Does anti-HPA-1a affect birthweight in fetal and neonatal alloimmune thrombocytopenia?

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) ensues from parental incompatibility for platelet alloantigens with maternal sensitization. HPA-1a/1b incompatibility is the most common cause of FNAIT in Caucasians. Placental villitis and lower birthweight in FNAIT suggest anti-HPA-1a may have effects beyond inducing thrombocytopenia. OBJECTIVES: Does FNAIT secondary to anti-HPA-1a result in smaller newborns and, the corollary, does antenatal management of FNAIT increase birthweight? STUDY DESIGN: Birthweights of 270 FNAIT-affected newborns from a randomized clinical trial and a NAITbabies.org survey (135 paired siblings) were compared with those of published controls and treated to untreated FNAIT-affected siblings. Birthweights were converted to percentiles to account for gestational age, sex, and role of birth order in birth weight. Body weights of FNAIT-affected and -unaffected pups in a mouse FNAIT model were analyzed. RESULTS: Untreated siblings in both the clinical trial and NAITbabies.org cohorts were not small, compared with normal controls. However, treated siblings in both cohorts had significantly higher birthweight percentiles compared with their previous untreated affected sibling. After accounting for gestational age, sex, and birth order, increased birthweight percentile in treated compared with the untreated siblings remained significant in both cohorts. FNAIT-affected neonatal mice had lower bodyweights than FNAIT-unaffected pups. CONCLUSIONS: Untreated FNAIT-affected newborns were not small; however, treatment of FNAIT-affected pregnancies increased newborn birthweights despite corrections to account for other factors that might have influenced the results. High dose IVIG is believed to "block" FcRn and lower maternal anti-HPA-1a levels, and thus increase birthweights by reducing levels of maternal anti-HPA-1a and reducing placental villitis.

Prophylactic administration of HPA-1a-specific antibodies prevents fetal/neonatal alloimmune thrombocytopenia in mice.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited human platelet alloantigens (HPAs) present on the surface of fetal and neonatal platelets. There are currently no approved therapies for the prevention of FNAIT. We report herein the ability of 2 human HPA-1a-specific therapeutic candidates, one a polyclonal, and the other a monoclonal antibody, to prevent alloimmunization in a novel preclinical mouse model of FNAIT. Both antibody preparations effected the rapid and complete elimination of HPA-1a+ platelets from circulation and prevented the development of HPA-1a alloantibodies. HPA-1a- female mice treated prophylactically with anti-HPA-1a antibody prior to exposure to HPA-1a+ platelets gave birth to HPA-1a+/- pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish both the potential and threshold exposure targets for prophylactic treatment with HPA-1a-specific antibodies for the prevention of FNAIT in humans.

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.

Preclinical evaluation of immunotherapeutic regimens for fetal/neonatal alloimmune thrombocytopenia.

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 ß3 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 when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.

SLC4A11 function: evidence for H+(OH-) and NH3-H+ transport.

Whether SLC4A11 transports ammonia and its potential mode of ammonia transport (NH4+, NH3, or NH3-2H+ transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 mediates significant conductive H+(OH-) transport is also controversial. The present study was performed to determine the mechanism of human SLC4A11 ammonia transport and whether the transporter mediates conductive H+(OH-) transport in the absence of ammonia. We quantitated H+ flux by monitoring changes in intracellular pH (pHi) and measured whole cell currents in patch-clamp studies of HEK293 cells expressing the transporter in the absence and presence of NH4Cl. Our results demonstrate that SLC4A11 mediated conductive H+(OH-) transport that was stimulated by raising the extracellular pH (pHe). Ammonia-induced HEK293 whole cell currents were also stimulated by an increase in pHe. In studies using increasing NH4Cl concentrations with equal NH4+ extracellular and intracellular concentrations, the shift in the reversal potential (Erev) due to the addition of ammonia was compatible with NH3-H+ transport competing with H+(OH-) rather than NH3-nH+ (n ≥ 2) transport. The increase in equivalent H+(OH-) flux observed in the presence of a transcellular H+ gradient was also compatible with SLC4A11-mediated NH3-H+ flux. The NH3 versus Erev data fit a theoretical model suggesting that NH3-H+ and H+(OH-) competitively interact with the transporter. Studies of mutant SLC4A11 constructs in the putative SLC4A11 ion coordination site showed that both H+(OH-) transport and ammonia-induced whole cell currents were blocked suggesting that the H+(OH-) and NH3-H+ transport processes share common features involving the SLC4A11 transport mechanism.

Generation of PECAM-1 (CD31) conditional knockout mice.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is an adhesion and signaling receptor that is expressed on endothelial and hematopoietic cells and plays important roles in angiogenesis, vascular permeability, and regulation of cellular responsiveness. To better understanding the tissue specificity of PECAM-1 functions, we generated mice in which PECAM1, the gene encoding PECAM-1, could be conditionally knocked out. A targeting construct was created that contains loxP sites flanking PECAM1 exons 1 and 2 and a neomycin resistance gene flanked by flippase recognition target (FRT) sites that was positioned upstream of the 3' loxP site. The targeting construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo-pregnant females. Resulting chimeric animals were bred with transgenic mice expressing Flippase 1 (FLP1) to remove the FRT-flanked neomycin resistance gene and mice heterozygous for the floxed PECAM1 allele were bred with each other to obtain homozygous PECAM1 flox/flox offspring, which expressed PECAM-1 at normal levels and had no overt phenotype. PECAM1 flox/flox mice were bred with mice expressing Cre recombinase under the control of the SRY-box containing gene 2 (Sox2Cre) promoter to delete the floxed PECAM1 allele in offspring (Sox2Cre;PECAM1 del/WT ), which were crossbred to generate Sox2Cre; PECAM1 del/del offspring. Sox2Cre; PECAM1 del/del mice recapitulated the phenotype of conventional global PECAM-1 knockout mice. PECAM1 flox/flox mice will be useful for studying distinct roles of PECAM-1 in tissue specific contexts and to gain insights into the roles that PECAM-1 plays in blood and vascular cell function.

Platelet Function Changes during Neonatal Cardiopulmonary Bypass Surgery: Mechanistic Basis and Lack of Correlation with Excessive Bleeding.

Thrombocytopenia and platelet dysfunction induced by extracorporeal blood circulation are thought to contribute to postsurgical bleeding complications in neonates undergoing cardiac surgery with cardiopulmonary bypass (CPB). In this study, we examined how changes in platelet function relate to changes in platelet count and to excessive bleeding in neonatal CPB surgery. Platelet counts and platelet P-selectin exposure in response to agonist stimulation were measured at four times before, during, and after CPB surgery in neonates with normal versus excessive levels of postsurgical bleeding. Relative to baseline, platelet counts were reduced in patients while on CPB, as was platelet activation by the thromboxane A2 analog U46619, thrombin receptor activating peptide (TRAP), and collagen-related peptide (CRP). Platelet activation by adenosine diphosphate (ADP) was instead reduced after platelet transfusion. We provide evidence that thrombocytopenia is a likely contributor to CPB-associated defects in platelet responsiveness to U46619 and TRAP, CPB-induced collagen receptor downregulation likely contributes to defective platelet responsiveness to CRP, and platelet transfusion may contribute to defective platelet responses to ADP. Platelet transfusion restored to baseline levels platelet counts and responsiveness to all agonists except ADP but did not prevent excessive bleeding in all patients. We conclude that platelet count and function defects are characteristic of neonatal CPB surgery and that platelet transfusion corrects these defects. However, since CPB-associated coagulopathy is multifactorial, platelet transfusion alone is insufficient to treat bleeding events in all patients. Therefore, platelet transfusion must be combined with treatment of other factors that contribute to the coagulopathy to prevent excessive bleeding.

Diacylglycerol kinase ζ is a negative regulator of GPVI-mediated platelet activation.

Diacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ζ isoform of DGK (DGKζ) has been reported to inhibit T-cell responsiveness by downregulating intracellular levels of DAG. However, its role in platelet function remains undefined. In this study, we show that DGKζ was expressed at significant levels in both platelets and megakaryocytes and that DGKζ-knockout (DGKζ-KO) mouse platelets were hyperreactive to glycoprotein VI (GPVI) agonists, as assessed by aggregation, spreading, granule secretion, and activation of relevant signal transduction molecules. In contrast, they were less responsive to thrombin. Platelets from DGKζ-KO mice accumulated faster on collagen-coated microfluidic surfaces under conditions of arterial shear and stopped blood flow faster after ferric chloride-induced carotid artery injury. Other measures of hemostasis, as measured by tail bleeding time and rotational thromboelastometry analysis, were normal. Interestingly, DGKζ deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGKζ as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo.

Frontline Science: PECAM-1 (CD31) expression in naïve and memory, but not acutely activated, CD8+ T cells.

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-ß-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+ , but not CD4+ T cells. These data demonstrate that PECAM-1 expression is dynamically regulated, albeit differently, in both CD4+ and CD8+ T cells. Furthermore, unlike memory CD4+ T cells, memory CD8+ T cells retain PECAM-1 expression and have the potential to be modulated by this inhibitory receptor.

Diacylglycerol kinase ζ (DGKζ) and Casitas b-lineage proto-oncogene b-deficient mice have similar functional outcomes in T cells but DGKζ-deficient mice have increased T cell activation and tumor clearance.

Targeting negative regulators downstream of the T cell receptor (TCR) represents a novel strategy to improve cancer immunotherapy. Two proteins that serve as critical inhibitory regulators downstream of the TCR are diacylglycerol kinase ζ (DGKζ), a regulator of Ras and PKC-θ signaling, and Casitas b-lineage proto-oncogene b (Cbl-b), an E3 ubiquitin ligase that predominantly regulates PI(3)K signaling. We sought to compare the signaling and functional effects that result from deletion of DGKζ, Cbl-b, or both (double knockout, DKO) in T cells, and to evaluate tumor responses generated in a clinically relevant orthotopic pancreatic tumor model. We found that whereas deletion of Cbl-b primarily served to enhance NF-κB signaling, deletion of DGKζ enhanced TCR-mediated signal transduction downstream of Ras/Erk and NF-κB. Deletion of DGKζ or Cbl-b comparably enhanced CD8+ T cell functional responses, such as proliferation, production of IFNγ, and generation of granzyme B when compared with WT T cells. DKO T cells demonstrated enhanced function above that observed with single knockout T cells after weak, but not strong, stimulation. Deletion of DGKζ, but not Cbl-b, however, resulted in significant increases in numbers of activated (CD44hi) CD8+ T cells in both non-treated and tumor-bearing mice. DGKζ-deficient mice also had enhanced control of pancreatic tumor cell growth compared to Cbl-b-deficient mice. This represents the first direct comparison between mice of these genotypes and suggests that T cell immunotherapies may be better improved by targeting TCR signaling molecules that are regulated by DGKζ as opposed to molecules regulated by Cbl-b.

Rotational Thromboelastometry Rapidly Predicts Thrombocytopenia and Hypofibrinogenemia During Neonatal Cardiopulmonary Bypass.

BACKGROUND: Thrombocytopenia and hypofibrinogenemia during neonatal cardiopulmonary bypass (CPB) contribute to bleeding and morbidity. Rotational thromboelastometry (ROTEM) is a viscoelastic assay with a rapid turnaround time. Data validating ROTEM during neonatal cardiac surgery remain limited. This study examined perioperative hemostatic trends in neonates treated with standardized platelet and cryoprecipitate transfusion during CPB. We hypothesized that ROTEM would predict thrombocytopenia, hypofibrinogenemia, and the correction thereof. METHODS: Forty-four neonates undergoing CPB were included in this prospective observational study. Blood samples were obtained at Baseline, On CPB, Post-CPB, and Postoperative. The ROTEM analysis included extrinsically activated (Extem) and fibrinogen-specific (Fibtem) assays. Platelet-specific thromboelastometry (Pltem) values were calculated. Platelet and cryoprecipitate transfusion was initiated prior to termination of CPB. RESULTS: Platelet count and Extem amplitude decreased significantly On CPB ( P < .0001), increased significantly Post-CPB ( P < .0001), and Postoperative values were not significantly different from Baseline. Extem amplitude at 10 minutes (A10) > 46.5 mm (AUC = 0.941) and Pltem A10 > 37.5 mm [area under curve (AUC) = 0.960] predicted platelet count > 100 × 103/µL, and they highly correlated with platelet count ( R = 0.89 and R = 0.90, respectively). Fibrinogen concentration and Fibtem amplitude decreased significantly On CPB ( P ≤ .0001) and normalized after cryoprecipitate transfusion. Fibtem A10 > 9.5 mm predicted fibrinogen >200 mg/dL (AUC = 0.817), but it correlated less well with fibrinogen concentration ( R = 0.65). CONCLUSIONS: ROTEM analysis during neonatal cardiac surgery is sensitive and specific for thrombocytopenia and hypofibrinogenemia, identifying deficits within 10 minutes. Platelet and cryoprecipitate transfusion during neonatal CPB normalizes platelet count, fibrinogen level, and ROTEM amplitudes.

CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1.

Na+-coupled acid-base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+-coupled acid-base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

Validation of a definition of excessive postoperative bleeding in infants undergoing cardiac surgery with cardiopulmonary bypass.

OBJECTIVE: To derive and validate an objective definition of postoperative bleeding in neonates and infants undergoing cardiac surgery with cardiopulmonary bypass. METHODS: Using a retrospective cohort of 124 infants and neonates, we included published bleeding definitions and cumulative chest tube output over different postoperative periods (eg, 2, 12, or 24 hours after intensive care unit admission) in a classification and regression tree model to determine chest tube output volumes that were associated with red blood cell transfusions and surgical re-exploration for bleeding in the first 24 hours after intensive care unit admission. After the definition of excessive bleeding was determined, it was validated via a prospective cohort of 77 infants and neonates. RESULTS: Excessive bleeding was defined as ≥7 mL/kg/h for ≥2 consecutive hours in the first 12 postoperative hours and/or ≥84 mL/kg total for the first 24 postoperative hours and/or surgical re-exploration for bleeding or cardiac tamponade physiology in the first 24 postoperative hours. Excessive bleeding was associated with longer length of hospital stay, increased 30-day readmission rate, and increased transfusions in the postoperative period. CONCLUSIONS: The proposed standard definition of excessive bleeding is based on readily obtained objective data and relates to important early clinical outcomes. Application and validation by other institutions will help determine the extent to which our specialty should consider this definition for both clinical investigation and quality improvement initiatives.

CRISPR-mediated deletion of the PECAM-1 cytoplasmic domain increases receptor lateral mobility and strengthens endothelial cell junctional integrity.

AIMS: PECAM-1 is an abundant endothelial cell surface receptor that becomes highly enriched at endothelial cell-cell junctions, where it functions to mediate leukocyte transendothelial migration, sense changes in shear and flow, and maintain the vascular permeability barrier. Homophilic interactions mediated by the PECAM-1 extracellular domain are known to be required for PECAM-1 to perform these functions; however, much less is understood about the role of its cytoplasmic domain in these processes. MAIN METHODS: CRISPR/Cas9 gene editing technology was employed to generate human endothelial cell lines that either lack PECAM-1 entirely, or express mutated PECAM-1 missing the majority of its cytoplasmic domain (∆CD-PECAM-1). The endothelial barrier function was evaluated by Electric Cell-substrate Impedance Sensing, and molecular mobility was assessed by fluorescence recovery after photobleaching. KEY FINDINGS: We found that ∆CD-PECAM-1 concentrates normally at endothelial cell junctions, but has the unexpected property of conferring increased baseline barrier resistance, as well as a more rapid rate of recovery of vascular integrity following thrombin-induced disruption of the endothelial barrier. Fluorescence recovery after photobleaching analysis revealed that ∆CD-PECAM-1 exhibits increased mobility within the plane of the plasma membrane, thus allowing it to redistribute more rapidly back to endothelial cell-cell borders to reform the vascular permeability barrier. SIGNIFICANCE: The PECAM-1 cytoplasmic domain plays a novel role in regulating the rate and extent of vascular permeability following thrombotic or inflammatory challenge.

Phospholipase Cγ1 is required for pre-TCR signal transduction and pre-T cell development.

Pre-T cell receptor (TCR) signaling is required for pre-T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre-TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate, that are important to mediate PKC activation and intracellular Ca2+ flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR-mediated signaling, development and T-cell activation, but the role of PLCγ1 in pre-TCR signal transduction and pre-T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre-T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre-TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre-T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre-TCR mediated Ca2+ flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre-TCR mediated signal transduction and pre-T cell development.

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.

Multifunctional ion transport properties of human SLC4A11: comparison of the SLC4A11-B and SLC4A11-C variants.

Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH2-terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H+ equivalents in both a Na+-independent and Na+-coupled mode. In both transport modes, SLC4A11-C H+ flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH2-terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH2-terminus functions as an autoactivating domain, enhancing Na+-independent and Na+-coupled H+ flux without significantly affecting the electrogenic NH3-H(n)+ cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11.

The adhesion molecule PECAM-1 enhances the TGF-ß-mediated inhibition of T cell function.

Transforming growth factor-ß (TGF-ß) is an immunosuppressive cytokine that inhibits the proinflammatory functions of T cells, and it is a major factor in abrogating T cell activity against tumors. Canonical TGF-ß signaling results in the activation of Smad proteins, which are transcription factors that regulate target gene expression. We found that the cell surface molecule platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitated noncanonical (Smad-independent) TGF-ß signaling in T cells. Subcutaneously injected tumor cells that are dependent on TGF-ß-mediated suppression of immunity for growth grew more slowly in PECAM-1(-/-) mice than in their wild-type counterparts. T cells isolated from PECAM-1(-/-) mice demonstrated relative insensitivity to the TGF-ß-dependent inhibition of interferon-γ (IFN-γ) production, granzyme B synthesis, and cellular proliferation. Similarly, human T cells lacking PECAM-1 demonstrated decreased sensitivity to TGF-ß in a manner that was partially restored by reexpression of PECAM-1. Co-incubation of T cells with TGF-ß and a T cell-activating antibody resulted in PECAM-1 phosphorylation on an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the recruitment of the inhibitory Src homology 2 (SH2) domain-containing tyrosine phosphatase-2 (SHP-2). Such conditions also induced the colocalization of PECAM-1 with the TGF-ß receptor complex as identified by coimmunoprecipitation, confocal microscopy, and proximity ligation assays. These studies indicate a role for PECAM-1 in enhancing the inhibitory functions of TGF-ß in T cells and suggest that therapeutic targeting of the PECAM-1-TGF-ß inhibitory axis represents a means to overcome TGF-ß-dependent immunosuppression within the tumor microenvironment.

Effect of 22q11.2 deletion on bleeding and transfusion utilization in children with congenital heart disease undergoing cardiac surgery.

BACKGROUND: Postsurgical bleeding causes significant morbidity and mortality in children undergoing surgery for congenital heart defects (CHD). 22q11.2 deletion syndrome (DS) is the second most common genetic risk factor for CHD. The deleted segment of chromosome 22q11.2 encompasses the gene encoding glycoprotein (GP) Ibß, which is required for expression of the GPIb-V-IX complex on the platelet surface, where it functions as the receptor for von Willebrand factor (VWF). Binding of GPIb-V-IX to VWF is important for platelets to initiate hemostasis. It is not known whether hemizygosity for the gene encoding GPIbß increases the risk for bleeding following cardiac surgery for patients with 22q11.2 DS. METHODS: We performed a case-control study of 91 pediatric patients who underwent cardiac surgery with cardiopulmonary bypass from 2004 to 2012 at Children's Hospital of Wisconsin. RESULTS: Patients with 22q11.2 DS had larger platelets and lower platelet counts, bled more excessively, and received more transfusion support with packed red blood cells in the early postoperative period relative to control patients. CONCLUSION: Presurgical genetic testing for 22q11.2 DS may help to identify a subset of pediatric cardiac surgery patients who are at increased risk for excessive bleeding and who may require more transfusion support in the postoperative period.