Distinct domains of alphaIIbbeta3 support different aspects of outside-in signal transduction and platelet activation induced by LSARLAF, an alphaIIbbeta3 interacting peptide.

The peptide LSARLAF causes alphaIIbeta3-dependent platelet activation exemplified by secretion, aggregation, spreading and adhesion on fibrinogen, and tyrosine phosphorylation. alphaIIIbeta3-dependent outside-in signal transduction induced by LSARLAF was investigated in variant thrombasthenic platelets which lack most of the cytoplasmic domain of the integrin beta3 subunit (alphaIIbbeta3 delta724). These studies revealed that only certain aspects of this alphaIIbbeta3-dependent outside-in signaling were affected by the beta3 truncation. Specifically, alphaIIbbeta3 delta724 supported LSARLAF-induced platelet aggregation, agglutination and secretion, but failed to trigger cytoskeletal reorganization and platelet spreading on fibrinogen, despite the fact that PMA-induced non alphaIIbbeta3 mediated signaling caused spreading of these platelets on fibrinogen. Thus, distinct domains of alphaIIbbeta3 are required to support different aspects of LSARLAF-induced platelet activation. Furthermore, these studies suggest that not all alphaIIbbeta3-dependent platelet responses require an intact beta3 cytoplasmic tail.

A naturally occurring mutation near the amino terminus of alphaIIb defines a new region involved in ligand binding to alphaIIbbeta3.

Decreased expression of functional alphaIIbbeta3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of alphaIIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P(145)A substitution, whereas a Chinese patient was doubly heterozygous for a P(145)L substitution. The mutations affect expression levels of surface alphaIIbbeta3 receptors on their platelets, which was confirmed by co-transfection of alphaIIb(P145A) and beta3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of alphaIIbbeta3 on affected platelets to interact with ligands. Moreover, when alphaIIb(P145A) and beta3 were stably coexpressed in Chinese hamster ovary cells, alphaIIbbeta3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after alphaIIbbeta3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to alphaIIb, induced PF4 secretion, indicating that the mutant alphaIIbbeta3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving alphaIIb(P145 )impair surface expression of alphaIIbbeta3 and that the alphaIIb(P145A) mutation abrogates ligand binding to the activated integrin. A comparative analysis of other alphaIIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alphaIIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)

Peptide LSARLAF activates alpha(IIb)beta3 on resting platelets and causes resting platelet aggregate formation without platelet shape change.

Adhesion of resting platelets to fibrinogen was enhanced by a peptide which was designed to bind near the presumptive fibrinogen gamma-chain binding site of the alpha subunit of the integrin alpha(IIb)beta3. This peptide, but not a scrambled control peptide, induced adhesion of resting platelets to fibronectin, vitronectin, von Willebrand factor, and monovalent (lacks one functional gamma-chain) fibrinogen. Resting platelets not treated with the agonist peptide did not adhere to these ligands. Agonist peptide induced adhesion of resting platelets to Fg was not secretion dependent and was inhibited by the monoclonal antibody 7E3. The agonist peptide caused aggregation of resting platelets on resting platelets adherent to immobilized Fg without causing platelet shape change. Therefore, the agonist peptide may activate alpha(IIb)beta3 by directly inducing a conformation change in the receptor on resting platelets.

The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin alphaIIbbeta3.

A novel peptide (designed to bind to alphaIIbbeta3) caused platelet aggregation and aggregation-independent secretion of the contents of alpha-granules in an alphaIIbbeta3-dependent fashion. The agonist peptide induced secretion in the presence of prostaglandin E1. In cell-free assays, alphaIIbbeta3 bound specifically to immobilized agonist peptide and the peptide enhanced the binding of fibrinogen to immobilized alphaIIbbeta3. The agonist peptide apparently activates alphaIIbbeta3 by directly inducing a conformational change in the receptor. This change activates the platelets and causes secretion in a manner independent of fibrinogen binding.

Characterization of adhesion of non-exogenously stimulated and resting platelets in normal plasma to fibrinogen and its fragments.

Platelet adhesion to various forms of fibrinogen was studied using platelets in plasma and washed platelets. The study was designed to determine if platelets prepared with minimal handling in plasma at physiological pH containing normal levels of Ca2+ have different requirements for adhesion to immobilized fibrinogen than do washed platelets tested in the absence of plasma. Exposure of platelets to citrate and low pH did not seem to affect the requirements of washed platelets for adhesion to fibrinogen. Nonetheless, behavioural differences between these two types of platelets were seen. Surprisingly, in the absence of exogenous activation normal platelets in plasma behaved qualitatively as stimulated washed platelets. That is, both types of platelets adhered to all forms of fibrinogen which possessed at least one gamma-chain carboxyl terminal platelet binding site. Platelets in plasma treated with prostaglandin E1 (resting platelets) adhered only to forms of fibrinogen which contained two gamma-chain platelet binding sites. These observations also demonstrate that the fibrinogen alpha-chain arginine-glycine-aspartic acid-phenylalanine and arginine-glycine-aspartic acid-serine sequences are not necessary or sufficient to mediate the adhesion of resting or stimulated platelets in plasma to fibrinogen. The presence of endogenous adenosine diphosphate appears to account, at least in part, for the ability of normal platelets in plasma to adhere to forms of fibrinogen which have only one gamma-chain platelet binding site.

Antibodies to GPIIb alpha (300-312) inhibit Fg binding, clot retraction, and platelet adhesion to multiple ligands.

We previously reported that a peptide with the sequence Gly-Ala-Pro-Leu (GAPL) found as residues 309-312 in glycoprotein IIb alpha (GPIIb) comprises at least part of a Fg binding site on GPIIb (1). Subsequent studies demonstrated that a peptide corresponding to residues 300-312 of GPIIb alpha can bind to Fg and Vn, and is a potent inhibitor of platelet aggregation and the adhesion of activated platelets to at least four adhesive ligands: Fg, Fn, Vn, and vWf (2). Here, the production and initial characterization of polyclonal antibodies against this peptide are described. ELISAs and dot-blot assays reveal the specificity of the antibodies for the peptide immunogen. In immunoblots, the antibodies recognize GPIIb under reducing conditions. The binding of Fg to immobilized GPIIb/IIIa, the rate of clot retraction and the adhesion of stimulated platelets to Fg, fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWf) were inhibited by these antibodies, but not by a control IgG. These results together with our earlier published data indicate that GPIIb alpha (300-312) comprises at least part of a common ligand binding site within the integrin alpha IIb subunit.

Characterization of adhesion of "resting" and stimulated platelets to fibrinogen and its fragments.

Adhesion of resting and stimulated platelets to immobilized fibrinogen (Fg) was characterized using various forms of Fg, receptor peptide mimics, and antibodies to glycoprotein (GP) IIb/IIIa and Fg. Resting platelets adhered to Fg, but to less than half the extent of the same platelets stimulated with epinephrine/ADP. The adhesion of resting and stimulated platelets to Fg was inhibited by a receptor peptide mimic (G13, a peptide corresponding to residues 300-312 of GPIIb), anti-GPIIb/IIIa antibodies, and a monoclonal antibody (4A5) against the carboxyl terminus of the gamma chain of Fg. The results presented here demonstrate that the alpha chain RGD platelet recognition sites are not required to mediate the adhesion of either stimulated or resting platelets to immobilized Fg. Although stimulated platelets can adhere extensively to monomeric Fg containing one functional gamma chain, resting platelets require bivalent Fg containing two functional gamma chains to mediate irreversible adhesion to Fg.