Niamh Moran

Integrins as therapeutic targets: lessons and opportunities

The integrins are a large family of cell adhesion molecules that are essential for the regulation of cell growth and function. The identification of key roles for integrins in a diverse range of diseases, including cancer, infection, thrombosis and autoimmune disorders, has revealed their substantial potential as therapeutic targets. However, so far, pharmacological inhibitors for only three integrins have received marketing approval. This article discusses the structure and function of integrins, their roles in disease and the chequered history of the approved integrin antagonists. Recent advances in the understanding of integrin function, ligand interaction and signalling pathways suggest novel strategies for inhibiting integrin function that could help harness their full potential as therapeutic targets.

Conformation-Specific Blockade of the Integrin GPIIb/IIIa: A Novel Antiplatelet Strategy That Selectively Targets Activated Platelets

Platelet activation causes conformational changes of integrin GPIIb/IIIa (&agr;IIb&bgr;3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

The Platelet Integrin αIIbβ3 Has an Endogenous Thiol Isomerase Activity

Integrins are cysteine-rich heterodimeric cell-surface adhesion molecules that alter their affinity for ligands in response to cellular activation. The molecular mechanisms involved in this activation of integrins are not understood. Treatment with the thiol-reducing agent, dithiothreitol, can induce an activation-like state in many integrins suggesting that cysteine-cysteine dithiol bonds are important for the receptors tertiary structure and may be involved in activation-induced conformational changes. Here we demonstrate that the platelet-specific integrin, αIIbβ3, contains an endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif, CXXC, in each of the cysteine-rich repeats of the β3 polypeptide. This motif comprises the active site in enzymes involved in disulfide exchange reactions, including protein-disulfide isomerase (EC 5.3.4.1) and thioredoxin. Intrinsic thiol isomerase activity is also observed in the related integrin, αvβ3, which shares a common β-subunit. Thiol isomerase activity within αIIbβ3 is time-dependent and saturable, and is inhibited by the protein-disulfide isomerase inhibitor, bacitracin. Furthermore, this activity is calcium-sensitive and is regulated in the EDTA-stabilized conformation of the integrin. This novel demonstration of an enzymatic activity associated with an integrin subunit suggests that altered thiol bonding within the integrin or its substrates may be locally modified during αIIbβ3 activation.

A Sequence within the Cytoplasmic Tail of GpIIb Independently Activates Platelet Aggregation and Thromboxane Synthesis

All integrin α subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989–995, of the platelet integrin subunit glycoprotein GpIIb (αIIb), palmitoyl-KVGFFKR (Ppep; 10 μm), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A2 (TXA2) synthesis (EC50 = 45 ± 5 μm), secretion of α-granules detected as enhanced surface expression of P-selectin (EC50 = 52 ± 8 μm), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC50 = 3.7 ± 1 μm). The TXA2 receptor antagonist, SQ29548, PGE1, and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991–994) as the critical residues in all peptide-mediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA2 synthesis and secretion.

Characterization of the kinetics of neural cell adhesion molecule homophilic binding

A solid‐phase assay has been developed for the investigation of the kinetics of neural cell adhesion molecule (NCAM) binding. Using this assay we can show that NCAM binds to itself in a time‐dependent and saturable manner. Binding constants (K B values) of 6.9 × 10−8 M and 1.23 × 10−6 M, respectively, were obtained for adult and newborn rat NCAM homophilic binding. Binding is specifically inhibited by Fab′ fragments of polyclonal anti‐NCAM antibodies but is unaffected by heparin or chondroitin sulphate. This indicates that the NCAM homophilic binding site is separate from and independent of the heparin‐binding site and that a developmental modification, probably polysialation, gives rise to marked differences in the adhesive properties of NCAM.

A novel and essential role for FcγRIIa in cancer cell–induced platelet activation

Platelets play a role in cancer by acting as a dynamic reservoir of effectors that facilitate tumor vascularization, growth, and metastasis. However, little information is available about the mechanism of tumor cell-induced platelet secretion (TCIPS) or the molecular machinery by which effector molecules are released from platelets. Here we demonstrate that tumor cells directly induce platelet secretion. Preincubation of platelets with human colon cancer (Caco-2), prostate cancer (PC3M-luc), or breast cancer cells (MDA-MB-231;MCF-7) resulted in a marked dose-dependent secretion of dense granules. Importantly, TCIPS preceded aggregation which always displayed a characteristic lag time. We investigated the role of platelet receptors and downstream molecules in TCIPS. The most potent modulators of TCIPS were the pharmacologic antagonists of Syk kinase, phospholipase C and protein kinase C, all downstream mediators of the immunoreceptor tyrosine-based activation motif (ITAM) cascade in platelets. Supporting this, we demonstrated a central role for the immune Fcγ receptor IIa (FcγRIIa) in mediating platelet-tumor cell cross-talk. In conclusion, we demonstrate that cancer cells can promote platelet dense-granule secretion, which is required to augment platelet aggregation. In addition, we show a novel essential role for FcγRIIa in prostate cancer cell-induced platelet activation opening the opportunity to develop novel antimetastatic therapies.

Continued Thromboxane A2 Formation Despite Administration of a Platelet Glycoprotein IIb/IIIa Antagonist in Patients Undergoing Coronary Angioplasty

Experimental data suggest that formation of thromboxane A2 may be suppressed during administration of a glycoprotein IIb/IIIa antagonist. We determined the dose of one such compound, fradafiban, required to provide > 80% occupancy of the platelet glycoprotein IIb/IIIa and examined its effects on thromboxane A2 formation in patients undergoing PTCA. The dose response to fradafiban and additional effects of aspirin were explored initially in patients with stable coronary artery disease. Fradafiban induced a dose-dependent inhibition of platelet aggregation that correlated with fibrinogen receptor occupancy and plasma drug concentration. Addition of aspirin 300 mg had no effect on these parameters. At the highest dose, mean fibrinogen receptor occupancy was 89.7 +/- 1.2% (n = 3) at 4 hours and platelet aggregation had decreased by 93.4 +/- 2.7%. Eighteen patients undergoing coronary angioplasty were randomized to receive either aspirin 330 mg or that dose of fradafiban producing > 80% fibrinogen receptor occupancy. Platelet aggregation was suppressed throughout the infusion of fradafiban to a greater extent than with aspirin. However, there was a marked increase in urinary excretion of 11-dehydrothromboxane B2 in patients treated with fradafiban: from 1973 +/- 889 to a peak of 9760 +/- 3509 pg/mg creatinine (P = .0046). Despite this evidence of continued platelet activation in vivo, there were no cases of coronary thrombosis. In conclusion, fradafiban suppresses platelet aggregation and may be a useful alternative to aspirin in the prevention of thrombotic events in patients undergoing PTCA. However, there is continued formation of thromboxane A2, which may continue to exert its effects as a potent vasoconstrictor and vascular smooth muscle mitogen.

A palmitylated peptide derived from the glycoprotein Ibβ cytoplasmic tail inhibits platelet activation

Summary.  The platelet receptor GPIb/IX/V mediates a crucial role in hemostasis, yet the signaling mechanisms involved are incompletely understood. The complex consists of four polypeptides GPIbα, GPIbβ, GPIX and GPV. We identified an amino acid sequence in the cytoplasmic tail of the GPIbβ subunit between residues R151 and A161 that is highly conserved across species and hypothesized that it has functional importance. To target this motif, we synthesized a corresponding cell‐permeable palmitylated peptide (Pal‐RRLRARARARA) and investigated its effect on platelet function. Pal‐RRLRARARARA completely inhibited low dose thrombin‐ and ristocetin‐induced aggregation in washed platelets but only partially inhibited collagen‐ and U46619‐induced aggregation. Thromboxane production in platelets stimulated with thrombin was significantly reduced by Pal‐RRLRARARARA compared with collagen. Activation of the integrin αIIbβ3 in response to thrombin was significantly reduced when platelets were preincubated with Pal‐RRLRARARARA. The adhesion of washed platelets to von Willebrand factor (VWF) under static conditions was significantly reduced by Pal‐RRLRARARARA. Under conditions of high shear, the velocity of platelets rolling on VWF was significantly increased when platelets are preincubated with Pal‐RRLRARARARA. This study defines a novel function for the RRLRARARARA motif of GPIbβ in platelet activation.

Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

BackgroundProtein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown.ResultsAs a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKAs primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma.ConclusionsPTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

Bacitracin reveals a role for multiple thiol isomerases in platelet function.

The platelet‐specific integrin αIIbβ3 has endogenous thiol isomerase activity associated with the CXXC motifs within the β subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited αIIbβ3 activation, as detected by PAC‐1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified αIIbβ3 with a 50% inhibitory concentration of 15·5 μmol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin‐independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of αIIbβ3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.