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.

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.

Regulation of endothelial cell barrier function by antibody-driven affinity modulation of platelet endothelial cell adhesion molecule-1 (PECAM-1).

PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane- proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.

Relative contribution of PECAM-1 adhesion and signaling to the maintenance of vascular integrity.

PECAM-1 (CD31) is a cellular adhesion and signaling receptor that is highly expressed at endothelial cell-cell junctions in confluent vascular beds. Previous studies have implicated PECAM-1 in the maintenance of vascular barrier integrity; however, the mechanisms behind PECAM-1-mediated barrier protection are still poorly understood. The goal of the present study, therefore, was to examine the pertinent biological properties of PECAM-1 (i.e. adhesion and/or signaling) that allow it to support barrier integrity. We found that, compared with PECAM-1-deficient endothelial cells, PECAM-1-expressing endothelial cell monolayers exhibit increased steady-state barrier function, as well as more rapid restoration of barrier integrity following thrombin-induced perturbation of the endothelial cell monolayer. The majority of PECAM-1-mediated barrier protection was found to be due to the ability of PECAM-1 to interact homophilically and become localized to cell-cell junctions, because a homophilic binding-crippled mutant form of PECAM-1 was unable to support efficient barrier function when re-expressed in cells. By contrast, cells expressing PECAM-1 variants lacking residues known to be involved in PECAM-1-mediated signal transduction exhibited normal to near-normal barrier integrity. Taken together, these studies suggest that PECAM-1-PECAM-1 homophilic interactions are more important than its signaling function for maintaining the integrity of endothelial cell junctions.

Residues within a lipid-associated segment of the PECAM-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation.

Immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors inhibit cellular responsiveness to immunoreceptor tyrosine-based activation motif (ITAM)-linked receptors. Although tyrosine phosphorylation is central to the initiation of both inhibitory ITIM and stimulatory ITAM signaling, the events that regulate receptor phosphorylation are incompletely understood. Previous studies have shown that ITAM tyrosines engage in structure-inducing interactions with the plasma membrane that must be relieved for phosphorylation to occur. Whether ITIM phosphorylation is similarly regulated and the mechanisms responsible for release from plasma membrane interactions to enable phosphorylation, however, have not been defined. PECAM-1 is a dual ITIM-containing receptor that inhibits ITAM-dependent responses in hematopoietic cells. We found that the PECAM-1 cytoplasmic domain is unstructured in an aqueous environment but adopts an α-helical conformation within a localized region on interaction with lipid vesicles that mimic the plasma membrane. The lipid-interacting segment contains the C-terminal ITIM tyrosine and a serine residue that undergo activation-dependent phosphorylation. The N-terminal ITIM is excluded from the lipid-interacting segment, and its phosphorylation is secondary to phosphorylation of the membrane-interacting C-terminal ITIM. On the basis of these findings, we propose a novel model for regulation of inhibitory signaling by ITIM-containing receptors that relies on reversible plasma membrane interactions and sequential ITIM phosphorylation.

An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling.

The Ig-ITIM family member PECAM-1 is expressed in vascular and endothelial cells, and its functions include suppression of mitochondria-dependent apoptosis. Previous studies have identified distinct PECAM-1 cytoplasmic domain splice variants at the mRNA, but not protein, level. Several relatively abundant mRNA isoforms lack exon 15 (Delta15) and would theoretically encode a protein with a truncated cytoplasmic domain and a unique C-terminal sequence. Using a novel rabbit polyclonal antibody that specifically recognizes Delta15 PECAM-1, we found that the Delta15 PECAM-1 isoform was expressed in human tissues, including brain, testes and ovary. This isoform was also expressed on the cell surface of human platelets, human umbilical vein endothelial cells (HUVECs) and the Jurkat T-cell leukemia, human erythroleukemia (HEL) and U937 histiocytic lymphoma cell lines. Furthermore, murine platelets and lung lysates demonstrated abundant amounts of exon-15-deficient PECAM-1. Functional studies revealed that Delta15 PECAM-1 retains both its homophilic binding capacity and its ability to signal by means of its immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. Delta15 PECAM-1 was unable, however, to protect against apoptosis induced by overexpression of Bax or treatment with the chemotherapy agent etoposide. These studies suggest a novel role for the PECAM-1 C-terminus in cytoprotective signaling and highlight a need for further characterization of expression of PECAM-1 isoforms in normal and malignant tissues.

The neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31).

Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration.

Palmitoylation at Cys595 is essential for PECAM-1 localisation into membrane microdomains and for efficient PECAM-1-mediated cytoprotection.

The Ig-ITIM superfamily member, PECAM-1 acts as a negative regulator of ITAM-signalling pathways in platelets involving GPVI/FcR gamma chain and Fc?RIIa. This negative feedback loop involves regulation of collagen and GPVI-dependent aggregation events, platelet-thrombus-growth on immobilised collagen under flow and Fc?RIIa-mediated platelet responses. In this study, we show that PECAM-1 is selectively palmitoylated involving a thioester linkage with an unpaired cysteine residue at amino acid position 595 in its cytoplasmic domain. As palmitoylation is known to target proteins to membrane microdomains, we investigated the microdomain localisation for PECAM-1 in platelets and nucleated cells. In unstimulated platelets, approximately 20% of PECAM-1 is localised to Triton-insoluble microdomain fractions and it does not increase with platelet activation by collagen, collagen-related peptide, thrombin- or human-aggregated IgG. PECAM-1 is in close physical proximity with GPVI in platelet microdomains. Removal of platelet cytoskeleton prior to sucrose-density-gradient separation showed that PECAM-1 was associated with both the Triton-soluble and membrane skeleton in microdomain-associated fractions. Disruption of microdomains by membrane-cholesterol depletion resulted in loss of PECAM-1 localisation to membrane microdomains. Mutational analysis of juxtamembrane cysteine residue to alanine (C595A) of human PECAM-1 resulted in loss of palmitoylation and a sixfold decrease in association with membrane microdomains. Functionally, the palmitoylated cysteine 595 residue is required, in part, for efficient PECAM-1-mediated cytoprotection. These results show that cysteine 595 is required for constitutive association of PECAM-1 with membrane microdomains and PECAM-1-mediated cytoprotection, where it may act as a crucial regulator of signaling and apoptosis events.

The cell-adhesion and signaling molecule PECAM-1 is a molecular mediator of resistance to genotoxic chemotherapy.

Defects in the regulation of apoptotic pathways have been implicated in the emergence of cancers resistant to chemotherapy-induced cell death. Identification of novel signaling molecules that influence cell survival has the potential to facilitate the development of new cancer therapies. The cell adhesion and signaling molecule, PECAM-1, is expressed in many hematopoietic and endothelial cell malignancies, and has previously been shown to suppress mitochondrial-dependent, Bax-mediated apoptosis. The ability of PECAM-1 to influence tumor cell survival following exposure to chemotherapeutic agents, however, is not known. Here we show that, when overexpressed in HEK293 and REN mesothelioma cells, PECAM-1 confers resistance to apoptosis induced by the DNA-damaging chemotherapeutic agent, etoposide. Surprisingly, PECAM-1-mediated cytoprotection was found to be largely independent of its ability to form a signaling complex with the protein-tyrosine phosphatase SHP-2, as virtually no tyrosine phosphorylation of, or SHP-2 association with, PECAM-1 could be detected after etoposide treatment. Furthermore, PECAM-1 retained its ability to protect against chemotherapy-induced apoptosis in cells with SHP-2 levels significantly reduced using SHP-2-specific siRNA, and in cells in which Erk1/2--a downstream effector of SHP-2--had been inhibited. Finally, to determine whether endogenous PECAM-1 confers resistance to chemotherapy-induced apoptosis in lymphoid malignancies and endothelial cells, we used a lentiviral vector to stably express PECAM-1-specific siRNA in the Jurkat leukemia cell line and human umbilical vein endothelial cells (HUVECs). siRNA-expressing Jurkat cells with a 70% reduction of PECAM-1 expression were significantly more sensitive to chemotherapy-induced apoptosis. HUVECs with PECAM-1 expression reduced 75% were also markedly more sensitive to chemotherapy-induced cell death. Taken together, these data demonstrate that endogenous PECAM-1 expression on lymphoid cancers confers resistance to apoptosis, and that lowering PECAM-1 expression in lymphoid malignancies can render them more susceptible to chemotherapy-induced apoptosis. In addition, reducing PECAM-1 levels in the tumor endothelium may aid in low-dose, anti-angiogenic therapy.

Anti-GPVI-associated ITP: an acquired platelet disorder caused by autoantibody-mediated clearance of the GPVI/FcRgamma-chain complex from the human platelet surface.

Platelet glycoprotein (GP) VI is a 62-kDa membrane glycoprotein that exists on both human and murine platelets in a noncovalent complex with the Fc receptor (FcR) gamma chain. The GPVI/FcRgamma-chain complex serves as the major activating receptor for collagen, as evidenced by observations that platelets genetically deficient in GPVI or the FcRgamma chain are highly refractory to collagen-induced platelet activation. Recently, several different rat anti-murine GPVI monoclonal antibodies, termed JAQs 1, 2, and 3, were produced that had the unique property of "immunodepleting" GPVI from the murine platelet surface and rendering it unresponsive to collagen or GPVI-specific agonists like convulxin or collagen-related peptide (CRP). Herein, we describe a patient with a mild bleeding disorder and a moderately reduced platelet count whose platelets fail to become activated in response to collagen or CRP and inefficiently adhere to and form thrombi on immobilized collagen under conditions of arterial shear. Although the amount of GPVI platelet mRNA and the nucleotide sequence of the GPVI gene were found to be normal, both GPVI and the FcRgamma chain were nearly absent from the platelet surface and were markedly reduced in wholeplatelet detergent lysates. Patient plasma contained an autoantibody that bound specifically to GPVI-positive, normal platelets, and cleared soluble GPVI from the plasma, suggesting that the patient suffers from a rare form of idiopathic thrombocytopenic purpura caused by a GPVI-specific autoantibody that mediates clearance of the GPVI/FcRgamma-chain complex from the platelet surface. Since antibody-induced GPVI shedding now has been demonstrated in both humans and mice, these studies may provide a rationale for developing therapeutic reagents that induce temporary depletion of GPVI for the treatment of clinical thrombosis.

Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) functions to control the activation and survival of the cells on which it is expressed. Many of the regulatory functions of PECAM-1 are dependent on its tyrosine phosphorylation and subsequent recruitment of the Src homology (SH2) domain containing protein tyrosine phosphatase SHP-2. The recent demonstration that PECAM-1 tyrosine phosphorylation occurs in cells exposed to the reactive oxygen species hydrogen peroxide (H2O2) suggested that this form of oxidative stress may also support PECAM-1/SHP-2 complex formation. In the present study, we show that PECAM-1 tyrosine phosphorylation in response to exposure of cells to H2O2 is reversible, involves a shift in the balance between kinase and phosphatase activities, and supports binding of SHP-2 and recruitment of this phosphatase to cell-cell borders. We speculate, however, that the unique ability of H2O2 to reversibly oxidize the reactive site cysteine residues of protein tyrosine phosphatases may result in transient inactivation of the SHP-2 that is bound to PECAM-1 under these conditions. Finally, we provide evidence that PECAM-1 tyrosine phosphorylation and SHP-2 binding in endothelial cells requires exposure to an "oxidative burst" of H2O2, but that exposure of these cells to sufficiently high concentrations of H2O2 for a sufficiently long period of time abrogates binding of SHP-2 to tyrosine-phosphorylated PECAM-1. These findings support a role for PECAM-1 as a sensor of oxidative stress, perhaps most importantly during the process of inflammation.

Disruption of the long-range GPIIIa Cys(5)-Cys(435) disulfide bond results in the production of constitutively active GPIIb-IIIa (alpha(IIb)beta(3)) integrin complexes.

The major platelet integrin alpha(IIb)beta(3), also known as the platelet glycoprotein (GP) IIb-IIIa complex, mediates platelet aggregation by serving as the receptor for fibrinogen and von Willebrand factor. In addition to its physiologic role, GPIIb-IIIa also bears a number of clinically important alloantigenic determinants. Previous studies have shown that disruption of the long-range Cys(5)-Cys(435) disulfide bond of the beta(3) subunit results in the production of isoforms that bind some, but not all, anti-Pl(A1) alloantibodies, suggesting that mutations in this so-called long-range disulfide bond can alter the conformation of GPIIIa. The purpose of this study was to examine the effects of either the Cys5Ala or Cys435Ala substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa complex. We found that both Ala5GPIIIa and Ala435GPIIIa were capable of associating with GPIIb and were expressed normally on the cell surface when cotransfected into Chinese hamster ovary (CHO) cells. CHO cells expressing GPIIb-Ala5GPIIIa or GPIIb-Ala435IIIa bound well-characterized, conformationally sensitive ligand-induced binding site (LIBS) antibodies, and were capable of constitutively binding the fibrinogen-mimetic monoclonal antibodies Pl-55 and PAC-1, as well as soluble fibrinogen. Both GPIIb-Ala5IIIa- and GPIIb-Ala435IIIa-transfected CHO cells also bound more avidly to immobilized fibrinogen and were capable of mediating the tyrosine phosphorylation of pp125(FAK) on cell adhesion. These data are consistent with the notion that these regions of GPIIIa participate in the conformational change associated with receptor activation. Additionally, these studies may provide a molecular explanation for the previously reported ability of mild reducing agents to activate the GPIIb-IIIa complex and promote platelet aggregation.