SummaryThe migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin αIIbβ3 (glycoprotein Ilb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the P3 subunit. The diffusion of P3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 X10'9 cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 μM) or α-thrombin (10 U/ml) at 22° C induced transient decreases in both parameters reducing D to 0.21 X 10‘9 cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, whereas cytochalasin D completely abolished the decrease in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile αIIbβ3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out.

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...

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