Human platelets are derived from megakaryocytes as anucleate cells, and thus contain only vestigial amounts of RNA capable of being transcribed into protein. This has greatly hampered efforts to study directly platelet-specific gene products and their associated polymorphisms. In this report, we describe direct amplification, using the polymerase chain reaction, of platelet-derived mRNA in amounts sufficient to permit detailed analysis, such as restriction mapping and nucleotide sequencing. The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders.

Integrin ␣ IIb ␤ 3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either "inside-out" or "outside-in" signaling through ␣ IIb ␤ 3 is the phosphorylation of focal adhesion kinase (pp125 FAK ) and the association of pp125 FAK with cytoskeletal components. To examine the role of pp125 FAK in these integrin-mediated events, pp125FAK phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of ␣ IIb ␤ 3 : ␣ IIb ␤ 3 (D723A/ E726A), a constitutively active integrin in which the putative binding site for pp125FAK is altered, and ␣ IIb ␤ 3 (F727A/K729E/F730A), in which the putative binding site for ␣-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing ␣ IIb ␤ 3 (D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing ␣ IIb ␤ 3 (F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125 FAK is recruited to focal adhesions in adherent cells expressing ␣ IIb ␤ 3 (D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing ␣ IIb ␤ 3 (F727A/K729E/F730A), pp125 FAK was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125 FAK can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.The cytoplasmic domains of integrins constitute an essential link between the extracellular, ligand-binding domain of the receptor and both signaling and structural mechanisms inside the cell. Ranging in length from 15 amino acids for the ␣ 1 subunit to over 1000 amino acids for the ␤ 4 subunit, the cytoplasmic domains extend inward from the plasma membrane toward various cytosolic components known to be involved in integrin-mediated signaling such as heterotrimeric G proteins, proteins involved in phospholipid metabolism, serine-threonine kinases, tyrosine kinases, calcium transport systems, and cytoskeleton components (1-7). Although structurally distinct, the different ␣ and ␤ cytoplasmic domains show areas of regional homology, suggesting a conserved tertiary structure. The nine known ␤ subunits contain a highly conserved membrane-proximal polar region with the consensus sequence HDRREFAKFEKEK, which is separated by a stretch of nine amino acids from a central NPXY motif and a stretch of 16 -25 amino acids from a distal NPXY motif. The NPXY sequence prescribes a signal for clathrin-coated pit-mediated internalization of integral membrane proteins (8) and is a recognition site for Shc, an SH2-containing adaptor protein (9). The spacing of the tyrosine residues in the ␣ 1 , ␤ 3 , and ␤ 6 cytoplasmic tails conforms to the spacing of tyrosine residues in immu...

Sindbis virions undergo a conformational rearrangement after attachment to cells but prior to entry, as detected by exposure of epitopes on virus-cell complexes which are not accessible to their cognate monoclonal antibodies on native virions (D. C.

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