Debra K. Newman

Signal transduction pathways mediated by PECAM-1: New roles for an old molecule in platelet and vascular cell biology

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.

Identification of FcγRIIa as the ITAM-bearing receptor mediating αIIbβ3 outside-in integrin signaling in human platelets

Immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins have recently been demonstrated in macrophages and neutrophils to be required for cell surface integrins to transmit activation signals into the cell. To identify ITAM-bearing proteins that mediate signaling via the platelet-specific integrin alphaIIbbeta3, fibrinogen binding was induced by (1) allowing platelets to spread directly on immobilized fibrinogen, or (2) activating the PAR1 thrombin receptor on platelets in suspension. Both initiated strong, ligand binding-dependent tyrosine phosphorylation of the ITAM-bearing platelet Fc receptor, FcgammaRIIa, as well as downstream phosphorylation of the protein tyrosine kinase Syk and activation of phospholipase Cgamma2 (PLCgamma2). Addition of Fab fragments of an FcgammaRIIa-specific monoclonal antibody strongly inhibited platelet spreading on immobilized fibrinogen, as well as downstream tyrosine phosphorylation of FcgammaRIIa, Syk, and PLCgamma2, and platelets from a patient whose platelets express reduced levels of FcgammaRIIa exhibited markedly reduced spreading on immobilized fibrinogen. Finally, fibrinogen binding-induced FcgammaRIIa phosphorylation did not occur in human platelets expressing a truncated beta3 cytoplasmic domain. Taken together, these data suggest that ligand binding to platelet alphaIIbbeta3 induces integrin cytoplasmic domain-dependent phosphorylation of FcgammaRIIa, which then enlists selected components of the immunoreceptor signaling cascade to transmit amplification signals into the cell.

PECAM-1: Conflicts of Interest in Inflammation

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a cell adhesion and signaling receptor that is expressed on hematopoietic and endothelial cells. PECAM-1 is vital to the regulation of inflammatory responses, as it has been shown to serve a variety of pro-inflammatory and anti-inflammatory functions. Pro-inflammatory functions of PECAM-1 include the facilitation of leukocyte transendothelial migration and the transduction of mechanical signals in endothelial cells emanating from fluid shear stress. Anti-inflammatory functions include the dampening of leukocyte activation, suppression of pro-inflammatory cytokine production, and the maintenance of vascular barrier integrity. Although PECAM-1 has been well-characterized and studied, the mechanisms through which PECAM-1 regulates these seemingly opposing functions, and how they influence each other, are still not completely understood. The purpose of this review, therefore, is to provide an overview of the pro- and anti-inflammatory functions of PECAM-1 with special attention paid to mechanistic insights that have thus far been revealed in the literature in hopes of gaining a clearer picture of how these opposing functions might be integrated in a temporal and spatial manner on the whole organism level. A better understanding of how inflammatory responses are regulated should enable the development of new therapeutics that can be used in the treatment of acute and chronic inflammatory disorders.

Site-Specific Effects of PECAM-1 on Atherosclerosis in LDL Receptor–Deficient Mice

Objective—Atherosclerosis is a vascular disease that involves lesion formation at sites of disturbed flow under the influence of genetic and environmental factors. Endothelial expression of adhesion molecules that enable infiltration of immune cells is important for lesion development. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31) is an adhesion and signaling receptor expressed by many cells involved in atherosclerotic lesion development. PECAM-1 transduces signals required for proinflammatory adhesion molecule expression at atherosusceptible sites; thus, it is predicted to be proatherosclerotic. PECAM-1 also inhibits inflammatory responses, on which basis it is predicted to be atheroprotective. Methods and Results—We evaluated herein the effect of PECAM-1 deficiency on development of atherosclerosis in LDL receptor–deficient mice. We found that PECAM-1 has both proatherosclerotic and atheroprotective effects, but that the former dominate in the inner curvature of the aortic arch whereas the latter dominate in the aortic sinus, branching arteries, and descending aorta. Endothelial cell expression of PECAM-1 was sufficient for its atheroprotective effects in the aortic sinus but not in the descending aorta, where the atheroprotective effects of PECAM-1 also required its expression on bone marrow–derived cells. Conclusion—We conclude that PECAM-1 influences initiation and progression of atherosclerosis both positively and negatively, and that it does so in a site-specific manner.

Heparin promotes platelet responsiveness by potentiating αIIbβ3-mediated outside-in signaling

Unfractionated heparin (UFH) is a widely used anticoagulant that has long been known to potentiate platelet responses to subthreshold doses of platelet agonists. UFH has been reported to bind and induce modest conformational changes in the major platelet integrin, αIIbβ3, and induce minor changes in platelet morphology. The mechanism by which UFH elicits these platelet-activating effects, however, is not well understood. We found that both human and murine platelets exposed to UFH, either in solution or immobilized onto artificial surfaces, underwent biochemical and morphologic changes indicative of a potentiated state, including phosphorylation of key cytosolic signaling molecules and cytoskeletal changes leading to cell spreading. Low molecular weight heparin and the synthetic pentasaccharide, fondaparinux, had similar platelet-potentiating effects. Human or mouse platelets lacking functional integrin αIIbβ3 complexes and human platelets pretreated with the fibrinogen receptor antagonists eptifibatide or abciximab failed to become potentiated by heparin, demonstrating that heparin promotes platelet responsiveness via its ability to initiate αIIbβ3-mediated outside-in signaling. Taken together, these data provide novel insights into the mechanism by which platelets become activated after exposure to heparin and heparin-coated surfaces, and suggest that currently used glycoprotein IIb-IIIa inhibitors may be effective inhibitors of nonimmune forms of heparin-induced platelet activation.

Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcγRIIa and the integrin β3 cytoplasmic domain

Thrombocytopenia and thrombosis following treatment with the integrin alphaIIbbeta3 antagonist eptifibatide are rare complications caused by patient antibodies specific for ligand-occupied alphaIIbbeta3. Whether such antibodies induce platelet clearance by simple opsonization, by inducing mild platelet activation, or both is poorly understood. To gain insight into the mechanism by which eptifibatide-dependent antibodies initiate platelet clearance, we incubated normal human platelets with patient serum containing an alphaIIbbeta3-specific, eptifibatide-dependent antibody. We observed that in the presence of eptifibatide, patient IgG induced platelet secretion and aggregation as well as tyrosine phosphorylation of the integrin beta3 cytoplasmic domain, the platelet FcgammaRIIa Fc receptor, the protein-tyrosine kinase Syk, and phospholipase Cgamma2. Each activation event was inhibited by preincubation of the platelets with Fab fragments of the FcgammaRIIa-specific mAb IV.3 or with the Src family kinase inhibitor PP2. Patient serum plus eptifibatide did not, however, activate platelets from a patient with a variant form of Glanzmann thrombasthenia that expressed normal levels of FcgammaRIIa and the alphaIIbbeta3 complex but lacked most of the beta3 cytoplasmic domain. Taken together, these data suggest a novel mechanism whereby eptifibatide-dependent antibodies engage the integrin beta3 subunit such that FcgammaRIIa and its downstream signaling components become activated, resulting in thrombocytopenia and a predisposition to thrombosis.

Neutrophil transmigration mediated by the neutrophil-specific antigen CD177 is influenced by the endothelial S536N dimorphism of platelet endothelial cell adhesion molecule-1.

The human neutrophil-specific adhesion molecule CD177 (also known as the NB1 alloantigen) becomes upregulated on the cell surface in a number of inflammatory settings. We recently showed that CD177 functions as a novel heterophilic counterreceptor for the endothelial junctional protein PECAM-1 (CD31), an interaction that is mediated by membrane-proximal PECAM-1 IgD 6, which is known to harbor an S536N single nucleotide polymorphism of two major isoforms V98N536G643 and L98S536R643 and a yet-to-be-determined region on CD177. In vitro transendothelial migration experiments revealed that CD177+ neutrophils migrated significantly faster through HUVECs expressing the LSR, compared with the VNG, allelic variant of PECAM-1 and that this correlated with the decreased ability of anti-PECAM-1 Ab of ITIM tyrosine phosphorylation in HUVECs expressing the LSR allelic variant relative to the VNG allelic variant. Moreover, engagement of PECAM-1 with rCD177-Fc (to mimic heterophilic CD177 binding) suppressed Ab-induced tyrosine phosphorylation to a greater extent in cells expressing the LSR isoform compared with the VNG isoform, with a corresponding increased higher level of β-catenin phosphorylation. These data suggest that heterophilic PECAM-1/CD177 interactions affect the phosphorylation state of PECAM-1 and endothelial cell junctional integrity in such a way as to facilitate neutrophil transmigration in a previously unrecognized allele-specific manner.

Axial heterogeneity of sodium-bicarbonate cotransporter expression in the rabbit proximal tubule

It is generally accepted that Na(HCO3) n cotransport is the most important mechanism mediating basolateral bicarbonate efflux in the early proximal tubule. The presence of basolateral Na(HCO3) n cotransport in the late proximal tubule (S3 segment) and in the juxtamedullary S1 and S2 segments has been controversial. The renal sodium-bicarbonate cotransporter (NBC) has been recently cloned from rat (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. J. Am. Soc. Nephrol. 7: 1259, 1996), salamander (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. Nature 387: 409-413, 1997), and human (C. E. Burnham, H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. J. Biol. Chem. 272: 19111-19114, 1997). The localization of NBC in the kidney is unknown. The present study was designed to localize NBC mRNA expression in the rabbit proximal tubule. In situ hybridization studies were combined with functional studies of basolateral Na(HCO3) n cotransport in superficial and juxtamedullary S1, S2, and S3 segments of the rabbit proximal tubule. The results demonstrate that NBC mRNA is localized predominantly to the cortex, with less expression in the outer medulla. NBC expression was not detected in the inner medulla. The highest level of NBC mRNA is in the S1 proximal tubule. NBC is expressed at a low levels in the S3 segment, with intermediate expression in the S2 segment. In bicarbonate-buffered solutions, the rate of base efflux mediated by Na(HCO3) n cotransport followed a similar pattern in superficial and juxtamedullary proximal tubule segments, i.e., S1 > S2 > S3. The juxtamedullary S1 segment had the greatest rate of basolateral Na(HCO3) n cotransport and the highest level of NBC expression in the proximal tubule.

Signal Transduction During Platelet Plug Formation

This chapter summarizes the major threads of what is currently known about platelet activation during the hemostatic response to injury. To put the information about signaling pathways in context, the chapter begins by considering the “macro” events of platelet activation before focusing on the molecular events that both propel and limit platelet activation and then considering how events outside the platelet impact those that occur within.

Oligomeric Structure and Minimal Functional Unit of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1-A

The electrogenic sodium bicarbonate cotransporter NBCe1-A mediates the basolateral absorption of sodium and bicarbonate in the proximal tubule. In this study the oligomeric state and minimal functional unit of NBCe1-A were investigated. Wild-type (wt) NBCe1-A isolated from mouse kidney or heterologously expressed in HEK293 cells was predominantly in a dimeric state as was shown using fluorescence energy transfer, pulldown, immunoprecipitation, cross-linking experiments, and nondenaturing perfluorooctanoate-PAGE. NBCe1-A monomers were found to be covalently linked by S-S bonds. When each of the 15 native cysteine residues were individually removed on a wt-NBCe1-A backbone, dimerization of the cotransporter was not affected. In experiments involving multiple native cysteine residue removal, both Cys630 and Cys642 in extracellular loop 3 were shown to mediate S-S bond formation between NBCe1-A monomers. When native NBCe1-A cysteine residues were individually reintroduced into a cysteineless NBCe1-A mutant backbone, the finding that a Cys992 construct that lacked S-S bonds functioned normally indicated that stable covalent linkage of NBCe1-A monomers was not a necessary requirement for functional activity of the cotransporter. Studies using concatameric constructs of wt-NBCe1-A, whose activity is resistant to methanesulfonate reagents, and an NBCe1-AT442C mutant, whose activity is completely inhibited by methanesulfonate reagents, confirmed that NBCe1-A monomers are functional. Our results demonstrate that wt-NBCe1-A is predominantly a homodimer, dependent on S-S bond formation that is composed of functionally active monomers.