Alpha IIb Beta Research Articles

Accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in thrombin-stimulated platelets. Different sensitivities to Ca2+ or functional integrin.

Differences in regulation of the accumulation of PtdIns(3,4)P2 versus that of PtdIns(3,4,5)P3 were noted in thrombin-stimulated human platelets. The rapid (within 20 s) response of PtdIns(3,4,5)P3 contrasted with a distinct lag in the accumulation of PtdIns(3,4)P2 that was followed by a pronounced increase by 90 s. The presence of 2.5 mM-CaCl2 further elevated PtdIns(3,4)P2 by 50-120%, but only at a late stage (after 90 s). Tetrapeptide RGDS (Arg-Gly-Asp-Ser), which blocks the interaction of ligands such as fibrinogen with platelet integrin alpha IIb beta 3 (GPIIb-IIIa), inhibited only the late-phase PtdIns(3,4)P2 accumulation that was associated with added Ca2+. Although stimulated tyrosine phosphorylation of platelet protein (total cell lysate) was altered by Ca2+ or RGDS, we could not identify any such proteins that were affected comparably to PtdIns(3,4)P2. In contrast to the PtdIns(3,4)P2 response, the accumulation of PtdIns(3,4,5)P3 was unaffected by Ca2+ or RGDS at any time.

Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids.

Soluble fibrinogen binding to the glycoprotein IIb-IIIa complex (integrin alpha IIb beta 3) requires platelet activation. The intracellular mediator(s) that convert glycoprotein IIb-IIIa into an active fibrinogen receptor have not been identified. Because the lipid composition of the platelet plasma membrane undergoes changes during activation, we investigated the effects of lipids on the fibrinogen binding properties of purified glycoprotein IIb-IIIa. Anion exchange chromatography of lipids extracted from platelets exposed to thrombin or other platelet agonists resolved an activity that increased fibrinogen binding to glycoprotein IIb-IIIa. A monoester phosphate was important for activity, and phosphatidic acid coeluted with the peak of activity. Purified phosphatidic acid dose-dependently promoted a specific interaction between glycoprotein IIb-IIIa and fibrinogen which possessed many but not all of the properties of fibrinogen binding to activated platelets. Phosphatidic acid appeared to increase the proportion of fibrinogen binding-competent glycoprotein IIb-IIIa complexes without altering their affinity for fibrinogen. The effects of phosphatidic acid were a result of specific structural properties of the lipid and were not mimicked by other phospholipids. Lysophosphatidic acid, however, was a potent inducer of fibrinogen binding to glycoprotein IIb-IIIa. These results demonstrate that specific lipids can affect fibrinogen binding to purified glycoprotein IIb-IIIa and suggest that the lipid environment has the potential to influence fibrinogen binding to its receptor.

Radiation-Induced Increase in Expression of the α IIb β 3 Integrin in Melanoma Cells: Effects on Metastatic Potential

We investigated the effects of nonlethal gamma radiation on the metastatic potential of the murine tumor cell line, B16 melanoma. The ability of B16 cells to adhere to fibronectin, which is in part mediated by the alpha IIb beta 3 integrin receptor, is predictive of metastatic potential. We determined that exposure to 0.25-2.5 Gy gamma radiation significantly enhanced B16 cell adhesion to fibronectin. The radiation-enhanced adhesion was dependent on enhanced expression of the alpha IIb beta 3 integrin. We observed that 15 min after 0.5 Gy radiation, 99% of irradiated B16 tumor cells were positively labeled with monoclonal antibodies directed against alpha IIb beta 3 compared to 22% of sham-irradiated cells. Radiation-enhanced expression of the alpha IIb beta 3 receptor is reversible and down-regulation begins within 2-4 h postirradiation. Finally, we found that irradiation significantly enhanced the ability of B16 cells to form metastases in a lung colony assay. It is concluded that a relationship exists between radiation effects on the B16 tumor cells, alpha IIb beta 3 receptor expression, adhesion in vitro, and metastasis in vivo. We suggest that low-dose radiation, at levels comparable to those used in fractionated or hyperfractionated radiotherapy, may alter the metastatic phenotype and potential of surviving tumor cells via a rapid alteration in their surface expression of alpha IIb beta 3 integrin receptors.

Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length.

The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha 1 at GPIIIa 63-87 and 303-350; gamma 1 at GPIIIa 9-37; alpha 2 at GPIIIa 151-191; and gamma 2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of platelet function through adhesion receptors. A dual role for glycoprotein IIb-IIIa (integrin alpha IIb beta 3) mediated by fibrinogen and glycoprotein Ib-von Willebrand factor.

We have found that the form of glycoprotein (GP) IIb-IIIa (integrin alpha IIb beta 3) expressed on nonstimulated platelets is a functional receptor that mediates selective and irreversible adhesion to immobilized fibrinogen. This occurs even in the presence of the elevated intracellular cAMP levels induced by prostaglandin E1 or after inhibition of protein kinase C activity by sphingosine. In the absence of inhibitors, platelets adhering to fibrinogen through GP IIb-IIIa become fully activated and aggregate with one another. Immobilized von Willebrand factor (vWF), in contrast, is recognized by nonstimulated platelets through another receptor, GP Ib. This interaction leads to a change in the ligand recognition specificity of GP IIb-IIIa that can then bind to immobilized vWF and mediate irreversible platelet adhesion and aggregation; this process, however, is inhibited by elevated intracellular cAMP levels or blockade of protein kinase C activity. Therefore, GP Ib and GP IIb-IIIa induce platelet activation through the selective recognition of immobilized vWF and fibrinogen, respectively, in the absence of exogenous agonists. Moreover, "nonactivated" and "activated" GP IIb-IIIa exhibits distinctly different reactivity toward surface-bound vWF, and the functional switch can be induced by the binding of vWF to GP Ib. These findings demonstrate the modulation of platelet function by two different adhesion receptors, GP Ib and GP IIb-IIIa, as well as the distinct dual role of the latter as the necessary common mediator of irreversible adhesion and aggregation on both fibrinogen and vWF.

Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased expression of alpha IIb beta 3 integrin in subpopulations of murine melanoma cells with high lung-colonizing ability.

Four subpopulations of B16 amelanotic melanoma cells, possessing different abilities to induce platelet aggregation (TCIPA) and to form lung colonies, were isolated by centrifugal elutriation. The expression of alpha IIb beta 3, alpha v beta 3 and alpha 5 beta 1 integrins was examined in the 4 subpopulations in order to determine the relationship between integrin receptor expression and tumor-cell metastatic potential. The mRNA of alpha IIb, alpha 5, beta 1 and beta 3 was detectable in the 4 subpopulations by Northern blotting. A gradual increase in mRNAs and cell-surface immunoreactivity of the alpha IIb beta 3 receptor, but not in their gene copies, was observed from the low to the high metastatic subpopulations. The ability of tumor cells to adhere to fibronectin and subendothelial matrix (SEM) increased in parallel. In the high metastatic cells, the alpha IIb beta 3 receptors, but not the alpha 5 beta 1 receptors, were localized to focal adhesion plaques. Incubation of the high metastatic cells with alpha IIb beta 3-specific antibodies reduced their matrix adhesion, TCIPA and lung-colonizing abilities. In contrast, in the low met- astatic cells, SEM adhesion and lung-colony formation were not affected by anti-alpha IIb beta 3 antibody treatment. Incubation of either the low or the high metastatic subpopulation with an alpha 5 beta 1-specific antibody had no effect in vitro and showed a slight inhibition of lung colonization in vivo. Our results suggest that several phenotypic characteristics of the enhanced metastatic potential of B16a subpopulations may be mediated by increased expression of alpha IIb beta 3 receptors and that expression of these receptors may be regulated at the transcriptional level.

The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb- IIIa on nonactivated platelets is accessible to high-affinity macromolecules

We have characterized a murine IgG monoclonal antibody, OP-G2, specific for platelet glycoprotein (GP) IIb-IIIa (alpha IIb beta 3). OP-G2 Fab fragments inhibit fibrinogen-mediated platelet aggregation and competitively inhibit adenosine diphosphate-induced binding of 125I-fibrinogen to washed platelets. OP-G2 binding to GPIIb-IIIa is specifically inhibited by RGD-containing peptides but not the fibrinogen gamma-chain carboxy-terminal peptide, and OP-G2 Fab fragments, like RGD-containing peptides, alter the conformation of GPIIb-IIIa resulting in the expression of a ligand-induced binding site (LIBS) recognized by PMI-1. OP-G2 fails to bind to the recombinant Cam variant of GPIIb-IIIa (alpha III beta 3Cam) wherein an Asp119 to Tyr119 substitution in GPIIIa abrogates the ability to recognize RGD. These data indicate that OP-G2 recognizes an epitope at or in very close proximity to the RGD recognition site of GPIIb-IIIa and that, in every aspect tested, OP-G2 behaves like a macromolecular RGD ligand. Interestingly, two-color flow cytometry shows that OP-G2 IgG can bind to nonactivated platelets. Quantitative binding assays indicate that nonactivated platelets bind approximately 50,000 125I-OP-G2 molecules/platelet. Furthermore, the affinity of OP-G2 for platelets activated with thrombin is roughly fivefold higher (nonactivated, kd = 24.8 nmol/L; activated, kd = 4.9 nmol/L). These results suggest that the RGD recognition site of GPIIb-IIIa is available to macromolecules that contain RGD even on nonactivated platelets, provided that the affinity of the ligand is adequate.

Is there a role for the tumor cell integrin alpha IIb beta 3 and cytoskeleton in tumor cell-platelet interaction?

In vitro tumor cell-platelet interaction was examined using B16 amelanotic (B16a) melanoma cells. These tumor cells express the alpha IIb beta 3-type cytoadhesin. Aggregation studies demonstrated that tumor cell surface alpha IIb beta 3 mediates the recognition of platelets since pretreatment of tumor cells with antibody against alpha IIb beta 3 prevents platelet-tumor cell interaction as well as platelet activation measured by aggregometry, platelet eicosanoid metabolism and ultrastructural analysis. In B16a cells, disruption of the microfilaments and intermediate filaments inhibits mobility of alpha IIb beta 3 on the cell surface. Microtubules do not play a role in receptor mobility, because B16a cells do not possess well-defined microtubules in interphase and colchicine does not affect receptor mobility. Disruption of microfilaments or intermediate filaments results in an inhibition of tumor cell-platelet interaction as evidenced by aggregometry studies and ultrastructural analysis. We suggest that platelet interaction with tumor cells begins with alpha IIb beta 3-mediated receptor recognition followed by not only platelet activation but also microfilament- and vimentin intermediate filament-dependent tumor cell activation.

A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site.

This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.

Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids.

Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids.

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin.

The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin.

Involvement of platelet glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin) in thrombin-induced synthesis of phosphatidylinositol 3‘,4‘-bisphosphate.

32P-Labeled human platelets were incubated with thrombin (1 unit/ml) for 5 min at 37 degrees C under conditions allowing maximal synthesis of [32P]phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2). Incorporation of 32P into the latter phosphoinositide was dose-dependently reduced (to a maximal level averaging 60%) by the tetrapeptide RGDS, an inhibitor of fibrinogen binding to activated glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin). Identical results were obtained with the fibrinogen gamma-chain dodecapeptide HHLGGAKQAGDV, whereas the tripeptide RGD and the tetrapeptide RGES displayed reduced or undetectable effects on 32P labeling of PtdIns(3,4)P2, respectively, in good correlation with their ability to inhibit platelet aggregation and fibrinogen binding to activated alpha IIb-beta 3 integrin. In addition, pathological platelets from three patients suffering thrombasthenia, which lack alpha IIb-beta 3 integrin and fail to aggregate in response to thrombin, displayed hardly detectable increases in the 32P labeling of PtdIns(3,4)P2. In contrast, thrombin-stimulated synthesis of PtdIns(3,4)P2 was unaltered in other deficient platelets lacking the glycoprotein Ib-IX complex (Bernard-Soulier syndrome). Although additional pathways seem to be involved in the regulation of phosphatidylinositol-3-kinase, these data indicate a strong relationship between platelet aggregation involving fibrinogen binding to activated alpha IIb-beta 3 integrin and the synthesis of the novel phosphoinositides phosphorylated at position D-3 of the inositol ring.

The lipoxygenase metabolite 12(S)‐hete induces a cytoskeleton‐dependent increase in surface expression of integrin αiibβ3 on melanoma cells

Integrin receptors are mediators of cell-extracellular matrix and cell-cell interactions. Biochemical and immunocytochemical evidence shows that the platelet integrin receptor alpha IIb beta 3 is present on the cell surface, at focal adhesion plaques and in the perinuclear region of metastatic B16a murine melanoma cells. Antibody to the fibronectin receptor alpha 5 beta i, inhibits basal adhesion by approx. 30%, whereas antibodies to alpha IIb beta 3 are ineffective. The surface immunoreactivity of tumor cells for alpha IIb beta 3 can be enhanced by pre-treatment (5 min) with a lipoxygenase metabolite of arachidonic acid [i.e. 12-(S)-HETE] in a dose-dependent manner (max. effect approx. 0.1 microM). Other lipoxygenase metabolites are ineffective. B16a cells possess a large intracellular pool of alpha IIb beta 3, from which the receptor complex translocates to the cell surface following 12-(S)-HETE pretreatment. This pre-treatment of tumor cells enhances their adhesion to fibronectin, which is mediated exclusively by alpha IIb beta 3 receptors. 12-(S)-HETE also facilitates the redistribution of alpha IIb beta 3 in the plasma membrane with localization at the focal adhesion plaques. The cytoskeleton of the B16a cell is characterized by an absence of distinct microtubules in interphase cells and the presence of prominent microfilaments and vimentin intermediate filaments. In B16a cells, the disruption of intermediate filaments and/or microfilaments prevents the 12-(S)-HETE-induced increase in plasma membrane alpha IIb beta 3 and enhanced tumor-cell adhesion to fibronectin. The microtubule-disrupting agent, colchicine, is ineffective in both respects. We conclude that the lipoxygenase metabolite of arachidonic acid, 12-(S)-HETE, regulates the surface expression and function of the alpha IIb beta 3 integrin in B16a cells. Further, these data support the hypothesis that microfilaments and intermediate filaments have a profound role in regulating the expression of a multifunctional integrin in B16a tumor cells.

Modulation of the Affinity of Integrin α IIb β 3 (GPIIb-IIIa) by the Cytoplasmic Domain of α IIb

Intracellular signaling alters integrin adhesive functions in inflammation, immune responses, hemostasis, thrombosis, and retinal development. By truncating the cytoplasmic domain of alpha IIb, the affinity of integrin alpha IIb beta 3 for ligand was increased. Reconstitution with the cytoplasmic domain from integrin alpha 5 did not reverse the increased affinity. Thus, the cytoplasmic domain of the alpha subunit of GPIIb-IIIa controls ligand binding affinity, which suggests mechanisms for inside-out transmembrane signaling through integrins. These findings imply the existence of hitherto unappreciated hereditary and acquired thrombotic disorders in humans.

Ultrastructural Similarities Between Epidermal Langerhans Cell Birbeck Granules and the Surface-Connected Canalicular System of EDTA-Treated Human Blood Platelets

Birbeck granules characterize under the electron microscope epidermal Langerhans cells. These distinctive pentalaminar organelles are indeed not detectable in the possible precursors of human Langerhans cells and tend to disappear in cultured human Langerhans cells. The mechanisms that lead to the appearance of Birbeck granules in epidermal Langerhans cells and to their later disappearance still remain unknown. In the present study we show that the more or less dilated elements of the surface-connected canalicular system of human blood platelets collapse after EDTA treatment. Made up of two parallel limiting membrane and central irregular striated density, these elements show great ultrastructural similarities with the Birbeck granules of human epidermal Langerhans cells. These platelet morphologic changes i) are directly dependent on the EDTA-induced dissociation of the glycoprotein GP IIb-IIIa, the platelet-specific calcium-dependent heterodimer complex, member of the beta 3 integrin subfamily (alpha IIb beta 3) and ii) apparently result from a cross-linking of the dissociated glycoproteins. These findings lead us to propose that in the same manner cells of the Langerhans lineage, on reaching the epidermis, will find themselves in contact with an epidermal specific ligand. Interactions between this epidermal ligand and Langerhans cell receptors could then induce, all along the circuit taken by the ligand-receptor complexes, morphologic modifications, i.e., appearance of structures of Birbeck granule type.

Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIB-IIIA) dependent homotypic and heterotypic cell-cell interactions.

The aggregation of cells bearing recombinant integrin alpha IIb beta 3 (platelet GPIIb-IIIa) has been analyzed by two-color flow cytometry. As in normal platelets, aggregation requires functional alpha IIb beta 3, "activation" of alpha IIb beta 3, and fibrinogen (fg) binding to alpha IIb beta 3. Cellular aggregation required that both interacting cells express functional alpha IIb beta 3, because a binding defective mutant, alpha IIb beta 3 (D119----Y), failed to support interaction with wild type alpha IIb beta 3-bearing cells. In addition, cells bearing resting alpha IIb beta 3 were incorporated into aggregates formed by cells bearing a constitutively active mutant, alpha IIb beta 3 (beta 1-2), indicating that only one of the cells in an interacting pair must be activated. Finally, heterotypic interactions occurred between cells bearing activated alpha IIb beta 3 and cells bearing alpha V beta 3, a fg-binding integrin present on endothelial and tumor cells. Thus, ligand bridging between fg-binding integrins represents a mechanism of cell-cell interaction, cells bearing resting alpha IIb beta 3 (e.g., resting platelets) may be incorporated into aggregates formed by cells bearing activated alpha IIb beta 3, and alpha IIb beta 3 mediates heterotypic interactions with cells bearing other fg receptors.

Monoclonal antibodies to ligand-occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function.

Occupancy of integrin receptors induces conformational changes in the receptor, resulting in exposure of novel interactive sites termed ligand-induced binding sites (LIBS). We report here that Fab fragments of certain antibodies against LIBS on integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) block platelet aggregation. Thus, certain LIBS or the regions surrounding them may participate in events required for platelet aggregation. In addition, certain anti-alpha IIb beta 3 LIBS Fab fragments stimulated platelet aggregation. This was due to induction of fg binding to alpha IIb beta 3, apparently by shifting a conformational equilibrium between a "resting" and an "activated" state of alpha IIb beta 3. Some of the activating anti-LIBS Fab fragments also induced high affinity fibronectin binding to alpha IIb beta 3, whereas others did not. Thus, changes in the conformation of this integrin modulate both the specificity and affinity of ligand recognition.

Aminopeptidase resistant Arg‐Gly‐Asp analogs are stable in plasma and inhibit platelet aggregation

Tetrapeptides containing the sequence Arg-Gly-Asp (RGD) antagonize fibrinogen binding to its platelet receptor (gp IIb/IIIa, integrin alpha IIb beta 3) and inhibit platelet aggregation in vitro. The peptides RGDS and RGDY(Me)-NH2 were rapidly degraded when incubated in human, rat, and dog plasma. HPLC analysis indicated that amino acids were sequentially removed from the peptide N-terminus, and this degradation was prevented by the aminopeptidase inhibitor bestatin. Analogs of RGDY(Me)-NH2 with an acetylated or deleted alpha-amino group were prepared. Both analogs were stable when incubated in plasma, blocked 125I-fibrinogen binding to activated platelets (IC50 = 10-30 microM) and inhibited ADP induced platelet aggregation (IC50 = 10-30 microM). This study concludes that aminopeptidase rapidly degrades RGD peptides in plasma, an important issue for in vivo testing of RGD peptides and analogs. RGD analogs intrinsically stabilized against aminopeptidase are stable in plasma and are important tools for antithrombotic studies involving antagonism of gp IIb/IIIa.

Effect of Single Amino Acid Substitutions on the Formation of the PIA and Bak Alloantigenic Epitopes

The subunits that comprise the platelet-specific integrin alpha IIb beta 3 are polymorphic in nature, with several allelic forms present in the human gene pool. Minor changes in the secondary and tertiary structures of platelet membrane glycoproteins (GP) IIb and IIIa encoded by these alleles can result in an alloimmune reaction after transfusion or during pregnancy. To better understand the molecular structure of the PlA alloantigen system, located on GPIIIa, and the Bak alloantigen on GPIIb, we used a heterologous mammalian expression system to express these integrin subunits in their known polymorphic forms. An expression vector containing the PlA1 form of a GPIIIa cDNA, which encodes a leucine at amino acid 33 (Leu33), was modified to express the PlA2-associated form encoding a proline at amino acid 33 (Pro33). Similarly, a Baka GPIIb cDNA expressing an isoleucine at amino acid 843 (IIe843) was modified to express the Bakb form containing a serine at the same position (Ser843). Transfection of these vectors into COS cells resulted in the synthesis of GPIIb and GPIIIa molecules that were identical in size to those present in platelet lysates. Immunoprecipitation of the GPIIIa-transfected COS lysates with PlA)-specific alloantisera indicated that the Leu33 form was recognized only by anti-PIA1 sera while the Pro33 form was bound only by anti-PlA2 sera, showing that single amino acid polymorphisms are necessary and sufficient to direct the formation of the PlA1 and PlA2 alloepitopes. Similar experiments with Bak allele-specific expression vectors indicated that while the amino acid polymorphism (IIe843 in equilibrium Ser843) was necessary, posttranslational processing of pro-IIb was required for efficient exposure of both the Baka and Bakb alloepitopes.