Platelet Integrin alphaIIbbeta3 Research Articles

Comparison of the RGD- and AGDV-Containing Peptide Interactions with the Platelet Integrin Alphaiibbeta3

Comparison of the RGD- and AGDV-Containing Peptide Interactions with the Platelet Integrin Alphaiibbeta3

The Platelet Integrin alphaIIbbeta3 Differentially Interacts with Fibrin and Fibrinogen

Fibrinogen binding to the integrin αIIbβ3 can mediate platelet aggregation and platelet spreading on fibrinogen-coated surfaces. However, in vivo, where blood clotting is catalyzed by thrombin, αIIbβ3 activation and fibrinogen conversion to fibrin occur simultaneously, although the relative contributions of fibrinogen versus monomeric and polymerized fibrin to αIIbβ3-mediated platelet aggregation and adhesion are unknown. The interaction of αIIbβ3 with fibrin is responsible for clot retraction. Nonetheless, differences in the efficacy of αIIbβ3 antagonists with regard to clot retraction versus platelet aggregation suggest that the interaction of αIIbβ3 with fibrinogen and fibrin is different. Here, we have compared nanomechanical measurements of the interaction of αIIbβ3 with fibrin and fibrinogen to explain these differential effects. Briefly, a microscopic bead covalently coated with purified αIIbβ3 was captured by a focused laser beamand repeatedly brought into contact with surface-attached fibrinogen, monomeric fibrin produced by treating the immobilized fibrinogen with thrombin, or a naturally-formed hydrated fibrin fiber at the edge of a plasma clot. When an αIIbβ3-ligand complex was detected, the force required to dissociate the complex was measured at piconewton resolution. By analyzing the distribution of these forces, we determined the overall reactivity and the binding strength of the interacting protein pairs. Besides native fibrinogen, experiments were performed with recombinant fibrinogen variants. Rupture force histograms for αIIbβ3-fibrinogen interactions displayed forces ranging from 20 pN to 140 pN and could be segregated into moderate (20-60 pN) and strong (>60 pN) interactions. The inhibitory effects of EDTA and αIIbβ3antagonists such as abciximab and eptifibatide confirmed that fully functional αIIbβ3 was required to obtain these force signals. Monomeric fibrin displayed a higher cumulative probability of interacting with αIIbβ3 and a greater binding strength. αIIbβ3-fibrin interactions were also less sensitive to inhibition by abciximab and eptifibatide, suggesting that they had a different binding specificity. Both fibrinogen- and fibrin- interactions with αIIbβ3 were partially inhibited by RGD-containing peptides, suggesting the existence of common RGD-containing binding motifs. This assumption was supported using the fibrin variants αD97E or αD574E whose mutated RGD motifs were less reactive with αIIbβ3 than those of wild type fibrin. Monomeric fibrin made from a homodimeric fibrinogen γ chain splice variant lacking the γC αIIbβ3 binding motif was more reactive with αIIbβ3 than the parent fibrinogen, suggesting that this binding motif is less important in fibrin. Polymeric fibrin displayed a rupture force profile similar to fibrinogen and monomeric fibrin with moderate (20-60 pN) and strong (>60 pN) forces that peaked at 70-80 pN. The αIIbβ3 antagonist eptifibatide, the αVβ3-specific cyclic RGD-containing peptide XJ735, a non-selective cyclic RGD-containing peptide, and a dodecapeptide corresponding to the C-terminus of the fibrinogen γ chain each produced a sharp drop in the medium-to-high force range. Interaction forces >60 pN were more effectively reduced by EDTA than by any of the inhibitory peptides, indicating their dependence on the structural integrity and functionality of αIIbβ3. The free γC dodecapeptide was also less efficient than the cRGD peptides or eptifibatide in preventing the stronger interactions. Taken together, these results demonstrate that surface-bound fibrinogen and monomeric, as well as polymeric fibrin, are highly reactive with the αIIbβ3. Fibrin is more reactive than fibrinogen in terms of its binding probability and it has greater binding strength. Fibrin binding to αIIbβ3 is also less sensitive to αIIbβ3 inhibitors, suggesting that fibrin and fibrinogen have distinct binding requirements. In particular, the maintenance of αIIbβ3-binding activity when the C-terminus of the fibrinogen γ chain and the α chain RGD sequences are mutated suggests that the αIIbβ3-binding sites in fibrin are not confined to its known γ chain and RGD motifs. DisclosuresNo relevant conflicts of interest to declare.

A Mechanism For Switch Of Integrin Signaling Direction and a New Anti-Thrombotic Strategy Through Selective Outside-In Signaling Inhibition

Antagonists of platelet integrin alphaIIbbeta3 are potent anti-thrombotics due to critical roles of integrins in thrombosis. However, integrins are also important in hemostasis, and thus integrin antagonists have potentially life-threatening bleeding side effect. It would be ideal if we can develop integrin antagonists without bleeding side effect. Integrins transmit signals bidirectionally. Intracellular signals activate integrin alphaIIbbeta3, leading to talin-dependent integrin ligation, which is required for platelet adhesion and initial hemostatic thrombus formation. Integrin ligation in turn mediates Galpha13/Src-dependent outside-in signaling that stabilizes and amplify thrombi, which is crucial for occlusive arterial thrombosis. Here we show that talin and Galpha13 bind to mutually exclusive but distinct sites in integrin beta3, and their bindings are dynamically regulated during integrin signaling. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for early phase outside-in signaling. Integrin ligation induces talin dissociation and Galpha13 binding, which selectively mediates early phase outside-in signaling. The second talin binding wave is associated with late phase outside-in signaling and clot retraction. Based on these findings, we have designed a selective inhibitor of Galpha13-integrin interaction, which specifically abolished outside-in signaling without affecting inside-out signaling and integrin ligation. Strikingly, this inhibitor potently inhibits occlusive thrombosis in vivo, but has no effect on tail bleeding time, in contrast to the current integrin antagonists. Thus, we have discovered a mechanism for switching the direction of integrin signaling and a new anti-thrombotic that does not cause bleeding. Disclosures:No elevant conflicts of interest to declare.

Mitochondrially-Mediated Platelet Integrin AlphaIIbbeta3 Inactivation Limits Platelet Recruitment and Thrombus Growth,

Abstract 3251Platelet stimulation with strong agonist(s) results in the formation of a phenotypically unique platelet subpopulation known as procoagulant platelets. All platelets stimulated in strongly-activating conditions undergo granule release, activation of integrin αIIbβ3, and spreading. However, minutes later a subpopulation of these activated platelets undergo additional phenotypic changes including phosphatidylserine (PS) exposure, adoption of a vesiculated and balloon-like morphology, and an altered integrin αIIbβ3 conformation that has a decreased binding affinity for activation-dependent antibodies such as PAC-1 or JON/A. Deletion of the mitochondrial protein cyclophilin D (CypD), a critical regulator of mitochondrial permeability transition pore (mPTP) formation, results in marked abrogation of procoagulant platelet formation. We have previously demonstrated remarkable impairment of dual-agonist-initiated PS exposure and procoagulant activity in CypD−/− platelets (Jobe et al, Blood 2008). In this study the functional importance of the morphological and adhesive changes that occur in procoagulant platelets were examined using CypD−/− platelets and mice with platelet-specific deficiency of CypD. (CypDplt−/−).The morphology and phenotype of fibrinogen-adherent CypD+/+ (WT) and CypD−/− platelets were examined following dual stimulation with thrombin and the GPVI agonist convulxin. In dual-stimulated WT platelets, platelets initially spread; then after three minutes, nearly half of the platelets demonstrated lamellopodial and filipodial retraction with platelet rounding and vesiculation, and these changes were associated with increased PS exposure and decreased binding of JON/A. These changes were delayed in CypD−/− platelets, but eventually started to be seen 30 minutes subsequent to stimulation. Adhesion and platelet recruitment to strongly-stimulated platelets were studied in flow conditions. Increased platelet accumulation was noted when unstimulated platelets were flowed over strongly-stimulated adherent CypD−/− platelets relative to WT platelets. Similarly, increased platelet accumulation of CypD−/− platelets was noted on a collagen surface compared to WT platelets. In platelet aggregation assays, dual stimulated WT platelets demonstrated a chaotic pattern of aggregation two to three minutes after activation consistent with aggregate disruption, a pattern that was not observed in CypD−/− platelets. Together these studies indicate that procoagulant platelet formation results in integrin inactivation. Since this process is impaired in the absence of CypD, CypD−/− platelets demonstrate increased platelet accumulation in flow assays and stable aggregate formation. Adhesive and cytoskeletal proteins were investigated in strongly-stimulated platelets. Western blot analysis demonstrated significant proteolytic cleavage of both talin and the cytoplasmic domain of integrin β3 in WT platelets stimulated with thrombin plus convulxin, and these events were only minimally observed in single-agonist stimulated platelets. Proteolytic cleavage of both talin and integrin β3 were markedly decreased in CypD−/− platelets and in WT platelets treated with calpain inhibitors.CypD is a ubiquitously expressed protein; therefore, in vivo thrombosis and hemostasis were tested in CypDplt−/− mice. Following photochemically mediated mesenteric endothelial injury, time to occlusion was shortened in CypDplt−/− mice (723 ± 111 sec in CypDplt+/− vs. 371 ± 106 sec in CypDplt−/− mice), and complete arterial occlusion was increased in CypDplt−/− mice (55% in CypDplt+/− vs. 86% in CypDplt−/−) (p=0.02). Our in vitro and in in vivo results are consistent with the hypothesis that in strongly-stimulated platelets mitochondrially-mediated events initiate proteolytic cleavage of talin and integrin β3 accompanied by platelet integrin αIIbβ3 inactivation, and that this process limits platelet recruitment and thrombus growth. Since these events are limited to platelets stimulated by strong agonist(s), these results suggest a novel negative feedback mechanism initiated by accumulation of multiple or strong agonist(s) that limits thrombotic occlusion in vivo. Disclosures:No relevant conflicts of interest to declare.

Exploring New Druggable Binding Sites of Platelet Integrin αIIbβ3

The major ligand binding site of platelet integrin alphaIIbbeta3, an essential receptor for hemostasis and thrombosis, lies between the head regions of its alpha and beta subunits. The Arginine-Glycine-Aspartic Acid (RGD) cell attachment sequence mediates the binding of several different adhesive plasma proteins to integrin alphaIIbbeta3. This sequence binds to both alphaIIb and beta3 residues in the ligand binding pocket, as well as to a divalent metal ion contained in the beta3 subunit. We recently identified a novel alphaIIbbeta3 antagonist (RUC-1) that binds exclusively to alphaIIb. In the present study we searched for additional new druggable binding sites of the integrin alphaIIbbeta3 headpiece along the conformational transition between its closed and open (swung-out) conformations. Specifically, we considered ten different alphaIIbbeta3 conformations resulting from a linear interpolation between the two extreme conformations of alphaIIbbeta3, and relaxed them by multi-nanosecond molecular dynamics simulations. Each trajectory was clustered by root mean square deviation over the protein Calpha atoms, and each cluster medoid was used as an input to a recent energy-based mapping algorithm (FTMAP) that searches an entire protein surface for consensus binding regions for a number of small organic probe molecules. Our results reveal new pockets at the beginning of the conformational transition whose structural information might be used as the basis for rational drug design of novel therapeutics targeting integrin alphaIIbbeta3.

Clopidogrel Pharmacogenetics and Its Clinical Implications

Pharmacogenetics encompasses a range of phenomena ranging from pharmacology to therapeutics to toxicology and generally focuses on the study of genetic factors related to interindividual variability in drug response. Individual differences in the rate of platelet reactivity markedly influence normal hemostasis and the pathologic outcome of thrombosis. Response to clopidogrel varies widely, with nonresponse rates in various studies ranging from 4%-30% at 24 hours. Polymorphism involved in the response to antithrombotic drugs has been described in components of the hemostatic and thrombotic systems, and these include genetic variations. Polymorphisms in the hepatic enzymes involved in the metabolism of clopidogrel (e.g., CYP 1A2, CYP3A4, CYP2C19) or within the platelet membrane receptor (P2Y12) and/or polymorphism of platelet integrin alphaIIbbeta3 or integrin alpha2beta1 may affect platelet responses and could influence response to clopidogrel administration. In this review, we discuss the pharmacogenetics of clopidogrel related to the phenomenon of response variability and its clinical implications.

No evidence for systemic platelet activation during or after orthotopic liver transplantation

Platelet function is thought to deteriorate during liver transplantation as a result of platelet activation and proteolysis of platelet receptors by plasmin following reperfusion. However, this hypothesis has never been formally tested. Twenty patients undergoing a first or second liver transplant were included in the study. Blood samples were taken at standardized time points during transplantation and up to 10 days after transplantation. Platelet activation was assessed by detection of the activation markers P-selectin and activated integrin alphaIIbbeta3 with flow cytometry. Proteolytic cleavage of platelet receptors was assessed by flow cytometry measurement of the constitutively expressed platelet receptors glycoprotein Ibalpha and integrin alphaIIbbeta3. In addition, using enzyme-linked immunosorbent assay techniques, we measured plasma levels of platelet activation products beta-thromboglobulin and platelet factor 4 and plasma levels of cleaved fragments of glycoproteins Ibalpha and V. Flow cytometry analyses provided no evidence of substantial platelet activation during transplantation. In fact, the expression of activated integrin alphaIIbbeta3 decreased postoperatively; this indicated that platelets were in a slightly activated state prior to surgery. Plasma levels of beta-thromboglobulin and platelet factor 4 also substantially decreased after transplantation. In addition, no changes were observed in the constitutively expressed platelet receptors or in the plasma levels of platelet receptor fragments, and this indicated a lack of substantial receptor proteolysis. In conclusion, no evidence was found for significant activation of circulating blood platelets or the proteolysis of key platelet receptors during liver transplantation. These findings suggest that the platelet functional capacity does not decrease during liver transplantation. Liver Transpl 15:956-962, 2009. (c) 2009 AASLD.

The beta3 integrin cytoplasmic tail: protein scaffold and control freak.

Platelet integrin alphaIIbbeta3 plays an essential role in thrombus formation through interactions with adhesive ligands. Successful parenteral blockade of these interactions has validated alphaIIbbeta3 as a therapeutic target in cardiovascular medicine. However, oral alphaIIbbeta3 antagonists have not been successful and there is an unmet need for more effective anti-platelet drugs. Growing evidence points to the cytoplasmic tails of alphaIIb and beta3, and the beta3 tail in particular, as scaffolds for intracellular proteins that mediate inside-out signaling and regulate alphaIIbbeta3 affinity for ligands. Intracellular protein interactions with the integrin cytoplasmic tails also regulate outside-in signals to the actin cytoskeleton. Here we focus on recent studies that illustrate the relevance of the beta3 cytoplasmic tail as a regulatory scaffold in vivo. We speculate that this scaffold or its interacting proteins may serve as therapeutic targets for the development of future anti-thrombotic drugs.

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated alpha2beta1 activation.

The platelet alpha2beta1 integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that alpha2beta1 function can be activated via inside-out signaling, similar to the prototypical platelet integrin alphaIIbbeta3. However, signaling molecules that regulate alpha2beta1 activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates alphaIIbbeta3 activation. We hypothesized that Rap1b positively regulates alpha2beta1 during agonist-induced platelet activation. To test whether Rap1b activates alpha2beta1 downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b-/- or wild-type mice with diverse agonists and measured alpha2beta1 activation using fluorescein isothiocyanate-labeled monomeric collagen. We also examined the role of Rap1b in outside-in signaling pathways by analyzing adhesion and spreading of Rap1b-/- or wild-type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b-mediated events. Rap1b-/- platelets displayed comparable ADP-induced or thrombin-induced alpha2beta1 activation as wild-type platelets, but reduced convulxin-dependent alpha2beta1 activation. Rap1b-/- platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild-type platelets. Rap1b-/- platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. Rap1b is required for maximal GPVI-induced but not ADP-induced activation of alpha2beta1 in murine platelets.

Risk Determinants of Arterial Thrombosis Increase the Risk of Intrauterine Growth Restriction (IGR)

Background: The HPA1-b allele of the beta3-subunit of the essential platelet integrin AlphaIIb-beta3 is a risk factor for increased platelet thrombogenicity, leading to arterial vascular occlusion also triggered by inflammatory endothelial alterations. We performed a case-control study to assess hereditary risk factor for arterial thrombosis in addition to the known risk determinants for venous thrombosis as risk determinants for fetal IGR.Materials and Methods: 116 women with severe fetal IGR (defined by birth weight below the 5th-percentile for gestational age and sex) and 300 normal women were evaluated. Women with other reasons of IGR (history of venous thrombosis, fetal loss, and preeclampsia, neonatale immunthrombocytopenia) were excluded. The fetuses were born alive after the 24th week of gestation.Results: A significant risk association could be shown for the HPA-1b/1b genotype OR 3.1 (95%CI 1.2–8.8), increased levels of lipoprotein (a) (>0.2g/l) OR 1.7 (95%CI 1.0–2.9), increased levels of fibrinogen (>median 284mg/dl) OR 2.8 (95%CI 1.8–4.4) increased levels of FVIII:C (>median 147%) OR 1.7 (95%CI 1.1–2.7), and increased levels of von-Willebrand-factor antigen (vWF-Ag) (>median) OR 2.7 (95%CI 1.1–2.7). The combination of risk determinants led to a further increase in risk (e.g. HPA-1b allele, increased lipoprotein (a) and vWF-Ag OR 14.0) There was no significant risk association for factor V Leiden G1691A OR 1.3 (95% CI 0.7–2.81), and prothrombin G20210A mutation. OR 2.0 (95% CI 0.6–6.5)Conclusions: Increased platelet thrombogenicity via interaction with an inflammatory vascular process and/or via plasma levels of components of hemostasis/receptor ligands appear to be more important in women with IGR than the established risk determinants for venous thrombosis.

Tickling the tails: cytoplasmic domain proteins that regulate integrin αIIbβ3 activation

Integrin alphaIIbbeta3 activation is essential for platelet aggregation and related hemostatic events. In recent years, intense effort has been put forward to understand the molecular mechanisms regulating platelet integrin alphaIIbbeta3 activation. Here we review the current models of alphaIIbbeta3 activation and highlight the potential regulatory roles of proteins that interact directly with the alphaIIbbeta3 cytoplasmic domains, with emphasis on the alphaIIb cytoplasmic domain binding protein, CIB1. Mutational and crystallographic studies reveal the importance of integrin transmembrane and cytoplasmic domains in propagating bidirectional signaling events. Proteins that interact directly with the integrin cytoplasmic domains may play important roles in mediating these signaling events. Of particular interest is the interaction between CIB1 and the alphaIIb tail which may function to negatively regulate alphaIIbbeta3 activation. In addition, a number of CIB1 interacting proteins have been identified, including p21-activated kinase and serum-inducible kinase, which may act in concert with CIB1 to regulate platelet function. Understanding the molecular mechanisms underlying integrin activation will be important in developing novel therapies to regulate platelet function in cardiovascular disease. Discussion of recent developments in elucidating the mechanism of integrin activation, with particular focus on the platelet integrin alphaIIbbeta3, is provided in this review.

PAR1, but Not PAR4, Activates Human Platelets through a Gi/o/Phosphoinositide-3 Kinase Signaling Axis

Thrombin-mediated activation of platelets is critical for hemostasis, but the signaling pathways responsible for this process are not completely understood. In addition, signaling within this cascade can also lead to thrombosis. In this study, we have defined a new signaling pathway for the thrombin receptor protease activated receptor-1 (PAR1) in human platelets. We show that PAR1 couples to G(i/o) in human platelets and activates phosphoinositide-3 kinase (PI3K). PI3K activation regulates platelet integrin alphaIIbbeta3 activation and platelet aggregation and potentiates the PAR1-mediated increase in intraplatelet calcium concentration. PI3K inhibitors eliminated these effects downstream of PAR1, but they had no effect on PAR4 signaling. This study has identified an important role for the direct activation of G(i/o) by PAR1 in human platelets. Given the efficacy of clopidogrel, which blocks the G(i/o)-coupled P2Y purinoceptor 12, as an antiplatelet/antithrombotic drug, our data suggest that specifically blocking only PAR1-mediated G(i/o) signaling could also be an effective therapeutic approach with the possibility of less unwanted bleeding.

Platelet adhesion onto immobilized fibrinogen under arterial and venous in-vitro flow conditions does not significantly differ between men and women

BackgroundGender-related differences in incidence of arterial thrombosis have been a focus of interest for years. The platelet integrin αIIbβ3 is primarily responsible for the interaction between platelets and fibrinogen and consecutive thrombus growth. In this study, we evaluated platelet adhesion onto immobilized fibrinogen under venous and arterial flow conditions in men and women.MethodsPlatelets in whole anticoagulated blood were labelled with the fluorescence dye Mepacrine and perfused through the rectangular flow chamber over glass cover slips coated with fibrinogen (shear rates of 50 s-1, 500 s-1 and 1500 s-1). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 seconds, 1 and 5 minutes after the start of perfusion.ResultsDuring perfusion, the platelet adhesion linearly increased in regard to exposition time and shear rate. After five minutes of perfusion the platelet adhesion onto immobilized fibrinogen showed no significant gender related difference, neither at 50 s-1 nor at 500 s-1 and 1500 s-1 (p > 0.05), respectively. No significant difference in platelet adhesion onto immobilized fibrinogen, in regard to the menopausal status, was either observed (p > 0.05).ConclusionIn our in vitro experimental system, hormonal differences between men and women did not influence platelet adhesion onto immobilized fibrinogen, neither under venous nor under arterial rheological conditions.

The Synergy Site of Fibronectin Is Required for Strong Interaction with the Platelet Integrin αIIbβ3

Integrins are a class of cell adhesion molecules that bind to ligands containing the RGD peptide sequence. There is increasing evidence that peptide sites other than the RGD site are required for optimal binding of integrins with their ligands. We have examined the sites on the protein fibronectin that are needed for optimal binding to the platelet integrin alphaIIbbeta3 using a strategy of site directed mutagenesis. Single amino acids near the RGD site or near the synergy site of fibronectin were mutated and the resultant proteins were expressed in a bacterial expression system. The purified protein was coated onto glass cover slips. Platelets, expressing alphaIIbbeta3 were perfused over the surface at physiologically relevant shear rates and the extent of adhesion was quantified. We found that the single amino acid substitution of the aspartic acid in the RGD sequence, D1495A, completely abolished adhesion. Surprisingly, the mutants R1445A and R1448Q that are near the RGD site also abolished adhesion of platelets under flow. Additionally, the synergy site mutants R1371A, R1374Q, or R1379A displayed only minimal adhesion of platelets. These results show that the binding site for alphaIIbbeta3 on fibronectin extends over a considerable distance from the RGD site and that these distant sites are required for optimal attachment of cells in the presence of physiologically relevant shear stress.

Diversity of Glanzmann thrombasthenia in southern India: 10 novel mutations identified among 15 unrelated patients.

Glanzmann thrombasthenia (GT) is a congenital bleeding disorder caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3. To determine the molecular basis of GT in patients from southern India. Fifteen unrelated patients whose diagnosis was consistent with GT were evaluated. Platelet surface expression of alphaIIbbeta3 was < 10%, 10%-50%, and > 50% of controls in five, nine, and one patient(s), respectively. Immunoblotting of the platelet lysates showed no alphaIIb in 14 patients, and no beta3 in 10 patients, although severely reduced in four patients. Platelet fibrinogen was undetectable in 13 patients, and severely reduced in one patient. One patient showed normal surface alphaIIbbeta3 expression, and normal alphaIIb, beta3 and fibrinogen levels in the lysate. Ten novel candidate disease-causing mutations were identified in 11 patients. The missense mutations included Gly128Ser, Ser287Leu, Gly357Ser, Arg520Trp, Leu799Arg in alphaIIb, and Cys575Gly in beta3. We have already shown that Gly128Ser, Ser287Leu, and Gly357Ser mutations variably affect alphaIIbbeta3 surface expression. The Cys575Gly mutation may disrupt the disulphide link with Cys586 to cause the GT phenotype. The molecular pathology of the other missense mutations is not clear. Two nonsense mutations, Trp-16Stop and Glu715Stop in alphaIIb, and a 7-bp deletion (330-336TCCCCAG) in beta3 are predicted to result in truncated proteins. An IVS15(-1)G --> A mutation in alphaIIb induced a cryptic splice site as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Thirteen polymorphisms were also identified (five in alphaIIb and eight in beta3), among which five were novel. While identifying a significant number of novel mutations causing GT, this study confirms the genetic heterogeneity of the disorder in southern India.

Wiskott–Aldrich syndrome protein is involved in αIIbβ3‐mediated cell adhesion

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the alphaIIb cytoplasmic tail of platelet integrin alphaIIb beta3, which has a primary role in platelet aggregation. We also show that the WASP-CIB complex is important in alphaIIb beta3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces alphaIIb beta3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding.

Intracellular signaling in platelets

Over the past few years, a large portion of platelet research has focused on intracellular signaling events that contribute to stable platelet adhesion and aggregation. Studies of knockout mice have suggested critical roles for several previously unappreciated signaling molecules including phosphatidylinositol 3-kinase, the exchange factor CalDAG-GEFI, and the small GTPase Rap1b. These proteins may function to remodel the platelet cytoskeleton and thereby regulate both adhesion and aggregation. The abundant cytoskeletal protein talin appears to be a key regulator of the platelet integrin alphaIIbbeta3. Recent evidence suggests that talin binding to the cytoplasmic tail of beta3 promotes integrin oligomerization, thereby increasing the binding avidity the alphaIIbbeta3 complex for fibrinogen. The identification of platelet signaling pathways not only has clinical implications for diagnosis, but perhaps more importantly for rationale drug design. Aspirin, dipyridamole (Persantine), and thienopyridines (ticlopidine and clopidogrel) are all examples of agents that specifically target discrete platelet signaling pathways. These drugs have already been proven to be beneficial in the treatment of cardiovascular disease. Novel agents that target newly identified signaling pathways hold promise of greater specificity and efficacy.

AFM Imaging of Ligand Binding to Platelet Integrin αIIbβ3 Receptors Reconstituted into Planar Lipid Bilayers

The platelet integrin alphaIIbbeta3 plays a key role in platelet adhesion, activation, and aggregation at the subendothelium and at protein-coated synthetic biomaterials. In this study, interactions between alphaIIbbeta3 and both protein and peptide ligands for the receptor were imaged under physiological conditions by high-resolution atomic force microscopy (AFM). To directly image the ligand-receptor interactions, alphaIIbbeta3 receptors were reconstituted into a supported lipid bilayer formed on a mica surface in the AFM fluid cell assembly and subsequently activated with Mn2+. Fibrinogen, the natural protein ligand for the integrin, as well as a nanogold-labeled peptide ligand (an RGD-containing heptamer) were infused into the AFM fluid cell, incubated with the reconstituted and activated receptors, and imaged under buffer. Height images illustrating topographical features showed the integrin reconstituted in the bilayer. Fibrinogen molecules binding to the receptors were easily observed in the height images, with fibrinogen showing its characteristic trinodular structure and occasionally bridging integrin receptors. Fibrinogen was observed to bind to integrins at the D-domain consistent with the location of the gamma-chain dodecapeptide, while fibrinogen bridging integrins bound to receptors on opposite sides of the protein consistent with a 2-fold axis of symmetry. Peptide ligands were not visible in height images; however, phase images that map the mechanical properties detected the nanogold labels and demonstrated the presence of peptide ligands bound to the receptors. The results demonstrate the ability of this high-resolution microscopy technique to directly visualize single ligand/receptor interactions in a dynamic and physiologically relevant environment, and establish a framework for future fundamental studies of single protein/receptor interactions during normal pathological processes as well as biomaterial surface-induced thrombosis.

Novel Integrin Antagonists Derived from Thrombospondins

Specific antagonists have been successfully developed for several different integrins. Clinical trials have been initiated to study therapeutic uses of these inhibitors in cancer, thrombosis, and inflammatory diseases. Most efforts to date have focused on the platelet integrin alphaIIbbeta3, endothelial alphavbeta3, and the leukocyte integrin alpha4beta1. However, the integrin family contains additional members with interesting tissue specificities and functional properties that could also be useful molecular targets for disease intervention. In many cases, specific recognition motifs for these integrins have not been identified, which has precluded development of specific antagonists. Our recent studies of thrombospondin-1 and thrombospondin-2 recognition by integrins have revealed novel motifs for alpha3beta1 and alpha6beta1 integrins as well as new motifs recognized by the well studied alpha4beta1 integrin. These three integrins play distinct roles in angiogenesis and its modulation by thrombospondins. This review will discuss recent insights into the specificities of alpha3beta1 and alpha6beta1 integrins, their functions in angiogenesis, and potential applications for antagonists of these integrins and of alpha4beta1 to control pathological angiogenesis and other diseases.

Confinement of Transmembrane Cell Receptors in Tunable Stripe Micropatterns

We report a simple method to confine transmembrane cell receptors in stripe micropatterns of a lipid/lipopolymer monolayer, which are formed as result of the transfer onto a solid substrate. The stripes are aligned perpendicular to the meniscus, whose periodicity can systematically be tuned by the transfer velocity. This strongly suggests the dominant role of the cooperative interaction between the film and substrate. Selective fluorescence labeling of lipids and lipopolymers confirms that the observed patterns coincide with the demixing of two species. Covalent coupling of polymer headgroups enables us to use the stripe patterns as a support for a lipid bilayer membrane. Spreading of lipid vesicles with platelet integrin alphaIIbbeta3 on a self-assembled membrane micropattern demonstrates that cell adhesion receptors are selectively incorporated into the lipopolymer-rich region. The method established here provides us with a tunable template for the confinement of receptor proteins to geometrically control the cell adhesion.