Sanford J. Shattil

Control of integrin affinity for ligands (integrin activation) is essential for normal cell adhesion, migration, and assembly of an extracellular matrix. Integrin activation is usually mediated through the integrin beta subunit cytoplasmic tail and can be regulated by many different biochemical signaling pathways. We report that specific binding of the cytoskeletal protein talin to integrin beta subunit cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that increase their affinity. Thus, regulated binding of talin to integrin beta tails is a final common element of cellular signaling cascades that control integrin activation.

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.

We have investigated the composition and function of membrane microparticles released from platelets exposed to the C5b-9 proteins of the complement system. Gel-filtered human platelets were incubated with sub-lytic amounts of the purified C5b-9 proteins and the distribution of surface antigens was analyzed using monoclonal antibodies and flow cytometry. C5b-9 assembly caused secretory fusion of the alpha-granule membrane with the plasma membrane and the release of membrane vesicles (approximately 0.1-micron diameter) that contained the plasma membrane glycoproteins (GP) GP Ib and GP IIb-IIIa as well as the alpha-granule membrane protein GMP-140. These microparticles were highly enriched in the C9 neoantigen of the C5b-9 complex. The apparent surface density of C5b-9 on the microparticles was approximately 10(3)-fold higher than on the platelet itself, suggesting that the vesicles were selectively shed from the plasma membrane at the site of C5b-9 insertion. C5b-9 induced the expression of an activation-dependent epitope (recognized by monoclonal antibody, PAC1) in GP IIb-IIIa on the platelet surface but not in GP IIb-IIIa on the microparticles. The surface of the microparticles was also highly enriched in alpha-granule-derived coagulation factor V (or Va), accounting for nearly half of all the membrane-bound factor V detected. The number of potential membrane binding sites for factor Va was probed by adding saturating concentrations of factor Va light chain. Under these conditions, the density of factor Va binding sites on the microparticle surface exceeded that on the C5b-9-treated platelet by three to four orders of magnitude. Moreover, the microparticles provided most of the membrane surface for conversion of prothrombin to thrombin by VaXa. These studies demonstrate that the microparticles shed by C5b-9-treated platelets (and not the platelets themselves) provide the principal binding sites for coagulation factor Va and the principal catalytic surface for the prothrombinase complex. Platelet-derived microparticles formed during complement activation in vivo could provide a membrane surface that facilitates the assembly and dissemination of procoagulant enzyme complexes.

Resting platelets contain about 80,000 surface copies of IIb 3 , with additional pools of