AlphaIIbbeta3 and its antagonism at the new millennium.

Because of its major role in regulating platelet functions and its prominence on the cell surface, integrin alphaIIbbeta3 has been the subject of intensive investigations. Such studies have provided substantial insights into its structure-function relationships and have led to the development of anti-thrombotic drugs that target the receptor. Nevertheless, recent findings have indicated that our understanding of the structure and function of alphaIIbbeta3 remains inadequate. This article addresses two aspects of still evolving alphaIIbbeta3 function: 1) the interface between alphaIIbbeta3 and the blood coagulation system, resulting from interaction of prothrombin with the receptor; and 2) the molecular basis for recognition of the RGD and the fibrinogen gamma-chain peptide ligands by alphaIIbbeta3. As illustrated by these two examples, there is still much to be learned about alphaIIbbeta3 if we are to fully appreciate its functions and its potential as a therapeutic target.

Peptide ligands can bind to distinct sites in integrin alphaIIbbeta3 and elicit different functional responses.

The spatial relationship between the binding sites for two cyclic peptides, cyclo(S,S)KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), high affinity analogs for the RGD and HLGGAKQAGDV peptide ligands, in integrin alphaIIbbeta3 (GPIIb-IIIa) has been characterized. For this purpose, cRGD and cHarGD were labeled with fluorescein isothiocyanate and tetramethylrhodamine 5-isothiocyanate, respectively. Both cyclic peptides were potent inhibitors of fibrinogen binding to alphaIIbbeta3, particularly in the presence of Mn2+; IC50 values for cRGD and cHarGD were 1 and <0.1 nM in the presence of Mn2+. Direct binding experiments and fluorescence resonance energy transfer analysis using the purified receptor showed that both peptides interacted simultaneously with distinct sites in alphaIIbbeta3. The distance between these sites was estimated to be 6.1 +/- 0.5 nm. Although cRGD bound preferentially to one site and cHarGD to the other, the sites were not fully specific, and each cyclic peptide or its linear counterpart could displace the other to some extent. The binding affinity of the cHarGD site was dramatically affected by Mn2+. cRGD, but not cHarGD, bound to recombinant beta3-(95-373) in a cation-dependent manner, indicating that the cRGD site is located entirely within this fragment. With intact platelets, binding of c-RGD and cHarGD to alphaIIbbeta3 resulted in distinct conformational alterations in the receptor as indicated by the differential exposure of ligand-induced binding site epitopes and also induced the opposite on membrane fluidity as shown by electron paramagnetic resonance analyses using 5-doxylstearic acid as a spin probe. These data support the concept the two peptide ligands bind to distinct sites in alphaIIbbeta3 and initiate different functional consequences within the receptor itself and within platelets.

Purification and crystallization of a novel membrane-anchored protein: the Schistosoma haematobium serpin.

A unique serine-protease inhibitor (serpin) of the blood fluke S. haematobium has been crystallized. It is an antitrypsin with an unusual residue (phenylalanine) at its reactive center. Unlike any known member of this gene family, it is a membrane-anchored protein on the surface of the parasite. The location of this serpin and immunological response to the protein indicate that it may play a important role in host-parasite interaction. The crystals belong to the trigonal space group P3221 or P3121 with unit-cell parameters a = b = 64.7, c = 186.7 A, alpha = 90.0, beta = 90.0, gamma = 120.0 degrees. There is one molecule per asymmetric unit and the crystals diffracted to 2.2 A.

Development of a structural model for the cytoplasmic domain of an integrin.

The cytoplasmic tails of integrin heterodimers play central roles in controlling the activation states of integrins and in transmitting intracellular signals. Despite their short length, no structure of any integrin cytoplasmic domain has been determined. Therefore, molecular models for the cytoplasmic domain of alpha(IIb)beta3, the major platelet integrin, were generated, including models for the individual cytoplasmic tails, the binary alphaIIb-calcium complex, and the ternary alphaIIb-beta3-calcium complex. Structural analysis of circular dichroism spectra were compiled with data obtained from short homologous sequences within crystallized proteins, and with secondary structural predictions to develop starting models for each subunit. These models were subjected to a series of energy minimization and molecular dynamic simulations to generate final models. AlphaIIb was predicted to be ordered at its N-terminus and its C-terminus could accommodate a cation in a multicoordinated complex. The structure of beta3 was dominated by a beta-turn at its NPXY motif (beta3 744-747). In docking of alphaIIb to different sites within beta3, the conformation of the beta3 juxta-transmembrane (beta3 716-721) was greatly altered. This region was confirmed to be a conformational 'hot-spot' by circular dichroism. The conformational flexibility of this juxta-transmembrane region, which is highly conserved amongst integrins, is ideally located to regulate signaling.

The cytoplasmic domain of alphaIIb beta3. A ternary complex of the integrin alpha and beta subunits and a divalent cation.

Peptides corresponding to the cytoplasmic tails of the alphaIIb (alphaIIb (985-1008)) and beta3 (beta3 (713-762)) subunits of the integrin receptor alphaIIb beta3 (glycoprotein IIb-IIIa) were synthesized and used to characterize their interaction with cations and with one another. alphaIIb (985-1008) was found to contain a functional cation binding site as assessed by both terbium luminescence and electrospray ionization mass spectroscopy. The binding of Tb3+ to alphaIIb (985-1008) was of high affinity (Kd = 8.8 +/- 5.2 nM), occurred with a 1:1 stoichiometry, and was mediated by its acidic carboxy] terminus (alphaIIb (999-1008), PLEEDDEEGE). The affinity of this site for divalent cations was in the micromolar range, suggesting that this site would be constitutively occupied in the intracellular environment. Incubation of alphaIIb (999-1008) with beta3 (713-762) resulted in the formation of a complex, both in the presence and absence of cations. The interactive site for alphaIIb (999-1008) in beta3 was mapped to beta3 (721-740), and complex formation was associated with a stabilization of secondary structure as assessed by circular dichroism. Both a binary (alphaIIb (985-1008).beta3 (721-740)) and a ternary (Tb3+.alphaIIIb (999-1008).beta3 (721-740)) complex were detected by mass spectroscopy, but the distribution and intensity of the mass/charge peaks were distinct. These difference may reflect the involvement of distinct cation coordination sites and the formation of salt bridges in stabilizing the ternary complex. These data demonstrate the formation of a novel entity composed of the cytoplasmic tails of alphaIIb and beta3 and a cation which may constitute a functional intracellular domain.

Ligand and cation binding are dual functions of a discrete segment of the integrin beta 3 subunit: cation displacement is involved in ligand binding.

The alpha IIb beta 3 integrin binds Arg-Gly-Asp-containing (RGD-containing) ligands in a cation-dependent interaction. A fourteen amino acid sequence, beta 3 (118-131), and an antibody to it, inhibited ligand binding functions of alpha IIb beta 3, and a 1:1 stoichiometric beta 3 (118-131)-RGD complex was detected by mass spectroscopy. Cation binding to beta 3 (118-131) was demonstrated by terbium luminescence and mass spectroscopy. Notably, ligand displaced cation from the beta 3(118-131) peptide and also from purified alpha IIb beta 3. Thus, beta 3 (118-131), a highly conserved region in integrin beta subunits, binds both ligand and cation. Formation of a ternary complex between cation, ligand, and receptor, with subsequent displacement of cation from beta 3 (118-131) and a second site within the receptor, may be central to the mechanism of ligand recognition by integrins.

Integrin-ligand interactions: a year in review.

Many cell-cell and cell-matrix interactions depend upon the engagement of specific ligands by members of the integrin family of cell-adhesion receptors. In concert with the identification of new integrins, the number of integrin ligands continues to expand dramatically. The diversity of the integrin ligands bridges many areas of cell and molecular biology. Ligand recognition by integrins requires not only the presence of the cognate primary sequence within an appropriate secondary structure, but also the correct tertiary and quaternary structure of the ligand. Presentation of an 'activated' ligand sequence to specific contact sites within the integrin under specified divalent-cation conditions is necessary for a productive and high-affinity interaction.

Characterization of cation-binding sequences in the platelet integrin GPIIb-IIIa (alpha IIb beta 3) by terbium luminescence.

The binding of cations to purified GPIIb-IIIa (alpha IIb beta 3) and synthetic peptides corresponding to the potential cation-binding sites within this integrin has been assessed by terbium luminescence spectroscopy. Tb3+ supported fibrinogen binding to purified GPIIb-IIIa, at lower concentrations than Ca2+, consistent with its higher affinity for cation-binding motifs. Titration analyses indicated the presence of five Tb(3+)-binding sites of relatively high affinity in the receptor. These sites also could be filled by divalent cations. Six sequences within GPIIb-IIIa have the appropriate spacing of five of the usual six coordination sites for cations in functional Ca(2+)-binding EF-hand motifs. Peptides containing Tyr and/or Trp at selected positions as fluorescence energy donors were synthesized, and their Tb(3+)-binding capacity was assessed. The four potential Ca(2+)-binding sequences in the GPIIb subunit, GPIIb 242-255, 296-309, 364-377, and 425-438, were functional, despite lacking the usual Glu residue at the terminal coordination position. These peptides bound Tb3+ with the same affinity as typical Ca(2+)-binding loop peptides and also bound Ca2+ and other divalent cations without preference. Of the two candidate GPIIIa sequences, 118-131 and 208-221, the former bound Tb3+ and divalent cations with an affinity similar to that of the GPIIb peptides, whereas the latter peptide was not functional. This functional difference, as well as data obtained with substituted peptides, emphasizes the importance of the first coordination position for interaction of synthetic peptide loops with cations. Together, these data identify the five cation-binding sites within intact GPIIb-IIIa.

Localization of 13-hydroxyoctadecadienoic acid and the vitronectin receptor in human endothelial cells and endothelial cell/platelet interactions in vitro.

Blood/vessel wall cell interactions depend, in part, on the expression of adhesion receptors on cell surfaces, such as expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells (ECs) for platelet/EC adhesion. However, it is unclear how receptor expression is regulated from within cells. In previous studies, we found that ECs metabolize linoleic acid into the lipoxygenase monohydroxide, 13-hydroxyoctadecadienoic acid (13-HODE), and that the intracellular level of 13-HODE correlates inversely with VnR expression and platelet adhesion to the EC apical surface. In this study, we determined the physical associations of 13-HODE and VnR in unstimulated and stimulated ECs, ie, at times when ECs were and were not adhesive for specific ligands and platelets, using double antibody immunofluorescent staining techniques and binding assays. 13-HODE and the VnR were colocalized within unstimulated ECs. When ECs were stimulated, 13-HODE was no longer detectable, either in or outside the ECs, and the VnR was detected on the apical surface of the ECs. These changes were paralleled by increased vitronectin binding and increased platelet adhesion to the ECs. We suggest that colocalization of 13-HODE with VnR reflects a 13-HODE/VnR interaction, confining the VnR in a nonadhesive form inside unstimulated ECs, and, as a result, the ECs are nonadhesive. When the ECs are stimulated, 13-HODE and VnR dissociate, allowing the VnR to relocate on the EC surface, where the VnR undergoes a conformational change resulting in increased EC adhesivity.

Interleukin 1-induced cancer cell/endothelial cell adhesion in vitro and its relationship to metastasis in vivo: role of vessel wall 13-HODE synthesis and integrin expression.

Previously, we have demonstrated that stimulation of endothelial cells (ECs) with interleukin-1 alpha (IL-1 alpha) enhances the synthesis and expression of the vitronectin receptor (VnR), promotes VnR-dependent adhesion of human A549 adenocarcinoma cells to ECs, and is associated with decreased EC 13-hydroxyoctadecadienoic acid (13-HODE) synthesis in vitro. To determine whether these observations are relevant in vivo, we examined the acute retention and subsequent metastasis of intravenously-injected B16F10 melanoma cells in murine lungs, in relation to vessel wall 13-HODE. In C57BL/6 mice pretreated with IL-1 alpha, vessel wall 13-HODE was decreased and B16F10 lung entrapment and metastasis were increased. The latter two events were blocked by pretreating the animals with the GRGDS peptide. These data suggest a relationship between vessel wall 13-HODE synthesis, adhesion molecule expression, and adhesion of B16F10 cells to the endothelium.

Platelet adhesion to exposed endothelial cell extracellular matrixes is influenced by the method of preparation.

The relative thrombogenicity of extracellular matrixes (ECMs) produced by cultured human umbilical endothelial cells (ECs) was studied under flow conditions. ECMs were prepared using a number of physical and chemical methods, and their reactivity toward platelets was morphometrically evaluated. von Willebrand factor (vWF), fibronectin (FN), and 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) were also determined. We found that platelet adhesion to ECMs differed significantly, both quantitatively and qualitatively, with the method of ECM preparation. Mechanically prepared ECM exposed a less thrombogenic surface compared with ECM prepared by chemical methods (platelet-covered surface of 20% and 50%, respectively). Evaluation of the ECM components vWF, FN, and 13-HODE showed significant changes, both in their concentrations and distribution patterns, depending on the method of ECM preparation. The decrease measured in the levels of ECM-associated vWF (from 108 to 9.2 ng/10(4) cells) and the minor changes observed in the distribution pattern of subendothelial FN did not appear to be sufficient to explain the altered platelet adhesion observed in our model. This suggests that the amount of 13-HODE probably associated to the remaining ECs present in the mechanically exposed ECM could be one factor that specifically contributed to the nonthrombogenic state of these preparations. We conclude that the degree of ECM reactivity toward platelets is dependent on the method of ECM preparation and that this is related to the removal of specific EC/ECM components that modulate their thromboresistant/thrombogenic properties. This fact should be taken into account when ECMs produced by cultured ECs are used in platelet adhesion studies.

Effects of endothelial cell treatment on 13-HODE and prostacyclin synthesis and its correlation with tumor cell-vascular endothelial cell adhesion.

Adhesion of tumor cells to vascular endothelial surfaces is one of the key steps in metastatic dissemination. Several factors are believed to be implicated in the regulation of the adhesive properties of tumor cells. We show that the adhesion of five different tumor cell lines, all of them of human origin, to human umbilical vascular endothelial cells (ECs) significantly increases following pretreatment of ECs with the cytokines interleukin-1 and tumor necrosis factor, whereas tumor cell/EC interactions remained unchanged after incubation with interferon-gamma. Significant augmentation in tumor cell adhesion was also observed when ECs were treated with the lipoxygenase inhibitors salicylate and the compound BW755C. In all cases, increased tumor cell adhesion was concomitant with significant decreases in the EC levels of linoleic acid, lipoxygenase-derived metabolite 13-hydroxy-octadecadienoic acid (13-HODE). On the contrary, pretreatment of the EC monolayers with aspirin did not result in any changes towards tumor cell adhesion. These results suggest that tumor cell/EC interaction is modulated, at least in part, by intracellular levels of 13-HODE and is independent of prostacyclin (PGI2) production by the ECs.

13-Hydroxyoctadecadienoic acid (13-HODE) metabolism and endothelial cell adhesion molecule expression: effect on platelet vessel wall adhesion.

Endothelial cells synthesize two important fatty acid metabolites, PGI2, which is synthesized from arachidonic acid via the cyclooxygenase pathway, and 13-HODE, which is synthesized from linoleic acid via the lipoxygenase pathway. PGI2 is synthesized following cell activation or injury while 13-HODE is synthesized in the unstimulated cell. While the role of PGI2 in platelet vessel wall interactions has been studied extensively, the role of 13-HODE in platelet vessel wall interactions is just now being understood. The present evidence suggests that 13-HODE is continuously synthesized in "resting" vessel wall cells and is in close juxtaposition with the ubiquous integrin adhesion molecule, the vitronectin receptor. The observation that the endothelial cell is not adhesive when 13-HODE and the vitronectin receptor are in close association and becomes adhesive when these two moieties dissociate and the vitronectin receptor relocates on the surface of the cell, provides further evidence that 13-HODE may induce conformational changes in the vitronectin receptor to reduce its ability to recognize its adhesive ligands. The additional observations that 13-HODE levels in both human and animal vessel walls are inversely related with vessel wall adhesivity, and that this adhesivity can be altered by altering 13-HODE synthesis, provides evidence that 13-HODE down-regulates the thrombogenecity of the injured vessel wall surface.

Selective effects of dietary fats on vascular 13-HODE synthesis and platelet/vessel wall interactions.

Fish oil (FO) diets are associated with decreased thrombosis, which is though to be related, in part, to changes in platelet and vessel wall prostanoid synthesis. Recently, we found that 13-hydroxyoctadecadienoic acid (13-HODE) synthesized in the vessel wall from linoleic acid (LA, 18:2 n-6) via the lipoxygenase pathway, also decreases platelet/vessel wall interactions. Thus, we determined whether diets containing fish oil, walnut oil (rich in linoleic acid), black currant seed oil (rich in both linoleic and gamma linolenic acids, 18:3 n-6), or lard influenced vessel wall 13-HODE synthesis and platelet/vessel wall adhesion in rabbits. In vivo, vessel wall thrombogenicity was decreased in animals fed the black currant seed oil rich diet for 4 weeks as compared to the control "LARD" diet. This latter effect was better obtained when gamma linoleic acid was present suggesting a secondary effect of this fatty acid. The decreased vessel wall thrombogenicity in those animals, was associated with increased vessel wall 13-HODE synthesis. In contrast, ex vivo platelet adhesivity was significantly decreased in the fish oil diet fed animals, as compared to the control "LARD" diet and correlated with decreased platelet 12-HETE synthesis. We conclude that both fish oil and black currant seed oil rich diets inhibit platelet/vessel wall adhesion; the black current seed oil diet by increasing the availability of linoleic acid for 13-HODE synthesis and inhibiting vessel wall thrombogenicity; the fish oil diet, by inhibiting platelet 12-HETE synthesis and subsequent platelet adhesion.

Regulation of tumor cell adhesion by intracellular 13-HODE: 15-HETE ratio.

We performed studies to determine whether tumor cells (TCs) produce 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE), and to determine the relationship between TC and endothelial cell (EC) 13-HODE and 15-HETE synthesis, and TC adhesion to ECs and their underlying extracellular matrix (ECM). We measured (1) the amounts and ratios of 13-HODE: 15-HETE in three different human TC lines and in three different murine TC lines under basal and stimulated conditions; and (2) the relationship between 13-HODE synthesis and cAMP levels in TCs and ECs. Under basal conditions, TCs produced both 13-HODE and 15-HETE, the intracellular ratios of which correlated with TC adhesivity. Stimulation of the TCs with the chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine, decreased 13-HODE synthesis, and increased 15-HETE synthesis and TC adhesion to ECs and to their ECM. Alternatively, enhancing 13-HODE synthesis in either TCs or ECs (by elevating the resting levels of intracellular cAMP) was associated with decreased TC adhesion to ECs and ECM. These results suggest that intracellular 13-HODE: 15-HETE ratio in TCs regulates TC adhesivity, and that an alteration in 13-HODE: 15-HETE ratio will markedly influence TC adhesion.

Relationship between vessel wall 13-HODE synthesis and vessel wall thrombogenicity following injury: influence of salicylate and dipyridamole treatment.

We performed studies to determine the relationship between injured vessel wall thrombogenicity, vessel wall 13-hydroxyoctadecadienoic acid (13-HODE) synthesis and cAMP levels in rabbit treated with salicylate or dipyridamole. Injured vessel wall thrombogenicity was measured as the number of 3H-adenine labelled platelets adhered to the subendothelial basement membrane exposed by air injury in carotid arteries of rabbits treated orally with salicylate or dipyridamole. Vessel wall 13-HODE was measured by HPLC and vessel wall cAMP was measured by RIA. Vessel wall thrombogenicity was increased two-fold in rabbits treated with salicylate and decreased by half in rabbits treated with dipyridamole. The levels of vessel wall cAMP levels were correlated both with the plasma dipyridamole levels and increases in 13-HODE synthesis. cAMP levels were unaffected by salicylate treatment, but 13-HODE synthesis was decreased. We conclude that there is a significant relationship between vessel wall cAMP levels and 13-HODE synthesis, which in turn, influences subsequent vessel wall thrombogenicity.

Cyclic AMP regulation of endothelial cell triacylglycerol turnover, 13-hydroxyoctadecadienoic acid (13-HODE) synthesis and endothelial cell thrombogenicity.

The 15-omega-lipoxygenase enzyme in endothelial cells metabolizes endogenous linoleic acid (18:2) into 13-hydroxyoctadecadienoic acid (13-HODE) under basal conditions, i.e., in unstimulated endothelial cells. 13-HODE is thought to regulate the non-adhesivity of the endothelium, contributing to vessel wall/blood cell biocompatibility. We performed experiments, therefore, to determine the relationship between basal levels of cAMP, 13-HODE synthesis, and platelet/endothelial cell adhesion. We found that 13-HODE synthesis increased with elevated cAMP levels and that the elevated 13-HODE levels correlated with increased 18:2 turnover in the triacylglycerol pool. In contrast, neither 18:2 nor arachidonic acid (20:4) turnover in the phospholipid nor prostacyclin (PGI2) production were changed with elevated cAMP levels. Platelet/endothelial cell adhesion was inversely proportional to 13-HODE synthesis. We conclude that intracellular 13-HODE influences platelet/vessel wall interactions, is synthesized from 18:2 released from the endogenous triacylglycerol pool, and that this pathway is modulated by intracellular cAMP levels.