P-selectin Glycoprotein Research Articles

Abstract A051: PSGL-1-deficiency promotes pancreatic ductal adenocarcinoma tumor control and synergy with immune checkpoint blockade

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer with increasing incidence rates while patient prognosis remains poor with a 5-year survival rate of only 12%. PDAC tumors are refractory to all currently available treatments, including existing immune checkpoint blockade (ICB) therapies, and demonstrate limited T cell infiltration. There is a critical need to develop novel approaches to improve patient outcomes. We previously demonstrated that deficiency of P-selectin glycoprotein 1 (PSGL-1) promotes enhanced T cell responses that clear chronic LCMV Cl13 virus infection and inhibit growth of PD-1 blockade resistant melanoma. Here we evaluated if targeting PSGL-1 promotes T cell responses to PDAC tumors to inhibit growth and metastases. KPC.4662 tumor cells were injected orthotopically into the tail of the pancreas of C57BL/6 (wildtype control) and PSGL-1−/− (C57BL/6 background) male or female mice and tumor growth and immune infiltration was evaluated by histology and flow cytometry. At 28 days post-orthotopic tumor cell injection, KPC.4662 tumor burden was reduced by 50% on average (1.1 g vs 0.45g) in PSGL-1−/− mice compared to controls. Total immune infiltration (CD45+) was increased in tumors from PSGL-1−/- mice (10% vs 24%), with a significant increase in CD3+ T cell infiltration. Moreover, fewer mice developed metastases in the peritoneal cavity, liver, and lungs. To assess if PSGL-1−/− mice had the capability to reduce metastatic burden, KPC 4662 tumor cells were injected intravascularly and tumor development in the lungs was assessed on day 17. PSGL-1−/− mice reproducibly developed fewer tumors in the lungs compared to wildtype control mice as determined by histological examination. To address if enhanced control of PDAC tumors was mediated by T cells, we depleted CD4+ and CD8+ T cells immediately prior to orthotopic implantation of KPC.4662 tumor cells. Depletion of T cells resulted in the loss of PDAC tumor growth control in PSGL-1−/− mice. Given our previous studies demonstrating an intrinsic role for PSGL-1 in the development of T cell exhaustion, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 immune checkpoint blockade, a therapy known to be ineffective as a monotherapy in PDAC patients and mouse models. When PD-1 blocking antibody was therapeutically administered on days 10, 12, and 14 post-tumor implantation, 85% of PSGL-1−/− animals demonstrated complete responses. From these studies, we conclude that in the absence of PSGL-1, we observe significantly reduced growth of primary orthotopic PDAC tumors and decreased metastases. These findings are associated with increased CD45+ immune cell infiltration in the tumors and notably functions in a T cell-dependent manner. PSGL-1 therefore represents a novel target for promoting immune responses to PDAC tumors. Citation Format: Jennifer L. Hope, Yijuan Zhang, Hannah A. Faso, Sreeja Roy, Michelle Lin, Ashley B. Palete, Swetha Maganti, Dennis C. Otero, Cosimo Commisso, Linda M. Bradley. PSGL-1-deficiency promotes pancreatic ductal adenocarcinoma tumor control and synergy with immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A051.

PSGL-1: a novel immune checkpoint driving T-cell dysfunction in obstructive sleep apnea.

Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Recently, the adhesion receptor P-selectin glycoprotein-1 (PSGL 1) has been identified as a novel immune checkpoint, which are recognized major hallmarks in several types of cancer and have revolutionized cancer therapy. The expression of PSGL-1 and its ligands VISTA and SIGLEC-5 was assessed in the leucocytes of OSA patients and control subjects exploring the role of intermittent hypoxia (IH) using in vitro models. In addition, PSGL-1 impact on T-cells function was evaluated by ex vivo models. Data showed PSGL-1 expression is upregulated in the T-lymphocytes from patients with severe OSA, indicating a relevant role of hypoxemia mediated by intermittent hypoxia. Besides, results suggest an inhibitory role of PSGL-1 on T-cell proliferation capacity. Finally, the expression of SIGLEC-5 but not VISTA was increased in monocytes from OSA patients, suggesting a regulatory role of intermittent hypoxia. In conclusion, PSGL-1 might constitute an additional immune checkpoint leading to T-cell dysfunction in OSA patients, contributing to the disruption of immune surveillance, which might provide biological plausibility to the higher incidence and aggressiveness of several tumors in these patients.

PSGL-1 deficiency eradicates orthotopic pancreatic ductal adenocarcinoma tumors in combination with anti-PD-1 therapy and enhances T cell anti-tumor immunity

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly refractory to all current immune checkpoint blockade (ICB) therapies and display limited CD8 +T cell infiltration. We previously showed that deficiency of P-selectin glycoprotein 1 (PSGL-1) promotes CD8 +T cell responses that clear chronic lymphocytic choriomeningitis virus (LCMV) Cl13 virus infection and dramatically inhibits the growth of anti-PD-1 blockade resistant melanoma. Here we evaluated if PSGL-1 regulates CD8 +T cell responses to PDAC tumors. Results: PSGL-1 −/−mice reproducibly showed T cell dependent, significant reduction of KPC.4662 primary PDAC tumors and reduced metastasis to lungs compared to control mice. Furthermore, anti-PD-1 treatment of PSGL-1 −/−mice completely obliterated tumor growth as opposed to control mice. Histology of primary tumors revealed marked increase of immune infiltration (CD45 +cells), including CD3 +T cells in PSGL-1 −/−mice. Flow cytometry and immunofluorescence staining confirmed a significant increase in tumor-infiltrating CD8 +T cells in PDAC tumors from PSGL-1 −/−mice. Importantly, PSGL-1 −/−CD8 +T cells were phenotypically and functionally less exhausted. Conclusion: PSGL-1 deficiency significantly reduced growth of primary orthotopic PDAC tumors and decreased metastases and this effect was enhanced in combination with anti-PD-1 ICB treatment. This protective effect was associated with increased immune cell infiltration in the tumors and notably, increased CD8 +T cell effector responses. PSGL-1 therefore represents a novel target for promoting immune responses to PDAC tumors and responses to PD-1 blockade. NCI Cancer Center Support Grant P30 CA030199

PSGL-1 attenuates early TCR signaling to suppress CD8+ T cell progenitor differentiation and elicit terminal CD8+ T cell exhaustion

PSGL-1 (P-selectin glycoprotein-1) is a Tcell-intrinsic checkpoint regulator of exhaustion with an unknown mechanism of action. Here, we show that PSGL-1 acts upstream of PD-1 and requires co-ligation with the Tcell receptor (TCR) to attenuate activation of mouse and human CD8+ Tcells and drive terminal Tcell exhaustion. PSGL-1 directly restrains TCR signaling via Zap70 and maintains expression of the Zap70 inhibitor Sts-1. PSGL-1 deficiency empowers CD8+ Tcells to respond to low-affinity TCR ligands and inhibit growth of PD-1-blockade-resistant melanoma by enabling tumor-infiltrating Tcells to sustain an elevated metabolic gene signature supportive of increased glycolysis and glucose uptake to promote effector function. This outcome is coupled to an increased abundance of CD8+ Tcell stem cell-like progenitors that maintain effector functions. Additionally, pharmacologic blockade of PSGL-1 curtails Tcell exhaustion, indicating that PSGL-1 represents an immunotherapeutic target for PD-1-blockade-resistant tumors.

Correlation between Platelet to Lymphocyte Ratio with C-Reactive Protein in COVID-19 Patients

Coronavirus 2019 (COVID-19) is an infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Inflammation occurs when the body is infected with the virus. Platelets play a role in immune response and immunomodulation by activating P-Selectin Glycoprotein (PSGL) to the site of inflammation. Lymphocytes play a role through CD4 T-cells, B-cells producing specific viral antibodies, and CD8 cytotoxic T-cells by directly killing the virus in infected cells. This study aimed to prove the correlation between PLR and CRP as inflammation markers in COVID-19 patients. This study was a retrospective observational study with the cross-sectional approach at Dr. Kariadi Hospital, Semarang, for the period March-August 2020. Spearman test performed for analyzing data with p<0.05 was significant. Thirty-three confirmed COVID-19 patients with median value of PLR 218 (103-1609) and CRP 15.94 (1.24-200) mg/L were tested for correlation with a value of p=0.013 and r=0.427. The increase of PLR and CRP in COVID-19 patients was caused by an inflammatory process mediated by the immune response. High values in the blood were associated with disease severity and poor prognosis. There was a statistically significant moderate positive correlation between PLR and CRP in COVID-19 patients.

PSGL-1-directed regulation of calcium signaling modulates PD-1 expression following T cell activation

Abstract We previously identified PSGL-1 (P-selectin glycoprotein-1) as a T cell-intrinsic, cell surface checkpoint inhibitor during chronic viral infection. In this model, we found that cross-linking PSGL-1 with antibody reduced signaling through the T cell receptor (TCR) in exhausted T cells and further upregulated PD-1 expression. However, the mechanism responsible for PD-1 regulation by PSGL-1 remained to be described. Here we show that, in naïve T cells, PSGL-1 ligation results in enhanced PD-1 expression following activation through the T cell receptor. Furthermore, naïve T cells lacking PSGL-1 fail to upregulate PD-1 to the same extent as wild type T cells following activation. Strikingly we have discovered that T cells lacking PSGL-1 showed reduced calcium signaling. In T cells, the calcium/calcineurin/NFAT pathway is important for induction of many genes including PD-1. Consequently, PSGL-1 deficient T cells also exhibit reduced nuclear translocation of NFAT explaining the reduced PD-1 expression. Conversely, we found that ligating PSGL-1 enhanced NFAT nuclear translocation with TCR signaling thus contributing to upregulation of PD-1. Therefore, by regulating calcium signaling through the T cell receptor, PSGL-1 modulates the extent of PD-1 expression early in activation and subsequently influencing the extent of T cell exhaustion. Further studies aim to elucidate how PSGL-1 regulates calcium signaling through the T cell receptor.

Preferential Homing of Tumor-specific and Functional CD8+ Stem Cell-like Memory T Cells to the Bone Marrow.

The bone marrow (BM) harbors not only hematopoietic stem cells but also conventional memory T and B cells. Studies of BM-resident memory T cells have revealed the complex relationship between BM and immunologic memory. In the present study, we identified CD122 stem cells antigen-1 (Sca-1), B-cell lymphoma protein-2 (Bcl-2), CD8 stem cell-like memory T cells (TSCMs) as a distinct memory T-cell subset preferentially residing in the BM, where these cells respond vigorously to blood-borne antigens. We found that the most TSCMs favorably relocate to the BM by adhesion molecules such as vascular cell adhesion protein 1, P-selectin glycoprotein 1, and P-selectin or E-selectin. Moreover, the BM-resident TSCMs exhibited much higher levels of antitumor activity than the spleen-resident TSCMs. These results indicate that the BM provides an appropriate microenvironment for the survival of CD8 TSCMs, thereby broadening our knowledge of the memory maintenance of antigen-specific CD8 T lymphocytes. The present findings are expected to be instructive for the development of tumor immunotherapy.

Dynamic interaction of PSGL-1 with the T cell receptor regulates the extent of T cell activation

Abstract We previously identified PSGL-1 (P-selectin glycoprotein-1) as a T cell-intrinsic, cell surface checkpoint inhibitor during chronic viral infection. In this model, we found that cross-linking PSGL-1 with antibody reduced signaling through the T cell receptor (TCR) in exhausted T cells and further upregulated PD-1 expression. However, the function of PSGL-1 in the early activation of naïve T cells has not been fully investigated. We found that, crosslinking PSGL-1 on naïve T cells augments TCR signaling, including enhanced Erk and Akt phosphorylation and Nur77 expression. In addition, T cells expressed more activation markers, including PD-1, proliferated to a greater extent, and secreted more cytokines when PSGL-1 was crosslinked compared to TCR stimulation alone, indicating a role as a costimulatory molecule. Strikingly, we have discovered that PSGL-1 co-localizes with the TCR upon stimulation. CD3 and PSGL-1 can be found in the same micro vesicles following anti-CD3 crosslinking, indicating a close connection between PSGL-1 and the TCR. However, the effects of PSGL-1 ligation are similar to what is seen in T cells that lack PSGL-1 expression, except with respect to PD-1 expression. We propose that decreased induction of PD-1 with PSGL-1 deficiency supports greater effector T cell responses. Further studies will address the function of PSGL-1 in cytoskeletal rearrangement and the formation of the immune synapse. Altogether, these data indicate that the extent of T cell activation is regulated by an association of PSGL-1 with the TCR.

Numerical Investigation of Leukocyte Rolling, Adhesion and Bond Formation on Surface Coated with Varying P-Selectin Density

Leukocyte attraction towards the site of inflammation, is modulated by selectins expressed on activated endothelial cells. Scientists have studied the role of P-selectin in the progression of systemic disease in mice; the experimental results have shown that, deficiency of P-selectin results in failure to respond to an inflammation. P-selectin molecules have been shown to be a key contributor of leukocytes recruitment to sites of inflammation. To date, there are limited theoretical investigations of the effects of changes in P-selectin density on leukocytes rolling, tethering and adhesion. To address this, we have employed a 3D numerical model of an elastic capsule coated with P-selectin glycoprotein-1 (PSGL-1) molecules (representative of a leukocyte) to simulate its motion on a surface decorated with P-selectins under linear shear flow (Jadhav et. al, 2005). We utilized an immersed boundary method to solve the Stokes equation governing the motion of an elastic capsule near a plane wall. The capsule membrane is modeled using Mooney-Rivlin constitutive equation. The stochastic nature of the bond formation and breakage between P-selectin receptors and PSGL-1 ligands is simulated using Monte Carlo method, and the binding kinetics from the Bell model (Bell 1978). We have examined the effects of changes in P-selectin density (NR) on the leukocyte rolling velocity, average force per bond, leukocyte footprints on the substrate by varying NR from 30-150 μm−2 on the substrate. The results indicate that the average rolling velocity of a leukocyte decreased from 2.6 to 2.1 μm/s and the average force per bond decreased from 11.3 to 2.35 pN with increase in NR values. Correspondingly elevated stresses are shown by the leukocyte footprint at lower NR values. We are currently simulating PMN rolling on substrates with P-selectin patterns.

The role of platelets in inflammation and immunity

In recent decades, significant progress has been made in understanding the mechanisms of platelet function and platelet hemostasis correction. Platelets are considered as the most important participants of both normal, and pathological thrombotic process characteristic of the most different diseases and states. In the present review pathophysiological mechanisms of platelet synthesis of various mediators with paracrine effects, which can influence the function of other cells, are consecrated. The physiology of platelets was considered in detail. The leading role of platelets in pathogenesis of the majority of diseases of cardiovascular system as modulators of inflammatory reactions of the immune response which are considered as the leading mechanism of development of atherosclerosis was shown. The ability of platelets to encode inflammatory proteins allowing them to influence adaptive immunity functions was discussed. The role of platelets as a key component of the innate immune system was presented, which is confirmed by the presence of Tolllike receptors (TLR) and glycoproteins, such as integrin αIIbβ3, glycoprotein Ib-IX and FcγRIIa, involved in interaction with bacterial cells. The pathogenesis of the formation of platelet-leukocyte aggregates due to the rapid reversible interaction of P-selectin (CD62P) on the platelet surface with ligand-1 glycoprotein P-selectin (PSGL-1) on the plasma of leukocytes and the mechanism of extracellular neutrophil traps (NETs), as well as the influence of platelets on the function of lymphocytes was presented. The role of platelets in cancer progression, metastasis and thrombosis is considered, and the interrelation of thrombosis and metastasis in malignant diseases was analyzed. The efficiency of the use of antithrombotic drugs in the prevention of thrombosis and, as a consequence, in the prevention of cardiovascular diseases and cancer was discussed.

Elevated plasma levels of P‐selectin glycoprotein ligand‐1‐positive microvesicles in patients with unprovoked venous thromboembolism

Background Microvesicles (MVs) express antigens from their parental cells and have a highly procoagulant surface. Animal studies suggest that P-selectin glycoprotein ligand-1-positive (PSGL-1+ ) MVs play a role in the pathogenesis of venous thromboembolism (VTE). Objective The aim of this study was to determine plasma levels, the cellular origin and the morphological characteristics of PSGL-1+ MVs in patients with unprovoked VTE. Methods We conducted a population-based case-control study in 20 patients with a history of unprovoked VTE and 20 age- and sex-matched healthy controls recruited from the general population. Plasma levels, the cellular origin and the morphological characteristics of PSGL-1+ MVs were evaluated using flow cytometry, electron microscopy and confocal microscopy. Results Plasma levels of PSGL-1+ MVs were associated with increased risk of VTE. The odds ratio per one standard deviation increase in PSGL-1+ MVs was 3.11 (95% confidence interval [CI], 1.41-6.88) after adjustment for age and sex, and 2.88 (95% CI, 1.29-6.41) after further adjustment for body mass index. The PSGL-1+ MVs originated mainly from monocytes and endothelial cells determined by double staining with markers of parental cells using flow cytometry and transmission electron microscopy. Scanning electron microscopy of PSGL-1-labeled plasma-derived MVs displayed dominantly spherical vesicles that varied between 50 and 300 nm in diameter. Conclusions Increased plasma levels of PSGL-1+ MVs are associated with the risk of unprovoked VTE. Large population-based prospective studies are required to validate our findings.

PSGL-1 regulates the extent of TCR-dependent activation in T cells and prevents their suppression by Tregs

Abstract We recently demonstrated that PSGL-1 (P-selectin glycoprotein-1) could act as a T cell-intrinsic checkpoint inhibitor during chronic viral infection. As such, PSGL-1 may be a viable target to control T cell responses in the setting of autoimmune disease. In this study, we investigated the role of PSGL-1 on the activation of T cells and in mouse models of autoimmune disease. Here we find that the lack of PSGL-1 on T cells results in clinically worse disease in both EAE and adoptive transfer colitis, which supports its function as a negative regulator of T cell activation. However, in vivo treatment with an anti-PSGL-1 antibody known to block binding of the ligand, P-selectin, from the time of disease initiation had little effect on disease severity in the transfer colitis model and led to exacerbated disease in the EAE model. Crosslinking PSGL-1 on naïve T cells in vitro in the presence of TCR signals results in up-regulation of PD-1 but also up-regulation of other activation markers, suggesting that PSGL-1 exerts regulatory effects during the early activation program in T cells. Surprisingly, however, in an in vitro assay, crosslinking PSGL-1 on effector T cells resulted in resistance to regulatory T cell suppression. Therefore, anti-PSGL-1 treatment results in enhanced T cell activation and resistance to Treg suppression, thereby leading to more severe autoimmune disease by altering/blocking PSGL-1 signaling to phenocopy PSGL-1-deficiency.

Better platelet function, less fibrinolysis and less hemolysis in re-transfused residual pump blood with the Ringer’s chase technique – a randomized pilot study

Residual pump blood from the cardiopulmonary bypass (CPB) circuit is often collected into an infusion bag (IB) and re-transfused. An alternative is to chase the residual blood into the circulation through the arterial cannula with Ringer's acetate. Our aim was to assess possible differences in hemostatic blood quality between these two techniques. Forty adult patients undergoing elective coronary artery bypass graft surgery with CPB were randomized to receive the residual pump blood by either an IB or through the Ringer's chase (RC) technique. Platelet activation and function (impedance aggregometry), coagulation and hemolysis variables were assessed in the re-transfused blood and in the patients before, during and after surgery. Results are presented as median (25-75 quartiles). Total hemoglobin and platelet levels in the re-transfused blood were comparable with the two methods, as were soluble platelet activation markers P-selectin and soluble glycoprotein VI (GPVI). Platelet aggregation (U) in the IB blood was significantly lower compared to the RC blood, with the agonists adenosine diphosphate (ADP) 24 (10-32) vs 46 (33-65), p<0.01, thrombin receptor activating peptide (TRAP) 50 (29-73) vs 69 (51-92), p=0.04 and collagen 24 (17-28) vs 34 (26-59), p<0.01. The IB blood had higher amounts of free hemoglobin (mg/L) (1086 (891-1717) vs 591(517-646), p<0.01) and D-dimer 0.60 (0.33-0.98) vs 0.3 (0.3-0.48), p<0.01. Other coagulation variables showed no difference between the groups. The handling of blood after CPB increases hemolysis, impairs platelet function and activates coagulation and fibrinolysis. The RC technique preserved the blood better than the commonly used IB technique.

The association between P selectin glycoprotein ligand 1 gene variable number of tandem repeats polymorphism and risk of thrombosis in Behçet's disease.

Behçet's disease (BD) has been recognized as an unclassified type of vasculitis with an accompanying tendency to thrombosis. No disease-specific pathology has been demonstrated so far to explain the prothrombotic state, and this predisposition is considered to be associated with endothelial activation/dysfunction. P-selectin glycoprotein ligand-1 (PSGL-1) variable number of tandem repeat (VNTR) polymorphism has an impact on the protein length, and heterozygosity affect of the PSGL-1 to P-selectin interaction, which has been found to be associated with an increased risk of thrombosis in patients with antiphospholipid syndrome. We aimed to analyze the association of PSGL-1 gene polymorphism, in a group of BD patients with and without thrombosis. The study group consisted of 136 BD patients (112 male, 24 female) with thrombosis, 120 BD patients without thrombosis (54 male, 66 female) during at least 5years disease course, and 190 healthy controls (103 male, 87 female) All patients fulfilled the International Study Group criteria for classification of BD. Genotyping for the PSGL-1 gene exon 2 VNTR polymorphism was carried out with the amplification of genomic DNA and running of the polymerase chain reaction product on agarose gel electrophoresis. The frequency of heterozygous genotypes (AB+AC+BC) was greater in BD patients with thrombosis compared to BD patients without thrombosis (33.1% vs. 20.8%, P=0.028, odds ratio=1.85). However, the increased frequency of heterozygous genotypes in BD patients with thrombosis did not reach a statistically significant level compared to healthy controls (33.1% vs. 32.6%). PSGL-1 VNTR polymorphism may have limited contribution to the thrombotic tendency in patients with BD.

Age-Related Changes in the Interaction of Acute Myeloid Leukemia with the Bone Marrow Microenvironment Correlate with Response to Treatment and Survival: A Role for L-Selectin

Introduction: Acute myeloid leukemia (AML) arises in the bone marrow microenvironment (BME), where cell-cell and cell-matrix adhesion promote AML survival. There is increased incidence of AML in older adults and age-related changes in the BME such as the senescence associated secretory phenotype (SASP) may promote myeloid disorders and cancer. Our objective is to further elucidate the influence of the BME on AML pathobiology.Methods: Using flow cytometry, we prospectively characterized 17 cell adhesion receptors expressed on AML blasts derived from 106 patient blood and bone marrow samples on an IRB approved protocol. For 55 patients, we correlated expression of these individual adhesion receptors with age, cytogenetics, complete remission (CR), relapse free survival (RFS) and overall survival (OS). We correlated expression of adhesion receptors with basal levels of DNA damage in AML blasts, as measured by phosphorylation of histone H2AX (γ-H2AX) by flow cytometry. mRNA microarray gene expression profiling (GEP) on 30 patients, we examined for age-associated differences in the gene expression of cell-cell and cell-matrix adhesion proteins. For 6 patients, we evaluated for changes in GEP that occur in AML blasts with adhesion to plates coated with alternative spliced fibronectin peptide CH296, RetronectinTM (RN) vs. bovine serum albumin (BSA) control.Results: We found a significant positive correlation between L-selectin cell surface expressed protein levels with advancing age (Spearman r = 0.35, p = 0.009) (Figure 1). The response to treatment (CR/CRi) rate, but not RFS, increased with greater surface expression of the following adhesion proteins: CD11a (αL), CD29 (β1), CD44, CD49a (α1) and CD49e (α5). OS improved with greater cell surface expressed protein levels of CD11a (αL) and CD29 (β1).There was a significant negative correlation in L-selectin mRNA levels with basal DNA damage in AML blasts, as measured by γ-H2AX (Spearman r = -0.6, p = 0.005). To further investigate the relationship between L-selection expression and DNA damage in AML, we cultured AML blasts on an L-selectin ligand, P-selectin glycoprotein-1 (PSGL-1) vs. BSA control. We observed increased blast survival and reduced apoptosis when adherent on PSGL-1 compared to BSA after treatment with cytarabine arabinoside (Ara-C) in 4/15 (27%) of samples tested (Figure 2).Ingenuity Pathway Analysis (IPA) results on the gene expression data showed age-associated changes in the expression of L-selectin and cell-matrix proteins involved in the SASP, such as tissue inhibitor of metalloproteinases (TIMP). Adhesion of AML blasts to RN vs. BSA control resulted in up-regulation of genes involved in protein ubiquitination and the PI3K/AKT, nuclear factor-kappa beta (NF-kβ) and IL-8 signaling pathways.Conclusions: Age-related changes in the expression of L-selectin, cell-cell and cell-matrix proteins in AML blasts appear to influence DNA damage signaling/response (DDR), promote Ara-C resistance, and influence patient response to treatment and survival. Adhesion of AML blasts to the BME results in up-regulation of cell survival pathways that confer resistance to apoptosis and maintenance of the SASP. Our results support disruption of L-selectin and other BME-mediated interactions as potential targets to enhance cytotoxicity to chemotherapy and improve age-specific outcomes in AML. [Display omitted] [Display omitted] DisclosuresBecker:Invivoscribe: Honoraria; CVS Caremark: Other: Accordant Health Services Medical Advisory Board; Millennium: Research Funding; Amgen: Research Funding; Abbvie: Research Funding; Glycomimetics: Research Funding; Pfizer: Other: Scientific Steering Committee for a post marketing study; JW Pharmaceutical: Research Funding.

Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes.

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell–cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1 also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1, which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development.

Determinants of Platelet-Leukocyte Aggregation and Platelet Activation in Stroke

Background: Platelet-leukocyte aggregation (PLA) and platelet activation are found to be on the higher side in ischemic stroke patients. The correlation of PLA with clinical features has not been intensively investigated and the influence of genetic factors on PLA is still unexplored. The interaction of platelets with leukocytes is mainly determined by the proteins encoded by six genes: P-Selectin (SELP encodes CD62P) on the thrombocyte binding to P-Selectin-Glycoprotein-Ligand-1 (PSGL1) on the leukocyte, intracellular-adhesion-molecule 2 (ICAM2) interacting with Integrin alpha M (ITGAM) and Glycoprotein 1b-alpha (GP1BA) binding to Integrin alpha L (ITGAL). Methods: Seventy-nine patients with acute ischemic stroke and 151 controls without vascular disease from a single German center were enrolled. A neurologist and a neuroradiologist ascertained clinical and radiological features. PLA and platelet activation were analyzed using flow cytometry with various antibodies. Coding as well as tagging SNPs in six genes determining PLA were genotyped. Three groups of parameters were correlated with each other: (i) clinical and radiological parameters, (ii) laboratory parameters, (iii) genetic parameters. For the comparisons, robust nonparametric statistical tests were applicable. Results: PLA and platelet activation were higher in ischemic stroke patients compared to controls. Both, anticoagulant and antiplatelet treatment in the patient group affected platelet activation but not PLA. PLA correlated weakly with measures of stroke severity but not with thrombus length or stroke etiology. The association of SNP rs2228315 in the P-Selectin Glycoprotein Ligand-1-gene (PSGL1) with ischemic stroke and platelet activation was significant before correction for multiple testing while a trend was observed for the association with PLA. Regression analysis revealed that (i) platelet activation was an independent determinant of stroke, (ii) that PLA correlated with stroke, sex, age and platelet activation and (iii) that platelet activation correlated only with stroke. None of the SNPs survived in the regression analysis for stroke, PLA or platelet activation as dependent variables. Conclusions: The most important result of our study is that PLA and platelet activation are independent of other vascular risk factors correlated with stroke in our sample. In addition, we identified the missense SNP rs2228315 in the PSGL1-gene as a candidate polymorphism for ischemic stroke-related PLA. Association between this SNP and stroke as well as coronary artery disease has also been shown by two other studies.

A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase.

Rapid β2-integrin activation is indispensable for leukocyte adhesion and recruitment to sites of infection and is mediated by chemokine- or P-selectin glycoprotein ligand-1-induced inside-out signaling. Here we uncovered a novel pathway for rapid activation of integrin-dependent leukocyte adhesion, triggered by toll-like receptor (TLR)-mediated signaling. TLR2 or TLR5 ligation rapidly activated integrin-dependent leukocyte adhesion to immobilized ICAM-1 and fibronectin. Consistently, in vivo administration of the TLR2-ligand Pam3CSK4 increased integrin-dependent slow rolling and adhesion to endothelium within minutes, as identified by intravital microscopy in the cremaster model. TLR2 and TLR5 ligation increased β2-integrin affinity, as assessed by the detection of activation-dependent neoepitopes. TLR2- and TLR5-triggered integrin activation in leukocytes required enhanced Rap1 GTPase activity, which was mediated by Rac1 activation and NADPH oxidase-2-dependent reactive oxygen species production. This novel direct pathway linking initial pathogen recognition by TLRs to rapid β2-integrin activation may critically regulate acute leukocyte infiltration to sites of pathogen invasion.

Platelet degranulation and glycoprotein IIbIIIa opening are not related to bleeding phenotype in severe haemophilia A patients

Recently we reported data suggesting that platelets could compensate for the bleeding phenotype in severe haemophilia A (HA). The aim of this study was to confirm these results in a larger population with a detailed characterisation of clinical phenotype. Patients with diagnostic severe HA (FVIII:C <1%) were scored for clinical phenotype by integrating data on age at first joint bleed, joint damage, bleeding frequency and FVIII consumption. Phenotype was defined as onset of joint bleeding-score + arthropathy-score + joint bleeding-score + (2* treatment intensity-score). After a washout period of three days, blood was collected for measurement of basal level of platelet activation, platelet reactivity, endothelial cell activation and presence of procoagulant phospholipids in plasma. Thirty-three patients with severe HA were included, 13 patients with a mild, 12 patients with an average and eight patients with a severe clinical phenotype. No relevant differences in basal level of platelet activation, platelet reactivity, endothelial cell activation and procoagulant phospholipids between all three groups were observed. The mean annual FVIII consumption per kg did not correlate with the platelet P-selectin expression and glycoprotein (GP)IIbIIIa activation on platelets. In conclusion, variability in clinical phenotype in patients with diagnostic severe HA is not related to platelet activation or reactivity, measured as platelet degranulation and platelet GPIIbIIIa opening.

Coordinated and unique functions of the E-selectin ligand ESL-1 during inflammatory and hematopoietic recruitment in mice

AbstractBeyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils.