Research Interests: Biochemistry, Chemistry, Molecular Biology, Biological Chemistry, Medicine, and 15 moreBiological Sciences, DNA, Platelet, Humans, Animals, Biological, Glycosylation, CHEMICAL SCIENCES, Cysteine, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, Blood platelets, Gene frequency, and Medical and Health Sciences
Research Interests: Biology, Cell Biology, Biological Chemistry, Medicine, Biological Sciences, and 15 moreHumans, Biological, Polymerase Chain Reaction, Gene, Genes, CHEMICAL SCIENCES, Introns, Receptor, Protein Conformation, Amino Acid Sequence, Base Sequence, Leukocytes, Cell Adhesion Molecules, Molecular Sequence Data, and Medical and Health Sciences
Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally inherited antigens present on the surface of fetal platelets. The human platelet... more
Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally inherited antigens present on the surface of fetal platelets. The human platelet alloantigen HPA-1a (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the ∼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and...
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The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large... more
The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool for the study of platelet-reactive antibodies, but previous attempts to produce such a construct have yielded only small quantities of the end product. Using a baculovirus expression system and the dual-promoter transfer vector P2Bac, we were able to express soluble GPIIb-IIIa complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in quantities of about 1,000 μg/L, about 40 times greater than reported previously. The high yield achieved may be related to inclusion of the entire extracellular region of the GPIIb light chain in the construct. srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction is totally inhibited by the peptid...
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Research Interests: Chemistry, Flow Cytometry, Medicine, Platelet, Fibrinogen, and 14 moreHumans, von Willebrand factor, Animals, Blood, P-glycoprotein, Clinical Sciences, Hemostasis, Cysteine, Integrin, Disulfide bond, CHO cells, Cricetinae, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
Inhibitory cell surface proteins on T cells are often dynamically regulated, which contributes to their physiologic function. PECAM-1 (CD31) is an inhibitory receptor that facilitates TGF-β-mediated suppression of T cell activity. It is... more
Inhibitory cell surface proteins on T cells are often dynamically regulated, which contributes to their physiologic function. PECAM-1 (CD31) is an inhibitory receptor that facilitates TGF-β-mediated suppression of T cell activity. It is well established in CD4+ T cells that PECAM-1 is expressed in naïve recent thymic emigrants, but is down-regulated after acute T cell activation and absent from memory cells. The extent to which PECAM-1 expression is similarly regulated in CD8+ T cells is much less well characterized. We evaluated T cells recovered from mice after infection with a model intracellular pathogen and determined that, in CD8+ T cells, PECAM-1 expression was strongly down-regulated during acute infection but re-expressed to intermediate levels in memory cells. Down-regulation of PECAM-1 expression in CD8+ T cells was transcriptionally regulated and affected by the strength and nature of TCR signaling. PECAM-1 was also detected on the surface of human activated/memory CD8+,...
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Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b... more
Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a–specific antibodies. Humanizing the PSI dom...
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Research Interests: Chemistry, Membrane Proteins, Medicine, Cardiac Surgery, Platelet, and 15 moreHumans, Platelet aggregation, Clinical, Glycoproteins, ENDOTHELIUM, Erythrocytes, Fixatives, Clinical Investigation, Leukocytes, Edetic Acid, Cardiopulmonary bypass, Cell Membrane, Blood platelets, Blood Specimen Collection, and Medical and Health Sciences
Research Interests: Chemistry, Biology, Cell Biology, Cell Signaling, Biological Chemistry, and 14 moreMedicine, Signal Transduction, Biological Sciences, Platelet, Humans, Platelet aggregation, Integrins, Protein tyrosine phosphatase, CHEMICAL SCIENCES, Amino Acid Sequence, Protein Tyrosine Phosphatases, Tyrosine, Molecular Sequence Data, and Medical and Health Sciences
Research Interests: Flow Cytometry, Biology, Cell Biology, Biological Chemistry, Medicine, and 15 moreAdhesion, Biological Sciences, Platelet, Humans, Endothelial Cells, Biological, Monoclonal Antibodies, CD, CHEMICAL SCIENCES, Neutrophils, Monocytes, Monoclonal Antibody, Protein Binding, Leukocytes, and Medical and Health Sciences
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The severity of nonalcoholic steatohepatitis (NASH) is determined by environmental and genetic factors, the latter of which are incompletely characterized. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane... more
The severity of nonalcoholic steatohepatitis (NASH) is determined by environmental and genetic factors, the latter of which are incompletely characterized. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed on blood and vascular cells. In the present study, we provide data for the novel finding that genetic deficiency of PECAM-1 potentiates the development and progression of NASH. We found that the rate of development and severity of diet-induced NASH are markedly enhanced in PECAM-1-deficient [knockout (KO)] mice relative to wild-type (WT) mice, as measured by histological and biochemical evaluation. Livers from KO mice exhibited typical histological features of NASH, including macrovesicular fat accumulation, hepatocyte injury with infiltration of inflammatory cells, fibrosis, and heightened oxidative stress. Alanine aminotransferase, a marker for liver injury, was also significantly higher in KO compared with WT mice. Consist...
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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... more
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 t...
Research Interests: Physiology, Chemistry, Biology, Cell Biology, Oxidative Stress, and 15 moreMedicine, Signal Transduction, Cell line, Humans, Kidney, Phosphatase, Reactive Oxygen Species, American, Phosphorylation, Medical Physiology, Protein tyrosine phosphatase, Hydrogen Peroxide, Protein Binding, Protein Tyrosine Phosphatases, and Oxidants
Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific... more
Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an im...
Research Interests: Biochemistry, Molecular Biology, Biology, Membrane Proteins, Cell Biology, and 15 moreMedicine, Biological Sciences, Humans, Glycoproteins, Affinity chromatography, Polyclonal Antibodies, The cell, Molecular weight, Monoclonal Antibody, Protein Binding, Sepharose, Electrophoretic Mobility, Gelsolin, Blood platelets, and Medical and Health Sciences
Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is... more
Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-α-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted...
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The molecular nature of cell adhesion mediated by platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) was examined using stably transfected L cells in a PECAM-dependent aggregation assay. This adhesion was temperature sensitive... more
The molecular nature of cell adhesion mediated by platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) was examined using stably transfected L cells in a PECAM-dependent aggregation assay. This adhesion was temperature sensitive and divalent cation dependent, with Mg2+ supporting aggregation to a greater degree than Ca2+. PECAM-dependent aggregation was heterophilic: PECAM-1 transfectants bound as readily to control-transfected L cells as to other PECAM-1 transfectants, demonstrating that a molecule endogenously expressed on the L cells serves as the ligand for PECAM in this system and presumably substitutes for the natural human ligand.
Research Interests: Chemistry, Kinetics, Cell Adhesion, Cell Biology, Medicine, and 15 moreAdhesion, Extracellular Matrix, Mice, Animals, Temperature, The, CD, Endothelial cell, Experimental Medicine, Transfection, Carcinoembryonic Antigen, Cations, Temperature Sensitive Polymer, Cellular system, and Medical and Health Sciences
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Interaction between the cytoplasmic domain of GPIbα with its cytoskeletal binding partner, filamin, is a major determinant of platelet size, and deficiency of either protein results in macrothrombocytopenia. To clarify the mechanism by... more
Interaction between the cytoplasmic domain of GPIbα with its cytoskeletal binding partner, filamin, is a major determinant of platelet size, and deficiency of either protein results in macrothrombocytopenia. To clarify the mechanism by which GPIbα-filamin interactions regulate platelet production, we manipulated the expression levels of filamin and GPIb in cultured embryonic stem cells (ESCs) that were subsequently differentiated into platelets. Knocking down filamins A and B resulted in the production of ESC-derived proplatelets with abnormally large swellings and proplatelet shafts that generated giant platelets in culture. Large platelets could also be generated by overexpressing GPIbα in ESCs, or by overexpressing in vivo a transgene encoding a chimeric protein containing the cytoplasmic domain of GPIbα. To identify the mechanism by which the GPIb:filamin ratio regulates platelet size, we manipulated filamin and GPIbα levels in HEK293T cells and examined the effects of overexpre...
Research Interests: Flow Cytometry, Biology, Cell Biology, Medicine, Platelet, and 15 moreCell Differentiation, Humans, Mice, Animals, Blood, Immunoprecipitation, Clinical Sciences, Cytoplasm, Protein Transport, Contractile Proteins, Filamin, Immunoblotting, Blood platelets, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the immunoglobulin (Ig) superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selected T cell subsets. It is also a... more
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the immunoglobulin (Ig) superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selected T cell subsets. It is also a major constituent of the endothelial cell intercellular junction (1–3), where up to 10 6 PECAM-1 molecules (4) are concentrated. With a few minor exceptions, PECAM-1 is not present on fibroblasts, epithelium, muscle, or other nonvascular cells. Since its cloning nearly 10 yr ago (5, 6), much has been learned about the structure of this cell adhesion receptor and its function in vascular cells. The purpose of this brief Perspective is to review progress in the field of PECAM-1 biology, and to bring the reader up to date on current concepts about ( a ) the function of PECAM-1 in the different vascular cells in which it is expressed; ( b ) the molecular mechanisms by which PECAM-1 mediates cell–cell interactions; and ( c ) its role in bidirectional tr...
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... more
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 substitutio...
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Recent studies of platelet endothelial cell adhesion molecule-1 (PECAM-1 (CD31))-deficient mice have revealed that this molecule plays an important role in controlling the activation and survival of cells on which it is expressed. In this... more
Recent studies of platelet endothelial cell adhesion molecule-1 (PECAM-1 (CD31))-deficient mice have revealed that this molecule plays an important role in controlling the activation and survival of cells on which it is expressed. In this review, we focus on the complex cytoplasmic domain of PECAM-1 and describe what is presently known about its structure, posttranslational modifications, and binding partners. In addition, we summarize findings that implicate PECAM-1 as an inhibitor of cellular activation via protein tyrosine kinase- dependent signaling pathways, an activator of integrins, and a suppressor of cell death via pathways that depend on damage to the mitochondria. The challenge of future research will be to bridge our understanding of the functional and biochemical properties of PECAM-1 by establishing mechanistic links between signals transduced by the PECAM-1 cytoplasmic domain and discrete cellular responses. (Arterioscler Thromb Vasc Biol. 2003;23:953-964.)
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Platelet endothelial cell adhesion molecule 1 (PECAM-1) is capable of transducing signals in endothelial cells exposed to shear; however, the biological consequences of this signal transduction are unknown. Because shear stress elicits... more
Platelet endothelial cell adhesion molecule 1 (PECAM-1) is capable of transducing signals in endothelial cells exposed to shear; however, the biological consequences of this signal transduction are unknown. Because shear stress elicits flow-mediated dilation (FMD), we examined whether steady-state FMD in mouse coronary arteries (MCAs) is affected in the PECAM-1 knockout (KO) mouse. MCAs were isolated from wild-type (WT) or KO mice and prepared for videomicroscopy, histofluorescence, Western blotting, and immunohistochemistry. FMD was examined in the absence and presence of Nω-nitro-l-arginine methyl ester (l-NAME) and l-NAME+indomethacin (INDO). FMD was reduced in KO relative to WT MCAs, but the l-NAME-inhibitable portion of FMD was similar between the two. The INDO-sensitive component of FMD was diminished in KO MCAs. In contrast, the residual component of dilation, presumably because of endothelium-derived hyperpolarizing factor (EDHF), was abolished in KO MCAs. Histofluorescence ...
Research Interests: Chemistry, Medicine, Biological Sciences, Mice, Nitric oxide, and 15 moreAnimals, Male, American, Peroxynitrite, Organoselenium Compounds, Hydrogen Peroxide, Prostaglandins, ENDOTHELIUM, Biological Factors, Knockout Mice, Tyrosine, Indomethacin, Medical and Health Sciences, Coronary Vessels, and Azoles
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Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor–ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed... more
Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor–ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1–PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking ...
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Research Interests: Molecular Biology, Polymorphism, Biology, Cell Adhesion, Medicine, and 14 morePlatelet, Humans, Polymerase Chain Reaction, P-glycoprotein, Genetic Polymorphism, Amino Acid Profile, Sialic Acid, Base Sequence, Clinical Investigation, Integrin, Molecular Sequence Data, Blood platelets, epitope, and Medical and Health Sciences
Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, IIbβ3. In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant IIbβ3 may interact normally with fibrin yet fail to... more
Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, IIbβ3. In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant IIbβ3 may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable IIbβ3, findings consistent with type II GT. Genotyping of LD revealed 2 novel β3 mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among β integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Proβ3 cotransfected with wild-type IIb into COS-7 cells showed delaye...
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PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown... more
PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown that the extracellular domain of PECAM-1, which is comprised of 6 Ig-like homology units, participates in mediating cell-cell adhesion, plays a role in initiating endothelial cell contact, and may later serve to stabilize the endothelial cell monolayer. PECAM-1 also has a relatively large 108 amino acid cytoplasmic domain, with potential sites for phosphorylation, lipid modification, and other posttranslational events that could potentially modulate its adhesive function or regulate its subcellular distribution. Virtually nothing is known about the contribution of the intracellular region of the PECAM-1 molecule to either of these cellular processes. Using human platelets as a model, we now demonstrate that PECAM-1 becomes highly phosphorylated in resp...
Research Interests: Biology, Cell Adhesion, Cell Biology, Cytoskeleton, Human, and 15 moreBiological Sciences, Humans, Homme, Activation, Endothelial cell, Amino Acid Profile, Cytoplasm, Hombre, Immunoglobulin, Cell Adhesion Molecules, Intracellular, Citoesqueleto, activación, Blood platelets, and Medical and Health Sciences
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Key Points HPA allele–specific HLA class I–negative MKs can be differentiated from CRISPR-edited human iPSCs. Such cells can be stored frozen and thawed to use in whole-cell flow cytometric assays to detect anti-HPA-3a, -3b, and -9b... more
Key Points HPA allele–specific HLA class I–negative MKs can be differentiated from CRISPR-edited human iPSCs. Such cells can be stored frozen and thawed to use in whole-cell flow cytometric assays to detect anti-HPA-3a, -3b, and -9b alloantibodies.
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Research Interests: Biology, Life Sciences, Inflammation, Cell Adhesion, Conflict of Interest, and 13 moreMedicine, Signal Transduction, Inflammatory Immune Response, Humans, Endothelial Cells, Animals, Endothelial cell, Adhesion molecules, Leukocytes, Biochemistry and cell biology, Inflammatory response, Pro-Inflammatory Cytokine, and Pharmacology and pharmaceutical sciences
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Research Interests: Immunology, Flow Cytometry, Biology, Immunology of the Gut, Medicine, and 11 moreCell separation, Humans, Endothelial Cells, Surface plasmon resonance, Neutrophils, Single Nucleotide Polymorphism, Protein isoforms, Base Sequence, Immunoblotting, Molecular Sequence Data, and reverse transcriptase polymerase chain reaction
The CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]/endothelial cell adhesion molecule [endoCAM]) molecule expressed on leukocytes, platelets, and endothelial cells is postulated to mediate adhesion to endothelial cells and... more
The CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]/endothelial cell adhesion molecule [endoCAM]) molecule expressed on leukocytes, platelets, and endothelial cells is postulated to mediate adhesion to endothelial cells and thereby function in immunity, inflammation, and wound healing. We report the following novel features of CD31 which suggests a role for it in adhesion amplification of unique T cell subsets: (a) engagement of CD31 induces the adhesive function of beta 1 and beta 2 integrins; (b) adhesion induction by CD31 immunoglobulin G (IgG) monoclonal antibodies (mAbs) is sensitive, requiring only bivalent mAb; (c) CD31 mAb induces adhesion rapidly, but it is transient; (d) unique subsets of CD4+ and CD8+ T cells express CD31, including all naive (CD45RA+) CD8 T cells; and (e) CD31 induction is selective, inducing adhesive function of beta 1 integrins, particularly very late antigen-4, more efficiently than the beta 2 integrin lymphocyte function-associated anti...
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Research Interests: Medicine, Stents, Signal Transduction, Antibodies, Platelet, and 15 moreHumans, Platelet aggregation, Heparin, Male, Thrombocytopenia, Peptides, Platelet activation mechanism, Aged, Clinical Investigation, Platelet Count, Integrin, immunoglobulin G, Blood platelets, Platelet Aggregation Inhibitors, and Medical and Health Sciences
Research Interests: Biochemistry, Chemistry, Flow Cytometry, Calcium, Adolescent, and 15 morePlatelet, Fibrinogen, Humans, Mutation, Female, Integrins, Amino Acid Profile, Amino Acid Sequence, Base Sequence, Structure activity Relationship, Clinical Investigation, Integrin, Molecular Sequence Data, Blood platelets, and Medical and Health Sciences
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... more
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 (Δ15) 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 Δ15 PECAM-1, we found that the Δ15 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 t...
Research Interests: Biology, Cell Biology, Confocal Microscopy, Apoptosis, Medicine, and 15 moreSignal Transduction, Biological Sciences, Cell line, Humans, Mice, Animals, Cell, Alternative splicing, Endothelial cell, Alternative Splicing, Relative Abundance, Amino Acid Sequence, Base Sequence, Molecular Sequence Data, and Medical and Health Sciences
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Key PointsThe study describes a potential novel treatment of fetal alloimmune thrombocytopenia by dissecting the effector activities of an epitope-specific IgG antibody. Neither the in vivo transplacental transport nor the inhibiting... more
Key PointsThe study describes a potential novel treatment of fetal alloimmune thrombocytopenia by dissecting the effector activities of an epitope-specific IgG antibody. Neither the in vivo transplacental transport nor the inhibiting properties of the blocking antibody are impaired by the N-glycan modification.
Research Interests: Biology, Polysaccharides, Medicine, Pregnancy, Humans, and 14 morePlacenta, Mice, Female, Animals, Blood, Monoclonal Antibodies, Glycosylation, Clinical Sciences, Phagocytosis, Protein Binding, Molecular Targeted Therapy, Blood platelets, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
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... more
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 int...
Research Interests: Chemistry, Medicine, Humans, Platelet aggregation, Micelles, and 12 moreCircular Dichroism, Blood, Phosphorylation, Platelet activation mechanism, Phosphotyrosine, Clinical Sciences, Recombinant Proteins, Cytoplasm, Cell Membrane, phosphorylcholine, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet–endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI)... more
Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet–endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI) collagen receptor; however, it is not known whether PECAM-1 and Lyn function in the same or different inhibitory pathways. In these studies, we found that, relative to wild-type platelets, platelets derived from PECAM-1–deficient, Lyn-deficient, or PECAM-1/Lyn double-deficient mice were equally hyperresponsive to stimulation with a GPVI-specific agonist, indicating that PECAM-1 and Lyn participate in the same inhibitory pathway. Lyn was required for PECAM-1 tyrosine phosphorylation and subsequent binding of the Src homology 2 domain–containing phosphatase-2, SHP-2. These results support a model in which PECAM-1/SHP-2 complexes, formed in a Lyn-dependent manner, suppress GPVI signaling.
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Drug-dependent antibodies (DDAbs) can cause the precipitous destruction of platelets if a patient is exposed to the drug for which the antibodies are specific. The molecular character of the epitopes recognized is poorly understood, and... more
Drug-dependent antibodies (DDAbs) can cause the precipitous destruction of platelets if a patient is exposed to the drug for which the antibodies are specific. The molecular character of the epitopes recognized is poorly understood, and the mechanism by which drugs promote tight binding of these antibodies to platelet glycoproteins without linking covalently to protein or antibody is not yet known. We studied a group of quinine-dependent antibodies that react with human glycoprotein IIIa (GPIIIa; β3-integrin subunit) but fail to recognize rat GPIIIa, despite close homology between the 2 proteins. By characterizing reactions of these antibodies with human/rat GPIIIa chimeras and selected GPIIIa mutants, we found that each of 3 quinine-dependent antibodies requires a 17-amino acid sequence in the newly recognized “hybrid” and PSI homology domains of GPIIIa for drug-dependent binding. Disulfide bonds are required to stabilize the target epitope. Monoclonal antibody AP3, which blocks th...