A general chemical principle for creating closure-stabilizing integrin inhibitors.

Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbß3 and α4ß1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbß3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4ß1.

Integrin α5ß1 contributes to cell fusion and inflammation mediated by SARS-CoV-2 spike via RGD-independent interaction.

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infects host cells by engaging its spike (S) protein with human ACE2 receptor. Recent studies suggest the involvement of integrins in SARS-CoV-2 infection through interaction with the S protein, but the underlying mechanism is not well understood. This study investigated the role of integrin α5ß1, which recognizes the Arg-Gly-Asp (RGD) motif in its physiological ligands, in S-mediated virus entry and cell-cell fusion. Our results showed that α5ß1 does not directly contribute to S-mediated cell entry, but it enhances S-mediated cell-cell fusion in collaboration with ACE2. This effect cannot be inhibited by the putative α5ß1 inhibitor ATN-161 or the high-affinity RGD-mimetic inhibitor MK-0429 but requires the participation of α5 cytoplasmic tail (CT). We detected a direct interaction between α5ß1 and the S protein, but this interaction does not rely on the RGD-containing receptor binding domain of the S1 subunit of the S protein. Instead, it involves the S2 subunit of the S protein and α5ß1 homo-oligomerization. Furthermore, we found that the S protein induces inflammatory responses in human endothelial cells, characterized by NF-κB activation, gasdermin D cleavage, and increased secretion of proinflammatory cytokines IL-6 and IL-1ß. These effects can be attenuated by the loss of α5 expression or inhibition of the α5 CT binding protein phosphodiesterase-4D (PDE4D), suggesting the involvement of α5 CT and PDE4D pathway. These findings provide molecular insights into the pathogenesis of SARS-CoV-2 mediated by a nonclassical RGD-independent ligand-binding and signaling function of integrin α5ß1 and suggest potential targets for antiviral treatment.

Atomic Level Dissection of the Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) Homophilic Binding Interface: Implications for Endothelial Cell Barrier Function.

OBJECTIVE: PECAM-1 (platelet endothelial cell adhesion molecule 1) is a 130 kDa member of the immunoglobulin (Ig) gene superfamily that is expressed on the surfaces of platelets and leukocytes and concentrated at the intercellular junctions of confluent endothelial cell monolayers. PECAM-1 Ig domains 1 and 2 (IgD1 and IgD2) engage in homophilic interactions that support a host of vascular functions, including support of leukocyte transendothelial migration and the maintenance of endothelial junctional integrity. The recently solved crystal structure of PECAM-1 IgD1 and IgD2 revealed a number of intermolecular interfaces predicted to play important roles in stabilizing PECAM-1/PECAM-1 homophilic interactions and in formation and maintenance of endothelial cell-cell contacts. We sought to determine whether the protein interfaces implicated in the crystal structure reflect physiologically important interactions. Approach and Results: We assessed the impact of single amino acid substitutions at the interfaces between opposing PECAM-1 molecules on homophilic binding and endothelial cell function. Substitution of key residues within the IgD1-IgD1 and IgD1-IgD2 interfaces but not those within the smaller IgD2-IgD2 interface, markedly disrupted PECAM-1 homophilic binding and its downstream effector functions, including the ability of PECAM-1 to localize at endothelial cell-cell borders, mediate the formation of endothelial tubes, and restore endothelial barrier integrity. CONCLUSIONS: Taken together, these results validate the recently described PECAM-1 IgD1/IgD2 crystal structure by demonstrating that specific residues visualized within the IgD1-IgD1 and IgD1-IgD2 interfaces of opposing molecules in the crystal are required for functionally important homophilic interactions. This information can now be exploited to modulate functions of PECAM-1 in vivo.

Hsp47 promotes cancer metastasis by enhancing collagen-dependent cancer cell-platelet interaction.

Increased expression of extracellular matrix (ECM) proteins in circulating tumor cells (CTCs) suggests potential function of cancer cell-produced ECM in initiation of cancer cell colonization. Here, we showed that collagen and heat shock protein 47 (Hsp47), a chaperone facilitating collagen secretion and deposition, were highly expressed during the epithelial-mesenchymal transition (EMT) and in CTCs. Hsp47 expression induced mesenchymal phenotypes in mammary epithelial cells (MECs), enhanced platelet recruitment, and promoted lung retention and colonization of cancer cells. Platelet depletion in vivo abolished Hsp47-induced cancer cell retention in the lung, suggesting that Hsp47 promotes cancer cell colonization by enhancing cancer cell-platelet interaction. Using rescue experiments and functional blocking antibodies, we identified type I collagen as the key mediator of Hsp47-induced cancer cell-platelet interaction. We also found that Hsp47-dependent collagen deposition and platelet recruitment facilitated cancer cell clustering and extravasation in vitro. By analyzing DNA/RNA sequencing data generated from human breast cancer tissues, we showed that gene amplification and increased expression of Hsp47 were associated with cancer metastasis. These results suggest that targeting the Hsp47/collagen axis is a promising strategy to block cancer cell-platelet interaction and cancer colonization in secondary organs.

Structural determinants of the integrin transmembrane domain required for bidirectional signal transmission across the cell membrane.

Studying the tight activity regulation of platelet-specific integrin αIIbß3 is foundational and paramount to our understanding of integrin structure and activation. αIIbß3 is essential for the aggregation and adhesion function of platelets in hemostasis and thrombosis. Structural and mutagenesis studies have previously revealed the critical role of αIIbß3 transmembrane (TM) association in maintaining the inactive state. Gain-of-function TM mutations were identified and shown to destabilize the TM association leading to integrin activation. Studies using isolated TM peptides have suggested an altered membrane embedding of the ß3 TM α-helix coupled with αIIbß3 activation. However, controversies remain as to whether and how the TM α-helices change their topologies in the context of full-length integrin in native cell membrane. In this study, we utilized proline scanning mutagenesis and cysteine scanning accessibility assays to analyze the structure and function correlation of the αIIbß3 TM domain. Our identification of loss-of-function proline mutations in the TM domain suggests the requirement of a continuous TM α-helical structure in transmitting activation signals bidirectionally across the cell membrane, characterized by the inside-out activation for ligand binding and the outside-in signaling for cell spreading. Similar results were found for αLß2 and α5ß1 TM domains, suggesting a generalizable mechanism. We also detected a topology change of ß3 TM α-helix within the cell membrane, but only under conditions of cell adhesion and the absence of αIIb association. Our data demonstrate the importance of studying the structure and function of the integrin TM domain in the native cell membrane.

Autonomous conformational regulation of ß3 integrin and the conformation-dependent property of HPA-1a alloantibodies.

Integrin α/ß heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of ß subunit induce the α/ß separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The ß3 integrins, including αIIbß3 and αVß3, are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the ß3 PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti-HPA-1a alloantibodies can distinguish the αIIbß3 from αVß3 and affect their functions with unknown mechanisms. Here we designed a single-chain ß3 subunit that mimics a separation of α/ß heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain ß3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the ß3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain ß3 integrin, we identified the conformation-dependent property of anti-HPA-1a alloantibodies, which enables them to differently recognize the ß3 in the bent state vs. the extended state and in the complex with αIIb vs. αV This study provides deeper understandings of integrin conformational activation on the cell surface.

Structure of an extended ß3 integrin.

Cells use adhesion receptor integrins to communicate with their surroundings. Integrin activation and cellular signaling are coupled with change from bent to extended conformation. ß3 integrins, including αIIbß3, which is essential for the function of platelets in hemostasis and thrombosis, and αVß3, which plays multiple roles in diverse cell types, have been prototypes in understanding integrin structure and function. Despite extensive structural studies, a high-resolution integrin structure in an extended conformation remains to be determined. The human ß3 Leu33Pro polymorphism, located at the PSI domain, defines human platelet-specific alloantigens 1a and 1b (HPA-1a/b), immune response to which is a cause of posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia. Leu33Pro substitution has also been suggested to be a risk factor for thrombosis. Here we report the crystal structure of the ß3 headpiece in either Leu33 or Pro33 form, both of which reveal intermediate and fully extended conformations coexisting in 1 crystal. These were used to build high-resolution structures of full-length ß3 integrin in the intermediate and fully extended states, agreeing well with the corresponding conformations observed by electron microscopy. Our structures reveal how ß3 integrin becomes extended at its ß-knee region and how the flexibility of ß-leg domains is determined. In addition, our structures reveal conformational changes of the PSI and I-EGF1 domains upon ß3 extension, which may affect the binding of conformation-dependent anti-HPA-1a alloantibodies. Our structural and functional data show that Leu33Pro substitution does not directly alter the conformation or ligand binding of ß3 integrin.

Naturally occurring point mutation Cys460Trp located in the I-EGF1 domain of integrin ß3 alters the binding of some anti-HPA-1a antibodies.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by the destruction of platelets in the fetus or newborn by maternal platelet alloantibodies, mostly against human platelet antigen (HPA)-1a. Recent studies indicate that two anti-HPA subtypes exist: Type I reacts with epitopes residing on the plexin-semaphorin-integrin (PSI) and type II with plexin-semaphorin-integrin/integrin epidermal growth factor 1 (I-EGF1) domains of the ß3 integrin. Here, we evaluated whether a Cys460Trp mutation in the I-EGF1 domain found in a patient with Glanzmann thrombasthenia can alter the binding of anti-HPA-1a. METHODS: Stable HEK293 cell lines expressing wild-type and mutant αIIbß3 and αvß3 were generated to prove the reactivity of different antibodies against HPA-1a. RESULTS: Flow cytometry analysis of wild-type (Cys460) and mutant (Trp460) expressed on HEK293 cells showed equal surface expression of αIIbß3 and αvß3. When tested with mutant αIIbß3 cells, reduced binding was observed in Type II but not in Type I anti-HPA-1a. These results could be confirmed with platelets carrying Cys460Trp mutation. Interestingly, reduced binding of Type I antibodies was detected with mutant αvß3 cells. Both antibody types were found in maternal sera from FNAIT cases by an antigen-capture assay with use of HEK293 transfected cells. CONCLUSIONS: These observations confirm the existence of Type I and Type II anti-HPA-1a. Furthermore, this study underlines different immunogenicity of HPA-1a antigen(s) residing on either αIIbß3 or αvß3. Further analysis of FNAIT cases from mothers having a fetus with and without intracranial bleedings with use of such an approach may highlight the functional relevance of different anti-HPA-1a subtypes.

A pivotal role for a conserved bulky residue at the α1-helix of the αI integrin domain in ligand binding.

The ligand-binding ßI and αI domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both ßI and αI domains, the α1- and α7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the α1-helix of ßI helps maintain the resting ßI conformation, whereas the homologous position in the αI α1-helix contains a conserved Phe. A functional role of this Phe is structurally unpredictable. Using αLß2 integrin as a model, we found that the residue volume at the Phe position in the α1-helix is critical for αLß2 activation because trimming the Phe by small amino acid substitutions abolished αLß2 binding with soluble and immobilized intercellular cell adhesion molecule 1. Similar results were obtained for αMß2 integrin. Our experimental and molecular dynamics simulation data suggested that the bulky Phe acts as a pawl that stabilizes the downward ratchet-like movement of ß6-α7 loop and α7-helix, required for high-affinity ligand binding. This mechanism may apply to other von Willebrand factor A domains undergoing large conformational changes. We further demonstrated that the conformational cross-talk between αL αI and ß2 ßI could be uncoupled because the ß2 extension and headpiece opening could occur independently of the αI activation. Reciprocally, the αI activation does not inevitably lead to the conformational changes of the ß2 subunit. Such loose linkage between the αI and ßI is attributed to the αI flexibility and could accommodate the αLß2-mediated rolling adhesion of leukocytes.

Structural basis for PECAM-1 homophilic binding.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily (IgSF) that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1-mediated homophilic interactions, known to be mediated by its 2 amino-terminal immunoglobulin homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to facilitate diapedesis, maintain vascular integrity, and transmit survival signals into the cell. Given the importance of PECAM-1-mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientation of the PECAM-1-PECAM-1 homophilic-binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic-binding domain, which is composed of amino-terminal immunoglobulin homology domains 1 and 2 (IgD1 and IgD2). The crystal structure revealed that both IgD1 and IgD2 exhibit a classical IgSF fold, having a ß-sandwich topology formed by 2 sheets of antiparallel ß strands stabilized by the hallmark disulfide bond between the B and F strands. Interestingly, despite previous assignment to the C2 class of immunoglobulin-like domains, the structure of IgD1 reveals that it actually belongs to the I2 set of IgSF folds. Both IgD1 and IgD2 participate importantly in the formation of the trans homophilic-binding interface, with a total buried interface area of >2300 Å(2). These and other unique structural features of PECAM-1 allow for the development of an atomic-level model of the interactions that PECAM-1 forms during assembly of endothelial cell intercellular junctions.

The Role of Sialylated Glycans in Human Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1)-mediated Trans Homophilic Interactions and Endothelial Cell Barrier Function.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) is a major component of the endothelial cell intercellular junction. Previous studies have shown that PECAM-1 homophilic interactions, mediated by amino-terminal immunoglobulin homology domain 1, contribute to maintenance of the vascular permeability barrier and to its re-establishment following inflammatory or thrombotic insult. PECAM-1 glycans account for ∼30% of its molecular mass, and the newly solved crystal structure of human PECAM-1 immunoglobulin homology domain 1 reveals that a glycan emanating from the asparagine residue at position 25 (Asn-25) is located within the trans homophilic-binding interface, suggesting a role for an Asn-25-associated glycan in PECAM-1 homophilic interactions. In support of this possibility, unbiased molecular docking studies revealed that negatively charged α2,3 sialic acid moieties bind tightly to a groove within the PECAM-1 homophilic interface in an orientation that favors the formation of an electrostatic bridge with positively charged Lys-89, mutation of which has been shown previously to disrupt PECAM-1-mediated homophilic binding. To verify the contribution of the Asn-25 glycan to endothelial barrier function, we generated an N25Q mutant form of PECAM-1 that is not glycosylated at this position and examined its ability to contribute to vascular integrity in endothelial cell-like REN cells. Confocal microscopy showed that although N25Q PECAM-1 concentrates normally at cell-cell junctions, the ability of this mutant form of PECAM-1 to support re-establishment of a permeability barrier following disruption with thrombin was significantly compromised. Taken together, these data suggest that a sialic acid-containing glycan emanating from Asn-25 reinforces dynamic endothelial cell-cell interactions by stabilizing the PECAM-1 homophilic binding interface.

The dual structural roles of the membrane distal region of the α-integrin cytoplasmic tail during integrin inside-out activation.

Studies on the mechanism of integrin inside-out activation have been focused on the role of ß-integrin cytoplasmic tails, which are relatively conserved and bear binding sites for the intracellular activators including talin and kindlin. Cytoplasmic tails for α-integrins share a conserved GFFKR motif at the membrane-proximal region and this forms a specific interface with the ß-integrin membrane-proximal region to keep the integrin inactive. The α-integrin membrane-distal regions, after the GFFKR motif, are diverse both in length and sequence and their roles in integrin activation have not been well-defined. In this study, we report that the α-integrin cytoplasmic membrane-distal region contributes to maintaining integrin in the resting state and to integrin inside-out activation. Complete deletion of the α-integrin membrane-distal region diminished talin- and kindlin-mediated integrin ligand binding and conformational change. A proper length and suitable amino acids in α-integrin membrane-distal region was found to be important for integrin inside-out activation. Our data establish an essential role for the α-integrin cytoplasmic membrane-distal region in integrin activation and provide new insights into how talin and kindlin induce the high-affinity integrin conformation that is required for fully functional integrins.

Structural basis for quinine-dependent antibody binding to platelet integrin αIIbß3.

Drug-induced immune thrombocytopenia (DITP) is caused by antibodies that react with specific platelet-membrane glycoproteins when the provoking drug is present. More than 100 drugs have been implicated as triggers for this condition, quinine being one of the most common. The cause of DITP in most cases appears to be a drug-induced antibody that binds to a platelet membrane glycoprotein only when the drug is present. How a soluble drug promotes binding of an otherwise nonreactive immunoglobulin to its target, leading to platelet destruction, is uncertain, in part because of the difficulties of working with polyclonal human antibodies usually available only in small quantities. Recently, quinine-dependent murine monoclonal antibodies were developed that recognize a defined epitope on the ß-propeller domain of the platelet integrin αIIb subunit (GPIIb) only when the drug is present and closely mimic the behavior of antibodies found in human patients with quinine-induced thrombocytopenia in vitro and in vivo. Here, we demonstrate specific, high-affinity binding of quinine to the complementarity-determining regions (CDRs) of these antibodies and define in crystal structures the changes induced in the CDR by this interaction. Because no detectable binding of quinine to the target integrin could be demonstrated in previous studies, the findings indicate that a hybrid paratope consisting of quinine and reconfigured antibody CDR plays a critical role in recognition of its target epitope by an antibody and suggest that, in this type of drug-induced immunologic injury, the primary reaction involves binding of the drug to antibody CDRs, causing it to acquire specificity for a site on a platelet integrin.

Antiendothelial αvß3 Antibodies Are a Major Cause of Intracranial Bleeding in Fetal/Neonatal Alloimmune Thrombocytopenia.

OBJECTIVE: Fetal/neonatal alloimmune thrombocytopenia is a severe bleeding disorder, which can result in intracranial hemorrhage (ICH), leading to death or neurological sequelae. In whites, maternal anti-human platelet antigen-1a (HPA-1a) antibodies are responsible for the majority of cases. No predictive factors for ICH are available to guide prophylactic treatment during pregnancy. In this study, we investigated antibodies from mothers with ICH-positive fetal/neonatal alloimmune thrombocytopenia and with ICH-negative fetal/neonatal alloimmune thrombocytopenia to identify serological and functional differences between the groups. APPROACH AND RESULTS: In an antigen capture assay, we observed a stronger binding of +ICH antibodies to endothelial cell (EC)-derived αvß3. By absorption experiments, we subsequently identified anti-HPA-1a antibodies of anti-αvß3 specificity in the +ICH but not in the -ICH cohort. Only the anti-αvß3 subtype, but not the anti-ß3 subtype, induced EC apoptosis of HPA-1a-positive ECs by caspase-3/7 activation, and mediated by reactive oxygen species. In addition, only the anti-αvß3 subtype, but not the anti-ß3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation. CONCLUSIONS: We conclude that the composition of the anti-HPA-1a antibody subtype(s) of the mother may determine whether ICH occurs. Analysis of anti-HPA-1a antibodies of the anti-αvß3 subtype in maternal serum has potential in the diagnostic prediction of ICH development and may allow for modification of prophylactic treatment in fetal/neonatal alloimmune thrombocytopenia.

The structure of a receptor with two associating transmembrane domains on the cell surface: integrin alphaIIbbeta3.

Structures of intact receptors with single-pass transmembrane domains are essential to understand how extracellular and cytoplasmic domains regulate association and signaling through transmembrane domains. A chemical and computational method to determine structures of the membrane regions of such receptors on the cell surface is developed here and validated with glycophorin A. An integrin heterodimer structure reveals association over most of the lengths of the alpha and beta transmembrane domains and shows that the principles governing association of hetero and homo transmembrane dimers differ. A turn at the Gly of the juxtamembrane GFFKR motif caps the alpha TM helix and brings the two Phe of GFFKR into the alpha/beta interface. A juxtamembrane Lys residue in beta also has an important role in the interface. The structure shows how transmembrane association/dissociation regulates integrin signaling. A joint ectodomain and membrane structure shows that substantial flexibility between the extracellular and TM domains is compatible with TM signaling.

Modulation of integrin activation and signaling by α1/α1'-helix unbending at the junction.

How conformational signals initiated from one end of the integrin are transmitted to the other end remains elusive. At the ligand-binding ßI domain, the α1/α1'-helix changes from a bent to a straightened α-helical conformation upon integrin headpiece opening. We demonstrated that a conserved glycine at the α1/α1' junction is crucial for maintaining the bent conformation of the α1/α1'-helix in the resting state. Mutations that facilitate α1/α1'-helix unbending rendered integrin constitutively active; however, mutations that block the α1/α1'-helix unbending abolished soluble ligand binding upon either outside or inside stimuli. Such mutations also blocked ligand-induced integrin extension from outside the cell, but had no effect on talin-induced integrin extension from inside the cell. In addition, integrin-mediated cell spreading, F-actin stress fiber and focal adhesion formation, and focal adhesion kinase activation were also defective in these mutant integrins, although the cells still adhered to immobilized ligands at a reduced level. Our data establish the structural role of the α1/α1' junction that allows relaxation of the α1/α1'-helix in the resting state and transmission of bidirectional conformational signals by helix unbending upon integrin activation.

TRAIL receptor deficiency sensitizes mice to dextran sodium sulphate-induced colitis and colitis-associated carcinogenesis.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (TRAIL-R) play important roles in immune regulation and cancer cell death. Although TRAIL has been shown to induce chemokine release in various tumour cells, the function of TRAIL-R in the development of colitis and colitis-associated carcinogenesis has not been explored. In this study, we found that TRAIL-R-deficient mice exhibited a higher incidence of colitis and colitis-associated cancer than that of wild-type (WT) mice, and TRAIL-R expression was down-regulated in WT mice that were fed dextran sulphate sodium. Chemokines, including CCL2 and CXCL1, were highly expressed in the serum and inflammatory colon tissues of TRAIL-R(-/-) mice compared with WT mice, and TRAIL-R(-/-) mice showed a marked infiltration of immune cells during colitis. Hyperactivation of Janus kinase and nuclear factor-κB in colon epithelial cells was also observed, which correlated with the severity of colonic inflammation in TRAIL-R(-/-) mice. These data suggest that TRAIL-R plays a protective role in chemical-induced colon injury and negatively regulates mucosal immune responses.

Antibodies causing thrombocytopenia in patients treated with RGD-mimetic platelet inhibitors recognize ligand-specific conformers of αIIb/ß3 integrin.

Arginine-glycine-aspartic acid (RGD)-mimetic platelet inhibitors act by occupying the RGD recognition site of α(IIb)/ß(3) integrin (GPIIb/IIIa), thereby preventing the activated integrin from reacting with fibrinogen. Thrombocytopenia is a well-known side effect of treatment with this class of drugs and is caused by Abs, often naturally occurring, that recognize α(IIb)/ß(3) in a complex with the drug being administered. RGD peptide and RGD-mimetic drugs are known to induce epitopes (ligand-induced binding sites [LIBS]) in α(IIb)/ß(3) that are recognized by certain mAbs. It has been speculated, but not shown experimentally, that Abs from patients who develop thrombocytopenia when treated with an RGD-mimetic inhibitor similarly recognize LIBS determinants. We addressed this question by comparing the reactions of patient Abs and LIBS-specific mAbs against α(IIb)/ß(3) in a complex with RGD and RGD-mimetic drugs, and by examining the ability of selected non-LIBS mAbs to block binding of patient Abs to the liganded integrin. Findings made provide evidence that the patient Abs recognize subtle, drug-induced structural changes in the integrin head region that are clustered about the RGD recognition site. The target epitopes differ from classic LIBS determinants, however, both in their location and by virtue of being largely drug-specific.

Sequence and structure relationships within von Willebrand factor.

In the present study, we re-annotated von Willebrand factor (VWF), assigned its entire sequence to specific modules, and related these modules to structure using electron microscopy (EM). The D domains are assemblies of smaller modules visible as lobes in EM. Modules in the D-domain assemblies include von Willebrand D, 8-cysteine, trypsin inhibitor-like, E or fibronectin type 1-like domains, and a unique D4N module in D4. The D1-D2 prodomain shows 2 large connected assemblies, each containing smaller lobes. The previous B and C regions of VWF are re-annotated as 6 tandem von Willebrand C (VWC) and VWC-like domains. These 6 VWC domains correspond to 6 elongated domains that associate in pairs at acidic pH in the stem region of VWF dimeric bouquets. This correspondence is demonstrated by binding of integrin α(IIb)ß(3) to the fourth module seen in EM, VWC4, which bears the VWF Arg-Gly-Asp motif. The C-terminal cystine knot domain dimerizes end-to-end in a manner predicted by homology to TGF-ß and orients approximately perpendicular to the VWC domains in dimeric bouquets. Homologies of domains in VWF to domains in other proteins allow many disulfide bonds to be tentatively assigned, which may have functional implications.