane after stimulation ionomycin had no effect [6]. 1 -(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene binds more specifically to the outer layer of the plasma membrane [6, 71. Such labeled platelets again showed a decrease in membrane fluidity at low doses of thrombin (G0.2 U/ml) [8, 91, but higher doses made the membrane more fluid [9]. A similar fall was found after stimulation with ADP (0.2-5 pM) [9] and a low concentration of ionomycin (100 n...