Anna Babinska

Signaling Pathways of the F11 Receptor (F11R; a.k.a. JAM-1, JAM-A) in Human Platelets: F11R Dimerization, Phosphorylation and Complex Formation with the Integrin GPIIIa

The F11 receptor (F11R) (a.k.a. Junctional Adhesion Molecule, JAM) was first identified in human platelets as a 32/35 kDa protein duplex that serves as receptor for a functional monoclonal antibody that activates platelets. We have sequenced and cloned the F11R and determined that it is a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. The signaling pathways involved in F11R-induced platelet activation were examined in this investigation. The binding of M.Ab.F11 to the platelet F11R resulted in granule secretion and aggregation. These processes were found to be dependent on the crosslinking of F11R with the FcγRII by M.Ab.F11. This crosslinking induced actin filament assembly with the conversion of discoidal platelets to activated shapes, leading to the formation of platelet aggregates. We demonstrate that platelet secretion and aggregation through the F11R involves actin filament assembly that is dependent on phosphoinositide-3 kinase activation, and inhibitable by wortmannin. Furthermore, such activation results in an increase in the level of free intracellular calcium, phosphorylation of the 32 and 35 kDa forms of the F11R, F11R dimerization coincident with a decrease in monomeric F11R, and association of the F11R with the integrin GPIIIa and with CD9. On the other hand, F11R-mediated events resulting from the binding of platelets to an immobilized surface of M.Ab.F11 lead to platelet adhesion and spreading through the development of filopodia and lammelipodia. These adhesive processes are induced directly by interaction of M.Ab.F11 with the platelet F11R and are not dependent on the FcγRII. We also report here that the stimulation of the F11R in the presence of nonaggregating (subthreshold) concentrations of the physiological agonists thrombin and collagen, results in supersensitivity of platelets to natural agonists by a F11R-mediated process independent of the FcγRII. The delineation of the two separate F11R-mediated pathways is anticipated to reveal significant information on the role of this cell adhesion molecule in platelet adhesion, aggregation and secretion, and F11R-dependent potentiation of agonist-induced platelet aggregation. The participation of F11R in the formation and growth of platelet aggregates and plaques in cardiovascular disorders, resulting in enhanced platelet adhesiveness and hyperaggregability, may serve in the generation of novel therapies in the treatment of inflammatory thrombosis, heart attack and stroke, and other cardiovascular disorders.

The F11 receptor (F11R/JAM-A) in atherothrombosis: Overexpression of F11R in atherosclerotic plaques

F11R is the gene name for an adhesion protein, called the F11-receptor, aka JAM-A, which under normal physiological conditions is expressed constitutively on the surface of platelets and localized within tight junctions of endothelial cells (EC). Previous studies of the interactions between human platelets and EC suggested that F11R/JAM-A plays a crucial role in inflammatory thrombosis and atherosclerosis. The study reported here obtained in-vivo confirmation of this conclusion by investigating F11R/JAM-A protein and mRNA in patients with aortic and peripheral vascular disease and in an animal model of atherosclerosis. Molecular and immunofluorescence determinations revealed very high levels of F11R/JAM-A mRNA and F11R/JAM-A protein in atherosclerotic plaques of cardiovascular patients. Similar results were obtained with 12-week-old atherosclerosis-prone apoE-/- mice, an age in which atherosclerotic plaques are well established. Enhanced expression of the F11R/JAM-A message in cultured EC from human aortic and venous vessels was observed following exposure of the cells to cytokines. Determinations of platelet adhesion to cultured EC inflamed by combined cytokine treatment in the presence of F11R/JAM-A - antagonists provided data indicating that de novo expression of F11R/JAM-A on the luminal surface of inflamed EC has an important role in the conversion of EC to a thrombogenic surface. Further studies of these interactions under flow conditions and under in-vivo settings could provide a final proof of a causal role for F11R/JAM-A in the initiation of thrombosis. Based on our in-vitro and in-vivo studies to date, we propose that therapeutic drugs which antagonize the function of F11R/JAM-A should be tested as novel means for the prevention and treatment of atherosclerosis, heart attacks and stroke.

Elevated Plasma Level of Soluble F11 Receptor/Junctional Adhesion Molecule-A (F11R/JAM-A) in Hypertension

BACKGROUND The F11 receptor (F11R, also known as junctional adhesion molecule A (JAM-A)) plays a role in the development of hypertension in rat. Genetic variants in the human F11R gene were demonstrated to influence systolic blood pressure. In the present study, we investigated the relationship between F11R and hypertension by examining the levels of a circulating soluble form of F11R (sF11R) in hypertensive patients. METHODS Plasma sF11R was measured by enzyme-linked immunosorbent assay in 152 hypertensive and 166 normotensive subjects in whom seven tagging single-nucleotide polymorphisms (SNPs) in the F11R gene had been genotyped. RESULTS Plasma sF11R levels were significantly higher in hypertensive subjects than in normotensive subjects (median (interquartile) range): 162.8 (85.5-293.2) vs. 116.5 (74.1-194.8) pg/ml, P = 0.004), which remained significantly higher after adjusting for age, sex, body mass index (BMI), and homeostasis model assessment of insulin resistance (HOMA-IR) (P = 0.028). In stepwise multiple logistic regression, sF11R level (log-transformed) (P = 0.040), triglycerides (log-transformed) (P = 0.024), and HOMA-IR (log-transformed) (P < 0.001) were independently associated with hypertension. Plasma sF11R level correlated with systolic and diastolic blood pressures (r = 0.15, P < 0.001, and r = 0.13, P = 0.024, respectively). In stepwise multiple linear regression, hypertension (P = 0.013) and fibrinogen levels (P = 0.027) were significant independent predictors of sF11R level. A seven-locus haplotype, present in 2.1% of the subjects, was associated with higher sF11R level (P = 0.024). CONCLUSIONS These results further support a role of F11 receptor in the pathophysiology of human hypertension.

Relationship between the soluble F11 receptor and markers of inflammation in hemodialysis patients.

Background The human F11 receptor (F11R) is an important cell adhesion molecule implicated in inflammatory thrombosis. We hypothesize that serum levels of the soluble released form of F11R (sF11R) are elevated in dialysis patients since these patients have higher cardiovascular disease burdens than the general population. In this study, we examined whether sF11R levels were elevated in hemodialysis (HD) patients and correlated with known inflammatory cytokines. Methods We used new and standard enzyme-linked immunosorbent assay techniques to measure levels of sF11R, as well as high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10), in a cross section of 52 HD patients and compared these with 15 healthy controls. Results The mean age of the patients was 56 ± 17.3 years; 60% were female, and 36% had diabetes mellitus. Serum levels of sF11R, hs-CRP, TNF-α, IL-6, and IL-10 were all significantly higher in patients than in control sera (p < .05). Within the patient group, there was a significant positive correlation between sF11R and TNF-α (r = .41, p = .003), IL-10 (r = .32, p = .023), and IL-6 (r = .32, p = .023), whereas hs-CRP showed no significant correlation (r = −.27, p = .052). Conclusion We conclude that the sF11R level is elevated in HD patients and correlates with known markers of cardiovascular disease. sF11R may be a novel cardiovascular risk marker, and longitudinal studies are needed to better assess its relationship with cardiovascular disease morbidity and mortality in this population.

Transcription and translation of human F11R gene are required for an initial step of atherogenesis induced by inflammatory cytokines

Background -The F11 Receptor (F11R; aka JAM-A, JAM-1) is a cell adhesion protein present constitutively on the membrane surface of circulating platelets and within tight junctions of endothelial cells (ECs). Previous reports demonstrated that exposure of ECs to pro-inflammatory cytokines causes insertion of F11R molecules into the luminal surface of ECs, ensuing with homologous interactions between F11R molecules of platelets and ECs, and a resultant adhesion of platelets to the inflamed ECs. The main new finding of the present report is that the first step in this chain of events is the de-novo transcription and translation of F11R molecules, induced in ECs by exposure to inflammatory cytokines.Methods -The experimental approach utilized isolated, washed human platelet suspensions and cultured human venous endothelial cells (HUVEC) and human arterial endothelial cells (HAEC) exposed to the proinflammatory cytokines TNF-alpha and/or IFN-gamma, for examination of the ability of human platelets to adhere to the inflamed ECs thru the F11R. Our strategy was based on testing the effects of the following inhibitors on this activity: general mRNA synthesis inhibitors, inhibitors of the NF-kappaB and JAK/STAT pathways, and small interfering F11R-mRNA (siRNAs) to specifically silence the F11R gene.Results -Treatment of inflamed ECs with the inhibitors actinomycin, parthenolide or with AG-480 resulted in complete blockade of F11R- mRNA expression, indicating the involvement of NF-kappaB and JAK/STAT pathways in this induction. Transfection of ECs with F11R siRNAs caused complete inhibition of the cytokine-induced upregulation of F11R mRNA and inhibition of detection of the newly- translated F11R molecules in cytokine-inflamed ECs. The functional consequence of the inhibition of F11R transcription and translation was the significant blockade of the adhesion of human platelets to inflamed ECs.Conclusion -These results prove that de novo synthesis of F11R in ECs is required for the adhesion of platelets to inflamed ECs. Because platelet adhesion to an inflamed endothelium is crucial for plaque formation in non-denuded blood vessels, we conclude that the de-novo translation of F11R is a crucial early step in the initiation of atherogenesis, leading to atherosclerosis, heart attacks and stroke.

Expression of a recombinant protein of the platelet F11 receptor (F11R) (JAM-1/JAM-A) in insect cells: F11R is naturally phosphorylated in the extracellular domain

The F11 receptor (F11R/JAM) is a member of the immunoglobulin superfamily localized on the membrane surface of human platelets and a component of tight junctions of endothelial and epithelial cells. F11R was demonstrated to participate in the adhesion of human platelets to cytokine-inflamed endothelial cells (EC), indicating an important role for F11R in inflammatory thrombosis and atherosclerosis. Domains responsible for the formation of tight junctions, the adhesion of platelets to EC, activation of platelets resulting in granule release, the activation of αIIb/β3 integrin and platelet aggregation, were identified in the external portion of F11R. To further examine critical sites of F11R, we utilized the baculovirus system to generate the F11R recombinant protein with the sequence of the extracellular domain, in two types of insect cells, Sf9 and H5. The F11R recombinant protein was detected in the cytoplasm of both infected Sf9 and H5 insect cells, but only infected H5 cells secreted a soluble F11R protein. The purified recombinant F11R proteins, obtained from both types of insect cells, were recognizeable by a conformation-dependent monoclonal antibody, M.Ab.F11, directed against domains within the N-terminus and the first Ig-like fold of F11R. Assessment of the phosphorylation state in the recombinant F11R protein revealed phosphorylation of serine, threonine and tyrosine amino acid residues within the external domain. Real-time biomolecular interaction analysis, performed to assess kinetic constants associated with the binding of active molecules to the purified recombinant F11R protein revealed high affinity binding of the phosphorylated recombinant protein by M.Ab.F11 with Ka of 5.47 × 106 and Kd of 1.83 × 10−7, comparable to values measured with intact human platelets. The findings reported here provide new information on specific domains of F11R that can lead to the generation of therapeutic agents expected to be useful in the treatment of cardiovascular diseases.

Colon Necrosis Due to Sodium Polystyrene Sulfonate with and without Sorbitol: An Experimental Study in Rats.

Introduction Based on a single rat study by Lillemoe et al, the consensus has been formed to implicate sorbitol rather than sodium polystyrene sulfonate (SPS) as the culprit for colon necrosis in humans treated with SPS and sorbitol. We tested the hypothesis that colon necrosis by sorbitol in the experiment was due to the high osmolality and volume of sorbitol rather than its chemical nature. Methods 26 rats underwent 5/6 nephrectomy. They were divided into 6 groups and given enema solutions under anesthesia (normal saline, 33% sorbitol, 33% mannitol, SPS in 33% sorbitol, SPS in normal saline, and SPS in distilled water). They were sacrificed after 48 hours of enema administration or earlier if they were very sick. The gross appearance of the colon was visually inspected, and then sliced colon tissues were examined under light microscopy. Results 1 rat from the sorbitol and 1 from the mannitol group had foci of ischemic colonic changes. The rats receiving SPS enema, in sorbitol, normal saline, distilled water, had crystal deposition with colonic necrosis and mucosal erosion. All the rats not given SPS survived until sacrificed at 48 h whereas 11 of 13 rats that received SPS in sorbitol, normal saline or distilled water died or were clearly dying and sacrificed sooner. There was no difference between sorbitol and mannitol when given without SPS. Conclusions In a surgical uremic rat model, SPS enema given alone or with sorbitol or mannitol seemed to cause colon necrosis and high mortality rate, whereas 33% sorbitol without SPS did not.

Isothermal titration calorimetry and inhibition of platelets aggregation by [D-Phe/(transcinnamoyl)-Pro-D-Arg-P1'-CONH2] peptides inhibitors of thrombin.

Introduction Thrombin is the major product of the plasma coagulation “cascade” of sequential “zymogen-to-protease” steps. As the result of a series of proteolytic cleavages, thrombin converts fibrinogen into fibrin, which deposits at the site of bleeding or thrombosis as the fibrinous portion of a haemostatic plug or thrombotic mass(1). Thrombin also stimulates the platelets through its protein G-coupled receptors PARs (protease activated receptors) (PAR1,4 in humans) being their most potent activator. Thrombin induced-platelets activation plays a critical role in the pathophysiology of thrombosis (1). Activated platelets bind to fibrinogen, causing platelets to aggregate at the site of a cardiovascular injury to form a thrombus that is further stabilized by thrombin-generated fibrin network (1). The discovery of new antithrombotic drugs was primarily focused on finding competitive inhibitors of thrombin, however new approaches target thrombin receptor PAR-1 (such as the PAR-1 structural antagonists) offering additional cardiovascular utility in the treatment of atherosclerosis and restenosis (1). We previously reported the discovery of new tetrapeptides with the sequence space D-Phe-Pro-D-Arg-P1’-CONH2 as reversible competitive inhibitors of thrombin (2) (were P1’ requires small, polar natural or unnatural aminoacids). To further evaluate their potential as antithrombotic drugs and as potential PAR-1 antagonists we synthesized new peptides based on the same sequence space but with D-configuration in P1’. The actual paper presents the structure-activity relationship (SAR) for some L/D tetrapeptides isomers and their evaluation as thrombin inhibitors using biophysical/biochemical approaches (isothermal titration calorimetry and kinetics of thrombin inhibition) and cell-based assays (thrombin mediated platelets aggregation). The synthesis of the D-isomers was performed only for the lead L-peptides (Ki (inhibitory constant) less then 20 μM) (2). Additional tetrapeptides were synthesized with the transcinnamoyl group at P3 position (instead of D-Phe) based on their prediction as high affinity competitive binders of thrombin (2).

Structure-Based Design, Synthesis and Evaluation of Novel Peptidic Inhibitors of Thrombin-Induced Activation of Platelets Aggregation

Janet Gonzalez, Anna Babinska, Ebenezer L.V. Ewul, Edem Timpo, Alhassan Jallow, Zhiyong Qiu, Radoslaw Bednarek, Maria Swiatkowska, Moro O. Salifu, Manfred Philipp, and Cristina C. Clement Department of Natural Sciences, LaGuardia Community College, New York, NY, 11104, USA; Division of Nephrology, Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, NY, 11203, USA; Department of Chemistry, Lehman College of the City University of New York, Bronx, NY, 10468, USA; Medical University of Lodz Department of Cytobiology and Proteomics, Lodz, Poland

Development of new antiatherosclerotic and antithrombotic drugs utilizing F11 receptor (F11R/JAM-A) peptides.

Peptides with enhanced resistance to proteolysis, based on the amino acid sequence of the F11 receptor molecule (F11R, aka JAM‐A/Junctional adhesion molecule‐A), were designed, prepared, and examined as potential candidates for the development of anti‐atherosclerotic and anti‐thrombotic therapeutic drugs. A sequence at the N‐terminal of F11R together with another sequence located in the first Ig‐loop of this protein, were identified to form a steric active‐site operating in the F11R‐dependent adhesion between cells that express F11R molecules on their external surface. In silico modeling of the complex between two polypeptide chains with the sequences positioned in the active‐site was used to generate peptide‐candidates designed to inhibit homophilic interactions between surface‐located F11R molecules. The two lead F11R peptides were modified with D‐Arg and D‐Lys at selective sites, for attaining higher stability to proteolysis in vivo. Using molecular docking experiments we tested different conformational states and the putative binding affinity between two selected D‐Arg and D‐Lys‐modified F11R peptides and the proposed binding pocket. The inhibitory effects of the F11R peptide 2HN‐(dK)‐SVT‐(dR)‐EDTGTYTC‐CONH2 on antibody‐induced platelet aggregation and on the adhesion of platelets to cytokine‐inflammed endothelial cells are reported in detail, and the results point out the significant potential utilization of F11R peptides for the prevention and treatment of atherosclerotic plaques and associated thrombotic events.