On the role of the platelet membrane skeleton in mediating signal transduction. Association of GP IIb-IIIa, pp60c-src, pp62c-yes, and the p21ras GTPase-activating protein with the membrane skeleton.
The platelet plasma membrane is lined with a membrane skeleton composed of short actin filaments, actin-binding protein, spectrin, vinculin, and other unidentified proteins. It is connected to the outside of the cell through association with the cytoplasmic domains of transmembrane receptors. In detergent-lysed platelets, cytoplasmic actin filaments are sedimented by centrifugation at 15,600 x g, but the sedimentation of membrane skeleton fragments requires higher g-forces (100,000 x g). In the present study, we show that the major platelet integrin, glycoprotein (GP) IIb-IIIa, sediments from detergent-lysed platelets at 100,000 x g together with fragments of the membrane skeleton that contain the cytoskeletal proteins spectrin, vinculin, and talin. In addition, this cell fraction contained the tyrosine kinases pp60c-src and pp62c-yes and the p21ras GTPase-activating protein (GAP). After thrombin-induced platelet aggregation mediated by fibrinogen binding to GP IIb-IIIa on adjacent platelets, we detected a redistribution of spectrin, talin, vinculin, pp60c-src, and pp62c-yes to the fraction that sediments at 15,600 x g. The redistribution of these proteins from the high-speed detergent-insoluble fraction to the low-speed fraction correlated with the extent of aggregation and was not detected in aggregation-defective thrombasthenic platelets (which lack the GP IIb-IIIa complex). In addition, many of the proteins phosphorylated on tyrosine in activated platelets were present in detergent-insoluble fractions. These results are consistent with the possibilities that 1) GP IIb-IIIa, pp60c-src, pp62c-yes, and GAP associate with a membrane skeleton fraction that contains spectrin, vinculin, and talin, 2) the association of GP IIb-IIIa with adhesive ligand in a platelet aggregate causes components of the membrane skeleton to undergo altered association with cytoplasmic actin filaments, and 3) many of the proteins phosphorylated on tyrosine residues in activated platelets are components of the cytoskeleton. The results imply that the membrane skeleton may play an important role in binding signaling molecules at sites of integrin-cytoskeleton interactions and in mediating signal transduction events in platelets. Further, GP IIb-IIIa-induced redistribution of components of the membrane skeleton and associated signaling molecules may represent an important step in regulating integrin-induced motile events in platelets.
Fox
J E
JE
Children's Hospital Oakland Research Institute, California 94609.
Lipfert
L
L
Clark
E A
EA
Reynolds
C C
CC
Austin
C D
CD
Brugge
J S
JS
D000199:Actins / Q000097:blood / Q000302:isolation & purification; D000328:Adult; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000097:blood* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins; D006801:Humans; D008840:Microfilament Proteins / Q000097:blood / Q000302:isolation & purification; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051575:Proto-Oncogene Proteins c-yes; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D012016:Reference Values; D015398:Signal Transduction; D013049:Spectrin / Q000302:isolation & purification / Q000378:metabolism; D013915:Thrombasthenia / Q000097:blood; D013917:Thrombin / Q000378:metabolism; D016596:Vinculin / Q000097:blood / Q000302:isolation & purification; D019061:src-Family Kinases
D000199:Actins; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; D008840:Microfilament Proteins; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016596:Vinculin; D013049:Spectrin; D011505:Protein-Tyrosine Kinases; D051575:Proto-Oncogene Proteins c-yes; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; D013917:Thrombin