Rodger P. McEver - US grants

The biosynthesis, structure, and function of membrane glycoproteins restricted to secretory granules have not been studied. These glycoproteins may control the packaging and secretion of granule contents. We have isolated a glycoprotein by immunoaffinity chromatography from human platelets which is found only in Alpha-granule membranes as determined by immunocytochemistry. The protein is also present in megakaryocytes, endothelial cells, and in the human erythroleukemia (HEL) cell line. We propose to characterize the biosynthesis and structure of this secretory granule membrane protein, designated glycoprotein IIa (GP IIa). First, we will determine its amino acid and carbohydrate composition. Next, pulse-chase studies in [35S] methionine-labelled HEL cells and endothelial cells will be performed to assess the role of proteolytic modifications and glycosylation in the processing of GP IIa. Partial amino acid sequencing of the NH2- and COOH-terminal ends of the protein will be used to prepare synthetic peptides and raise anti-peptide antibodies in rabbits. Poly(A)+ mRNA isolated from HEL cells will be used to direct in vitro translation of [35S]methionine-labelled GP IIa in the presence of microsomal membranes. The orientation of the protein in the membrane will be examined by protease treatment followed by immunoprecipitation with antibodies to the intact protein and to the NH2- and COOH-terminal peptides. Cyanogen bromide cleavage products of GP IIa will be sequenced and used to prepare synthetic 16-mer oligonucleotide probes from segments of limited codon degeneracy. Size-fractionated mRNA enriched for GP IIa will be used to prepare a cDNA library in the expression vector Lamda gt11 or in the Okayama-Berg modified plasmid pBR322. The library will be screened with polyclonal antibodies to GP IIa or with the synthetic oligonucleotide probes. The identity of cloned cDNAs for GP IIa will be confirmed by hybrid-selected in vitro translation. If the sequence is incomplete, full length cDNA will be constructed using a nucleotide restriction fragment as a primer for extension with poly(A)+ mRNA and reverse transcriptase. Restriction fragments of full length cDNA will be cloned in M13 phage and overlapping DNA segments sequenced. The deduced complete amino acid sequence of the protein will be used to make predictions about its structure and orientation in the membrane. The structural data will also be compared with that found in glycoproteins of other membrane domains, with particular attention to segments which might serve as sorting signals. Ultimately, the information obtained will clarify the function of secretory granule membrane glycoproteins as well as the mechanisms by which proteins are directed to secretory granules.

The biosynthesis, structure, and function of membrane glycoproteins restricted to secretory granules have not been studied. These glycoproteins may control the packaging and secretion of granule contents. We have isolated a glycoprotein by immunoaffinity chromatography from human platelets which is found only in Alpha-granule membranes as determined by immunocytochemistry. The protein is also present in megakaryocytes, endothelial cells, and in the human erythroleukemia (HEL) cell line. We propose to characterize the biosynthesis and structure of this secretory granule membrane protein, designated GMP-140. First, we will determine its amino acid and carbohydrate composition. Next, pulse-chase studies in [35S]methionine-labelled HEL cells and endothelial cells will be performed to assess the role of proteolytic modifications and glycosylation in the processing of GMP-140. Partial amino acid sequencing of the NH2- and COOH-terminal ends of the protein will be used to prepare synthetic peptides and raise anti-peptide antibodies in rabbits. Poly(A)+ mRNA isolated from HEL cells will be used to direct in vitro translation of [33S]methionine-labelled GMP-140 in the presence of microsomal membranes. The orientation of the protein in the membrane will be examined by protease treatment followed by immunoprecipitation with antibodies to the intact protein and to the NH2- and COOH-terminal peptides. Cyanogen bromide cleavage products of GMP-140 will be sequenced and used to prepare synthetic 16-mer oligonucleotide probes from segments of limited codon degeneracy. Size-fractionated mRNA enriched for GMP-140 will be used to prepare a cDNA library in the expression vector Lambdagt11 or in the Okayama-Berg modified plasmid pBR322. The library will be screened with polyclonal antibodies to GMP-140 or with the synthetic oligonucleotide probes. The identity of cloned cDNAs for GMP-140 will be confirmed by hybrid-selected in vitro translation. If the sequence is incomplete, full length cDNA will be constructed using a nucleotide restriction fragment as a primer for extension with poly(A)+ mRNA and reverse transcriptase. Restriction fragments of full length cDNA will be cloned in M13 phage and overlapping DNA segments sequenced. The deduced complete amino acid sequence of the protein will be used to make predictions about its structure and orientation in the membrane. The structural data will also be compared with that found in glycoproteins of other membrane domains, with particular attention to segments which might serve as sorting signals. Ultimately, the information obtained will clarify the function of secretory granule membrane glycoproteins as well as the mechanisms by which proteins are directed to secretory granules.

Little is known about the structure and function of integral membrane proteins in secretory storage granules or about the signals which direct these proteins to granules. We have identified a protein, named GMP-140, which is localized to the membranes of storage granules in platelets and vascular endothelial cells. This protein is rapidly translocated to the plasm membrane when these cells are activated and it may be reinternalized from the surface of endothelial cells. We propose to examine the properties of this unique molecule through four approaches: 1) The primary structure of GMP-140 will be determined by sequencing cDNA clones isolated from endothelial cell and megakaryocytic tumor cDNA libraries. The sequence will be compared with known peptide sequence and analyzed for features providing clues as to function. 2) The movements of GMP-140 in human endothelial cells will be studied by a combination of immunocytochemistry and binding of 125I-labeled monoclonal antibodies. 3) Cloned GMP-140 will be expressed in heterologous cells which contain secretory storage granules. If the expressed protein is localized in granule membranes, site-directed mutagenesis will be employed to locate the signals which specify its delivery to these organelles. 4) The function of GMP-140 will be investigated by measuring its binding to candidate ligands and by introducing antibodies to peptides derived from the sequence of the cytoplasmic domain into cells. These studies will clarify the role of GMP-140 in the vascular system as well as the mechanisms by which integral membrane proteins are directed to secretory storage granules.

DESCRIPTION: (Adapted from investigator's abstract). Interactions of leukocytes with platelets and endothelium may be fundamental events that link the hemostatic and inflammatory responses to tissue injury. GMP-140, a membrane glycoprotein of platelet and endothelial cell secretory granules, is rapidly redistributed to the plasma membrane during cellular activation, where it serves as an inducible receptor for neutrophils and monocytes. GMP-140, along with ELAM-1 and the Mel 14/Leu 8 antigen, comprise the selectin family of cell surface molecules that mediate interactions of leukocytes with other blood or vascular cells. Each selectin contains an N-terminal domain homologous to Ca2+-dependent lectins, followed by an EGF-like domain, a variable number of consensus repeats similar to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. The lectin and EGF domains of the selectins show particularly striking sequence homology. We have found that the lectin domain of GMP-140 is involved in neutrophil recognition and that this interaction is modulated by at least two divalent-cations binding sites. We postulate that; (1) the EGF-like domain enhances the binding affinity and specificity of the lectin domain for leukocytes; (2) separate functionally relevant binding sites for divalent cations reside on both the lectin and EGF domains, and (3) oligosaccharides comprise part of the receptor on leukocytes that interacts with the lectin domain of GMP-140. We propose to; (1) correlate divalent cation-dependent conformational changes in GMP-140 with ability of the protein to bind to neutrophils; (2) Perform detailed structure-function analyses of the individually expressed lectin and EGF-like domains and characterize potential interactions between the domains; (3) Isolate and characterize the leukocyte receptor for GMP-140. These studies should advance our understanding of the molecular basis for interactions of leukocytes with platelets and endothelium and may provide one of the first examples of a biologically significant, highly specific recognition event between a cell surface lectin and a cell surface oligosaccharide.

The objective of this project is to define the biochemical and biophysical mechanisms through which the selectins initiate rolling of leukocytes on the vessel wall under shear forces. Rolling is a prerequisite for stable adhesion and, ultimately, for emigration of leukocytes into the tissues in response to infection or injury. The work will focus on the interactions of P- and E-selectin, which are expressed on activated platelets and/or endothelial cells, with PSGL-l, a mucin- like glycoprotein expressed on leukocytes. We have shown that these interactions are critical for neutrophil rolling on P- or E-selectin. We have purified native or recombinant forms of PSGL-l and the selectins, and have developed monoclonal antibodies to each molecule. We will determine whether the consensus repeats and EGF domains of E and P- selectin affect their kinetics and affinities of binding to PSGL-l, and we will explore the structural basis for high affinity binding of Ca2+ to the lectin domains of the selectins. We will map the binding sites for mAbs and for the selectins on PSGL- 1, and seek to express a recombinant PSGL-1 that is glycosylated such its function resembles that of the native myeloid cell glycoprotein. We will examine the structural requirements for E- and P-selectin to mediate rolling of myeloid cells under shear stresses. Finally, we will examine the mechanical strength of binding of purified PSGL-1 incorporated into liposomes to P-selectin incorporated into planar membranes, under shear forces or using micromanipulation techniques. This project is closely linked with Project 4, where the structures of the glycans of PSGL-I will be determined. Within the context of the program project, this work is designed to extend our knowledge of protein-carbohydrate interactions, biomechanical factors affecting cell-cell adhesion under shear stress, and the recruitment of inflammatory cells to the vessel wall.

The goal of this SCOR application is to identify and characterize key risk factors for thrombosis that will improve methods for its prevention and treatment. The SCOR is specifically designed to promote synergy between basic and clinical research. It includes a randomized, prospective clinical trial to address the optimal duration of oral anticoagulant therapy for venous thrombosis (Project 1), a physiological study of the role of IL-6 in augmenting thrombosis (Project 4), and biochemical projects that examine the role of phospholipids and anti- phospholipid antibodies in the protein C pathway (Project 2), the membrane requirements for the tissue factor/factor VII pathway (Project 3), and the role of cytokines and other mediators in regulating the expression of P-selectin (Project 5). There is significant overlap in the scientific goals of the laboratory-based projects. In addition, an essential link for all SCOR components is the Assay Core, which will perform coagulation, ELISA, and DNA assays on blood samples from the patients in Project 1. The data from these assays will help answer mechanistic questions in the laboratory-oriented projects, which, in turn, will help define specific risk factors for recurrent thrombosis in the patients of Project 1. The SCOR is organized to facilitate generation and testing of hypotheses that will move between the basic and clinical arenas. The long-term objective is better understand the etiology of thrombosis, thereby allowing design of better treatment strategies.

P-selectin, an adhesion receptor for leukocytes, is constitutively synthesized by megakaryocytes and endothelial cells, where it is sorted into membranes of secretory granules. Upon cellular activation by agonists such as thrombin, P-selectin is rapidly redistributed from secretory granules to the cell surface, where it is then internalized. Inflammatory mediators such as TNFalpha, LPS, IL-3, IL-4, IL-6, and oncostatin M also increase synthesis of P-selectin over its basal levels. We have identified a short 5' flanking region of the human P-selectin gene that confers specific expression of a reporter gene in cultured endothelial cells. This sequence contains several regulatory domains that include a functional GATA motif, a kappaB element, sequences that bind members of the STAT protein family, and at least three elements that bind unknown nuclear proteins. Unlike kappaB elements in genes encoding proteins such as E-selectin, the kappaB element in the P-selectin gene binds p50 and p52 homodimers but not p65 homodimers or p65/p50 heterodimers. We propose to (1) Compare the effects of agonists on expression of P- and E-selectin in a panel of endothelial and megakaryocytic cells, (2) Determine the effects of agonists on expression of reporter genes containing P-selectin 5' flanking sequences in transfected cells, (3) Correlate the sequence requirements for nuclear protein binding with those for reporter gene expression for each putative regulatory element in the P-selectin gene, (4) Clone and characterize cDNAs for transcription factors that bind to these regulatory elements, (5) Determine whether the purified transcription factors function cooperatively through protein-protein or protein-DNA interactions, and (6) Determine the expression patterns of reporter genes containing P- selectin regulatory elements in transgenic mice. Because P-selectin mediates the adhesion of leukocytes to both activated platelets and endothelial cells, it links the hemostatic and inflammatory responses to tissue injury. These studies should clarify how the expression of P-selectin is controlled by inflammatory mediators, under physiological conditions and in pathological conditions such as thrombosis and atherogenesis. The findings may also contribute to our understanding of how gene expression is restricted to megakaryocytes and endothelial cells.

DESCRIPTION: (Adapted from investigator's abstract) Flowing leukocytes use the glycoprotein PSGL-1 to tether to and roll on P-selectin on activated endothelial cells and platelets. These two proteins are a novel prototype for a lectin-glycoprotein interaction that mediates cell adhesion and signaling. The investigator and coworkers have obtained preliminary data suggesting that P-selectin and PSGL-1 dimerize in the membrane and interact with cytoplasmic proteins. They hypothesize that the transmembrane and cytoplasmic domains mediate these interactions, which in turn regulate the cell surface distributions, adhesive functions, and signaling capabilities of P-selectin and PSGL-1. There are five specific aims: 1) determine whether the cytoplasmic domain of P-selectin regulates its cell surface distribution and its adhesive function, 2) determine whether P-selectin dimerizes in the membrane, and if so, whether dimerization affects its adhesive function or internalization efficiency, 3) determine whether the cytoplasmic or transmembrane domain of PSGL-1 regulates its dimerization, cell surface distribution, or adhesive function, 4) identify proteins that are phosphorylated upon PSGL-1 engagement and determine whether the transmembrane and cytoplasmic domains of PSGL-1 are required for signaling, and 5) characterize proteins that bind to the cytoplasmic domains of PSGL-1.

In shear flow, leukocytes use the mucin PSGL-1 to tether to and roll on P-selectin on activated platelets and endothelial cells and on L-selectin on adherent leukocytes. P-and L-selectin bind to a small N-terminal region of PSGL-1 that requires both tyrosine sulfate (TyrSO3) and a core-2 O-glycan capped with sialyl Lewis x (C2-0-sLeX). A synthetic glycosulfopeptide modeled after this region binds with the same affinity as native PSGL-1 to P-selectin. The investigator will use four approaches to determine the biochemical and biophysical features that enable selectin-PSGL-1 interactions to mediate cell adhesion in shear flow: (1) Investigator will compare the affinity, kinetics, and thermodynamics of binding of soluble monomers of P-, L-, and E-selectin to native PSGL-1 and recombinant soluble PSGL-1 and to synthetic glycosulfopeptides modeled after the N terminus of PSGL-1. Binding will be measured by surface plasmon resonance, fluorescence, and microcalorimetry. (2) The investigator will use Xray crystallography to determine the three-dimensional structures of the lectin and EGF domains of P-selectin and L-selectin, alone or complexed with a glycosulfopeptide modeled after the N terminus of PSGL-1. The structural information will be used to test contributions of specific amino acids to the ligand-binding characteristics of each selectin. (3) The investigator will determine whether PSGL-1 on some cells uses posttranslational modifications other than TyrSO3 and C2-0-sLeX to confer selectin binding. (4) The investigator will examine the kinetic and mechanical properties of selectin-PSGL-1 bonds that mediate tethering and rolling adhesion of cells in shear flow. The investigator will study interactions of cells expressing wild-type or altered selectins and PSGL-1, and the investigator will perfuse microspheres coupled with glycosulfopeptides modeled after PSGL-1 over immobilized selectins A key component will be coupling of flow chamber data to mathematical models, which will allow evalluation of physiochemical properties and prediction of adhesiive behaviors. These studies will provide new insigrhts into the mechanisms by which selectins initiate leukocyi:e adhesion in shear flow.

The goal of this SCORE renewal application is to identify and characterize key risk factors for thrombosis that will improve methods for its prevention and treatment. The SCOR is specifically designed to promote synergy between basic and clinical research. The SCOR is specifically designed to promote synergy between basic and clinical research. It includes randomized clinical trials to address optimal anticoagulant therapy for venous thrombosis (Project 1), biochemical projects that examine the membrane requirements for activated protein C function (Project 2), the structural requirements for the cofactor and signaling functions of the endothelial protein C receptor, the membrane requirements of the tissue factor/factor VIIa complex, and the use of gene-targeted mice to study the functions of vascular adhesion and signaling molecules in vivo. There is scientific overlap in the scientific goals of the laboratory-based projects. In addition, an essential link for all SCOR components is the Assay Core, which performs coagulation, ELISA, and DNA assays on blood samples from the patients in Project 1. The data from these assays will help answer mechanistic questions in the laboratory-oriented projects, which in turn, will help to define specific risk factors for thrombosis in the patients of Project 1. The SCOR is organized to facilitate generation and testing of hypotheses that will move between the basic and clinical arenas. The long-term objective is to better understand the etiology of thrombosis, thereby allowing design of better treatment strategies.

(Applicant?s Abstract) The trainers of this Post Doctoral Training program have trained 119 trainees over the last ten years. 55 of whom are active in academic research, 44 are laboratory directors in industry and 20 perform service and research. We propose to continue a postdoctoral training program in hemostasis and thrombosis on the Campus of the University of Oklahoma Health Sciences Center,(OUHSC) for M.D. and Ph.D. candidates who seek careers in hematological research and who wish to study on lymphocyte, function and mechanisms of hemostasis including the interaction of plasma proteins with vascular endothelium/platelets. This program is divided into three tracks. 1. Hemostatic proteins (structure and function) including thrombomodulin, endothelial protein C receptor, proteins C and S, factors V and VIII/vWF, tissue factor/factor VII and platelet p-selectin. 2. Role of receptor/ligands in platelet, endothelial cell and lymphocyte function. Early events in differentiation of marrow stromal cells and lymphocytes. 3. Animal/clinical studies, including protein C and S deficiencies, and factor VIla in patients with thrombotic and vascular degenerative disorders, the role of the protein C network in thrombotic and neoplastic diseases, and in the response of baboons infused with E.coli. This program will prepare the trainee for a career in academic medicine and hemostasis research. Trainees are selected from physicians who have had at least two years of postdoctoral training in straight medical, surgical or pediatric residency programs and from PhD?s in biochemistry, pathology, physiology, or microbiology. In accordance with the principle of affirmative action, we actively seek female and minority applicants. The criteria which we use for successful completion of training are the ability to run their own laboratory, write their own grant proposals and carry out their own independent research in basic science and research orientated clinical departments. Two to three years of training are allowed on the basis of performance. M.D. trainees do not have any formal patient care responsibilities while on this training program. This training program is implemented by the Cardiovascular Biology Research group of the Oklahoma Medical Research Foundation, (OMRF) on the OUHSC campus. This group is located on the lst and 2nd floors of the Acree/Woodworth Building, OMRF. This serves as the administrative and conference center for the training grant. Training in laboratory research also is conducted in the laboratories of the Department of Medicine and St. Francis Fdt. (OUHSC). All members of the faculty work closely together under Specialized Center of Research (SCOR) and Program Project (PP).

B. Background and Significance Leukocyte trafficking into sites of infection and tissue injury is a multistep process that is regulated by the coordinated expression of adhesion and signaling molecules (1). Flowing leukocytes tether to and roll on the vessel wall, then adhere more firmly, and finally emigrate between endothelial cells into the underlying tissues. Binding of selectins to cell-surface glycoconjugates initiates tethering and rolling (2-4). Binding of leukocyte integrins to immunoglobulin (Ig)-like molecules and other ligands slows rolling velocities, promotes firm adhesion, and directs migration (5). Cytokines, thrombin, histamine, and other mediators initiate the inflammatory cascade by stimulating endothelial cells to express adhesion molecules, chemokines, and lipid autacoids (6). Rolling leukocytes, in turn, receive signals from engagement of selectins, selectin ligands, and chemokines that promote integrin-dependent adhesion, signaling, and migration. Leukocytes also use selectins and integrins to interact with activated platelets and with other leukocytes (2). These multicellular interactions at the vascular surface link the hemostatic and inflammatory responses to tissue injury. Combinatorial diversity in expression of adhesion and signaling molecules controls the number and type of leukocytes recruited to a particular site, as well as the duration of the response. Dysregulated expression of these molecules promotes excessive accumulation of leukocytes, whose effector responses contribute to acute and chronic inflammatory diseases, thrombosis, and atherosclerosis (2).

DESCRIPTION (provided by the applicant): A BIAcore 3000 instrument is requested for inclusion in the BIAcore core facility at the Oklahoma Medical Research Foundation (OMRF). Our core facility currently has one shared first-generation BIAcore 1000 instrument. This instrument has been extensively used over the past few years since its installation, resulting in several publications in high quality journals. The BIAcore 1000 continues to be heavily used for pilot experiments, high-affinity quantitative analyses, and qualitative analyses. However, many scientists have expressed the need for a more sophisticated instrument that can reliably measure low-affinity binding and reproducibly measure binding of small peptides and growth factors. The BIAcore 3000 can accurately measure binding of small molecules (>200 Da), and its fully automated, multi-channel system configuration allows the simultaneous measurement of surface plasmon resonance (SPR) signals in multiple flow cells. This system also includes real-time signal subtraction of non-specific binding and refractive index changes in a control channel. The increased capabilities of the BIAcore 3000, which will be available to both new and current users of the core facility, will facilitate numerous NIH-funded projects that require the more advanced SPR technology. The entire package includes the BIAcore 3000 instrument, a complete computer system (CPU, monitor, printer) for instrument control as well as data acquisition, processing and analysis, a two-day training course, assorted system accessories, and a follow-up consultation. In addition we will purchase three additional evaluation software (BIAevaluation 3.0) packages to install on departmental personal computers. These packages will ensure that the computer dedicated to the BIAcore 3000 is always available for data acquisition.

Oklahoma Medical Research Foundation (OMRF) is a private, not-for-profit institution that is composed of 44 principal investigators (PI) and over 500 total employees. OMRF has operated an Institutional Review Board (IRB) since inception of these boards in the early 1970s. At present the OMRF IRB has over 100 active protocols arising from the work of about one-half of the PIs on the faculty. The protocols range from simple blood drawing from normal volunteers to placebo-controlled, multi-armed pharmacology trials as well as studies of the genetics of complex diseases. At present three nationwide, NIH-funded disease registries or repositories are included among these ongoing protocols. This proposal will enhance the supervision by the OMRF IRB with three specific aims. First, an interactive web-based education module will be established to ensure that all PIs, IRB members and staff, and other appropriate personnel maintain knowledge of, and comply with, relevant ethical principles, relevant Federal Regulations, OHRP guidance, other applicable guidance, state and local laws, and institutional policies for the protection of human subjects in research. This will include both commercially available educational modules as well as locally developed educational tools that are specific to the institution's needs. Although funded by OMRF, access to this module will be shared by the PIs, IRB members and staff and other appropriate personnel at another private, not-for-profit institution here in Oklahoma City. Operated by a group of Catholic orders known as SSMOK doing business as St. Anthony Hospital, this institution has 1700 employees including about a dozen research investigators. The St. Anthony IRB has approximately 30 active protocols. As a further aspect of this aim, equipment will be purchased to enhance the present technology in OMRF's Wileman Learning Center in order to present interactive educational seminars and meetings available only through teleconferencing. These activities will be available to St. Anthony personnel and, with available space, other interested institutions. Other aims include (a) developing existing software to monitor and track IRB activities such as approval of consent forms used in multiple institutions and/or multi-center trials, (b) strengthening other activities such as initial, continuing and expedited reviews, adverse events, and study compliance, and (c) revising online forms used to request IRB review and approval.

DESCRIPTION (provided by applicant): This proposal for a Center for Biomedical Research Excellence (COBRE) originates from the Oklahoma Medical Research Foundation (OMRF). It includes key collaborations with the adjacent University of Oklahoma Health Sciences Center (OUHSC) in Oklahoma City and with the main campus of the University of Oklahoma (UO), located 20 miles south of Oklahoma City in Norman. The scientific theme is the role of glycosylation in host defense. Virtually all membrane and secreted proteins undergo post-translational modifications, particularly glycosylation. Yet the functions of these extremely diverse modifications remain poorly understood. Oklahoma has developed major strengths in this area. The proposal aims to build on these strengths by interfacing glycobiology with existing and emerging expertise in cardiovascular biology, bioengineering, and immunology. This interdisciplinary approach centers on active participation of productive, senior investigators in mentoring a group of promising junior investigators. New Core facilities will further enhance their research. The success of these junior investigators will enlarge the critical mass of talent in overlapping scientific areas, a synergistic method to expand the biomedical research infrastructure in Oklahoma.

DESCRIPTION (provided by applicant): This Program Project Grant proposal employs an interdisciplinary approach to study the functions of protein glycan interactions in the vascular system. The three thematically related projects are led by investigators with complementary expertise and a strong published record of collaborative research. Project 1 uses gene targeted mice to study the interplay of adhesion and signaling molecules in the vasculature. The emphasis is on processes that regulate the interactions of selectins with their glycoconjugate ligands, a critical early response during inflammation and thrombosis. Mice with altered expression of selectins, selectin ligands, or glycosyltransferases will be studied. Project 2 studies the interactions of galectins, another class of lectins, with neutrophils. The galectins, some of which are expressed in vascular endothelial cells, induce exposure of phosphatidylserine on activated neutrophils without causing apoptosis. This represents a novel mechanism for clearing activated neutrophils at sites of inflammation that will be explored further in the proposal. Project 3 studies how core 1-derived O-glycans contribute to angiogenesis. Major tools are mice with tissue-specific or inducible deletions of the gene encoding T-synthase, a glycosyltransferase that constructs the precursor for all core 1 O-glycans. There is unusually high synergy among the projects that results from the intellectually overlapping themes and the sharing of reagents and methods. An administrative core cements the interactions, in particular through maintenance of a server for data exchange among the projects and through computer support for processing images and other complex data. The data obtained will enhance our understanding of the functions of lectins and glycoconjugates in the vascular system during inflammation, thrombosis, and angiogenesis. This information may suggest new approaches to treat heart attacks, strokes, and other cardiovascular disorders.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Administrative core coordinates, assists, and monitors the progress of the research projects, pilot studies, and scientific cores. The Core operates at OMRF with a subcontract to OUHSC to support Dr. Chris West's participation as mentor. The COBRE principal investigator, Dr. Rodger McEver, directs the Administrative Core. Day-to-day operation flows through the program administrator, Ms. Anita James. She arranges meetings and seminars held by the COBRE, organizes the meetings of the External Advisory Committee, and deals with other administrative issues that arise with the projects and cores. She manages the budgets of each of the projects and cores. She also prepares the annual reports to NCRR. Mr. Todd Walker, a programmer/analyst, supports information technology for all projects and cores. The Core provides partial salary support for the mentors who constitute the Internal Advisory Committee. The core also provides financial support for travel of the External Advisory Committee to OMRF for its annual reviews. The Administrative core functions within the leadership structure of OMRF. Dr. McEver and other senior OMRF members of the Internal Advisory Committee participate in the OMRF Scientific Council, which charts scientific policy in conjunction with OMRF President Dr. Stephen Prescott. As principal investigator, Dr. McEver makes strategic decisions about changes in COBRE allocations to project leaders and core directors, with input from the Internal and External Advisory Committees and subject to administrative approval by NCRR.

DESCRIPTION (provided by applicant): This proposal employs a multidisciplinary approach to elucidate how platelets and leukocytes overcome kinetic and mechanical constraints to adhere to vascular surfaces under flow. The focus is the interaction of the three selectins (P-selectin, E- selectin, and L-selectin) with P-selectin glycoprotein ligand-1 (PSGL-1) and other cell- surface glycoconjugate ligands. These interactions mediate rolling adhesion of leukocytes on activated platelets, endothelial cells, and adherent leukocytes. Our overall hypothesis is that important kinetic properties (e.g., catch and slip bonds) for binding of selectins to their ligands result from specific atomic-level interactions that are dictated by the structures of these molecules. Force regulates function by inducing conformational changes and/or forming new atomic-level interactions in the structures. Transport parameters influence how intrinsic docking rates affect molecular interactions. Since these kinetic properties determine cellular function under flow (e.g., tethering, rolling, and aggregation), relatively minor structural differences that alter these atomic-level interactions have major consequences for physiology and pathology. The proposal is integrated into four specific aims. The first three aims use crystal structures, molecular modeling, and biochemical and biophysical assays to define how specific structural features of selectins and their ligands govern tethering, rolling, and aggregation of flowing cells. The fourth aim uses knock-in mice expressing a mutant selectin to reveal the biological functions of flow-enhanced cell adhesion in vivo. The information obtained from this integrated study will clarify how molecular structure fulfills the biophysical requirements for blood cells to adhere in a hydrodynamic environment, and may suggest new therapeutic approaches to inhibiting pathological cell adhesion during inflammation and thrombosis. Project Narrative: In response to infection or injury, circulating white blood cells and platelets adhere to blood vessel surfaces, the first step in controlling infection or bleeding. This project addresses how specific "adhesion molecules" control this process. This information obtained may offer new methods to treat excessive blood cell adhesion in heart attacks, strokes, deep venous thrombosis, and other disorders.

DESCRIPTION (provided by applicant): The objective of this proposal is to elucidate the mechanical regulation of molecular interactions between selectins and glycoconjugate ligands, which mediate the first step of a multistep adhesion and signaling cascade for circulating leukocytes to attach to and migrate across vascular endothelium at sites of tissue injury or infection. These interactions are crucial, because their malfunction can result in a number of inflammatory and thrombotic disorders. Selectin-ligand interactions are regulated mechanically as they take place in the hydrodynamic environment of the circulation. Our hypothesis is that mechanical regulation of selectin-ligand binding kinetics results from specific atomic-level interactions that are dictated by the structures of these molecules. Force regulates bond dissociation by changing the energy landscape of these interactions and/or forming new interactions during force-induced fit and/or conformational changes, thereby eliciting slip and catch bonds. Transport regulates bond formation by influencing collision frequency and encounter time between interacting molecules, modulating the dependence of association kinetics on intrinsic docking. Since selectin-ligand binding kinetics determines cellular function under flow, including tethering, rolling, and aggregation, relatively minor structural differences that alter atomic-level interactions may have major consequences for physiology and pathology. This broad hypothesis will be tested in three integrated specific aims: 1) Define impact of structural variations in selectins and ligands on their interactions, 2) Define selectin-ligand interactions at the atomic level by molecular dynamics simulations, and 3) Define the consequences of ligand-specific alterations in L-selectin-dependent adhesion in vivo. The three specific aims combine experimental, theoretical and computational approaches, include in silico, in vitro, and in vivo studies, and span multiple scales from atomic-level mechanisms, single-cell and single-molecule kinetic/mechanics experiments, to whole animal physiology. This systematic study will clarify how the mechanical regulation of selectin-ligand binding kinetics enables leukocytes to adhere to blood vessel wall in the hydrodynamic environment of the circulation. Decoding how molecular structure determines this regulation will provide key insights into vascular physiology and pathology. As a result, the data may offer new therapeutic approaches to inhibiting pathological cell adhesion during inflammation and thrombosis. PUBLIC HEALTH RELEVANCE: We propose to elucidate the mechanical regulation of molecular interactions between selectins and glycoconjugate ligands, which mediate circulating white blood cells to adhere to vascular surface at sites of tissue injury or infection. This regulation is crucial because malfunction of selectin-ligand interactions can result in a number of inflammatory and thrombotic disorders. The data may offer new therapeutic approaches to inhibiting pathological cell adhesion during inflammation and thrombosis.

? DESCRIPTION (provided by applicant): In this COBRE Phase III application, we will move to independence a world-class center of excellence in vascular biology at the Oklahoma Medical Research Foundation (OMRF). In Phases I and II, we fostered vascular biology research through a multidisciplinary approach. Mentoring launched the independent careers of outstanding junior investigators, who developed innovative new technologies, published papers in top-tier journals, and competed successfully for multiple grants from NIH, DOD, NSF, and other sources. We supported development of core facilities that enhanced the research projects. Several of our scientific cores are already financially independent. In Phase III we will expand and transition to sustainability two scientific cores that support cardiovascular research for all COBRE investigators and for non-COBRE investigators. The Microscopy Core provides unique expertise to image tissues in vitro and in vivo using state-of-the-art microscopes. The Phenotyping Core provides unique expertise and equipment to assess cardiovascular physiology and pathology. The Pilot Core will identify and support innovative pilot projects from COBRE- eligible junior investigators. These will expand the scope and depth of research within the Center and further improve competitiveness for funding. The Administrative Core will coordinate the scientific cores, assist with regulatory requirements, evaluate the pilot projects, and, most importantly, nurture our strong mentoring environment to ensure its sustainability. OMRF will continue to partner with the COBRE through commitments to salaries, capital equipment, laboratory space, and faculty recruitment. Phase III funding will ensure our transition to a self-sustaining research center in vascular biology of the highest caliber.

DESCRIPTION (provided by applicant): During inflammation, flowing leukocytes roll on vascular surfaces through interactions of selectins with their glycosylated ligands. Activated endothelial cells express P-selectin and E-selectin. Their major ligands on leukocytes are P-selectin glycoprotein ligand-1 (PSGL-1), which binds to P- and E-selectin, and CD44, which binds to E-selectin. Data obtained during the previous funding period demonstrated that the organization of selectins and their ligands on cell surfaces has major impact on functions. We hypothesize that selectins and their ligands form homodimers through self-associations of their transmembrane domains. They localize in specialized membrane regions (e.g. microvilli, clathrin-coated pits, or lipid rafts) through interactions of their transmembrane or cytoplasmic domains with lipids, adaptor proteins, or cytoskeletal elements. Dimerization and membrane-domain targeting cooperate to enhance adhesive and signaling functions. We propose to test these hypotheses by making monomeric and dimeric forms of P-selectin, E-selectin, PSGL-1, and CD44 that do or do not target to lipid rafts or clathrin-coated pits. Other variants will test interactions with cytoskeletal and signaling proteins. Cells from knockout mice lacking signaling components will also be used. These tools will probe how cells organize selectins and their ligands to regulate leukocyte adhesion and signaling in vitro and in vivo. Because selectins and their ligands are major contributors to pathological inflammation and thrombosis, understanding how they function in their cellular environments may suggest new opportunities for therapeutic intervention.

.Importance of Defining Pathways for Recruitment and Turnover of Leukocytes in Inflammation -Inflammation is an immune cell response that leads to the accumulation of white blood cells (leukocytes) at sites of infection, injury or irritation. The extravasation of polymorphonuclear leukocytes (PMNs), primarily neutrophils, through selectin and integrin-mediated pathways, to a site of inflammation is followed by the emigration of monocytes/macrophages (Fig. 1). Released granule proteins from activated neutrophils induce further emigration of inflammatory cells to such sites, involving selectins, P2-integrins and formyl-peptide receptors. Accompanying changes in the chemokine network create a favorable local microenvironment for extravasation of additional inflammatory monocytes. Thus, for this positive feedback loop to end, inflammation must be resolved by the removal of the inflammatory insult (bacteria or wound detritus), along with eventual removal of the emigrated inflammatory cells. While resolution is clearly the key to limiting tissue injury in all types of inflammation, it is remarkable that so little is known about the mechanism of cellular turnover and the factors promoting the resolution of inflammation. It has been thought that resolution occurs through spontaneous apoptosis of neutrophils and their subsequent phagocytosis and engulfment(4). Surprisingly, however, neutrophil apoptosis in vivo is not a major factor in regulating their numbers in inflammation. Unlike T cells, which accumulate following perturbation of pathways involved in leukocyte apoptosis(5,6), inhibition of neutrophil apoptosis does not greatly alter neutrophil turnover in vivo, but the mechanism(s) of targeting viable neutrophils for removal is unknown (5-8). Thus, neutrophil turnover is possibly regulated by nonapoptotic factors.

Podoplanin (Pdpn) is a type 1 transmembrane mucin-type O-glycoprotein [1, 2]. It consists of 172 amino acids in mice and 163 amino acids in humans. It is expressed in lymphafic endothelial cells (LECs) as well as many other cell types including alveolar type I epithelial cells, podocytes, osteoblast cells, and several tumor cell types [1-4]. Hence, it is also known as Tia, OTS-8, gp36 and Aggrus, based on the cell type in which it has been identified [2, 5-8]. Pdpn has an extracellular domain, a single transmembrane domain, and a short cytoplasmic tail (Fig. 1 A). It is highly consen/ed between rodents and humans (Fig. 1 A). Protein homology is particulariy evident in the cytoplasmic carboxy-terminal tail of Pdpn, suggesfing important functions. Indeed, the cytoplasmic domain of Pdpn has been shown to interact with members ofthe ERM (ezrin, radixin, moesin) proteins in epithelial cells, and to subsequently activate RhoA and promote cell transdifferentiation [9]. A striking feature of the extracellular domain of Pdpn is a high content of serine and threonine residues that could potenfially be O-glycosylated [10,11] (Fig. IA). Mucin-type O-glycosylation is a prevalent form of post-translational modification of membrane and secreted proteins [12-15]. It occurs in the Golgi apparatus via sequential reactions catalyzed by specific glycosyltransferases (Fig. IB). The core of all mucin-type O-glycans is serine/threonine-linked Nacetylgalactosamine (GalNAcal-Ser/Thr), also known as Tn antigen, which is normally further modified to form distinct subtypes of Oglycans. Among them, core 1 O-glycans are a predominant form. Core 1 O-glycans are synthesized by adding galactose (Gal) to Tn antigen, a reaction catalyzed solely by the T-synthase (core 1 synthase, Cigaltl) [13-16). Core 1 structure can be further branched to form extended core 1, core 2 structures, or can be modified by adding sialic acids. These glycans are known as core 1-derived O-glycans [15,16]. Core 1-derived O-glycans are present in most cell types, especially in epithelial cells and endothelial cells [15]. Altered O-glycosylafion can affect numerous processes such as glycoprotein conformafion, trafficking, sorting, or degradafion [14,17,18]. Moreover, the O-glycosylafion state of glycoproteins may also dictate changes in cell-cell interacfions and/or cell signaling [19]. The extracellular domain of mouse Pdpn contains 24 potential sites of O-glycosylafion (Fig. 1A). The molecular weight of core Pdpn protein is about 17 kDa, however, Pdpn isolated from different tissues has molecular weight ranging from 37 to 41 kDa, suggesting extensive O-glycosylafion. Our recent study provides the first evidence in vivo that O-glycosylation is essential for fhe cell surface expression of Pdpn [14], although how O-glycosylafion regulates Pdpn expression/funcfion remains to be determined. Our study also revealed a critical contribution of core 1-derived O-glycans and Pdpn to lymphatic vascular development.

DESCRIPTION (provided by applicant): Interactions with P- and E-selectin cause flowing neutrophils to roll on activated platelets and endothelial cells. Rolling neutrophils activate b2 integrins that interact with intercellular adhesion molecule-1 (ICAM-1) to slow rolling, induce arrest, and enable emigration into surrounding tissues. Neutrophil adhesion and signaling are interdependent. In the past funding period we explored mechanisms for selectin-triggered, partial aLb2 activation to slow rolling and for chemokine-triggered, full aLb2 activation to mediate arrest. We discovered that chemokines also induce aLb2-dependent slow rolling. We distinguished talin-docking requirements for partial and full activation of aLb2. We adopted a system to express WT or mutant proteins in immortalized myeloid progenitors that are differentiated into neutrophils to probe their functions. In the next grant cycle we will further explore the molecular basis for signal-dependent neutrophil adhesion under flow. We will use biochemical assays, flow-chamber experiments, and intravital microscopy to distinguish components used for inside-out activation of aLb2 by selectins or chemokine and for outside-in signaling by integrins. To define how the cytoplasmic domain of P-selectin glycoprotein ligand-1 (PSGL-1) initiates signaling, we will generate PSGL-1 cytoplasmic-domain mutants in differentiated neutrophils from PSGL-1-deficient mice. We will screen for mutants that do not activate kinases or trigger aLb2-dependent slow rolling. We will use mass spectrometry to Identify cytosolic proteins that bind to the WT PSGL-1 tail but not to signal-defective tail mutants. We will define steps in talin1-dependent b2 integrin activation by expressing WT or mutant talin1 or b2 in neutrophils differentiated from talin1- or b2-deficient myeloid progenitors. These studies will yield new insights into connections between neutrophil adhesion and signaling that may offer improved methods to treat inflammatory and thrombotic diseases.

? DESCRIPTION (provided by applicant) Principal signs of inflammation include edema and bleeding, indicators of impaired vascular integrity. Vascular leakage often leads to impaired wound healing, and delayed clearance of pathogens, even hypotension and organ failure during severe systemic inflammation. Platelets prevent microvessels from leakage during inflammation. However, how platelets safeguard vascular integrity remains unclear. Our preliminary studies show that mice lacking platelet C-type lectin-like receptor 2 (CLEC-2) and its ligand podoplanin have increased vascular leakage in a model of lipopolysaccharide (LPS)-induced endotoxemia. These data support the novel hypothesis that interaction of platelet CLEC-2 with podoplanin protects vascular integrity in systemic inflammation. In this proposal, we will test this hypothesi by determining: 1) whether and if so, how platelet CLEC-2 interacts with perivascular podoplanin to protect vascular integrity during inflammation. Our preliminary data on confocal imaging detected staining for platelets on the abluminal side of venules and staining for podoplanin on perivascular macrophages during inflammation. These data suggest that interactions of CLEC-2 on extravasated platelets with podoplanin on perivascular macrophages limit vascular leakage during inflammation. In addition, platelets interact with leukocytes during inflammation. We will use spinning-disk and two-photon confocal intravital microscopy to determine whether platelets emigrate with or follow emigrating leukocytes through venules to interact with perivascular podoplanin. Models of systemic inflammation (such as cecal ligation and puncture) will be used to determine whether platelet CLEC-2 and perivascular podoplanin protect vascular integrity in different organs. 2) How platelets activated by podoplanin maintain vascular integrity during inflammation. We hypothesize that podoplanin-CLEC-2 interactions induce local platelet secretion of S1P that activates S1PR1 on endothelial cells to stabilize endothelial adherens junctions during inflammation. We will examine whether deficient podoplanin-CLEC-2 interactions exacerbate or platelet S1P ameliorates vascular leakage and organ failure in systemic inflammation. We will determine whether platelet S1P protects vascular integrity by maintaining endothelial adherens junctions during inflammation. Our preliminary data show that platelets, after podoplanin-CLEC-2-mediated activation, also release angiopoietin 1 (Ang1), another mediator of vascular integrity. Therefore, we will determine whether platelet S1P and Ang1 function cooperatively to protect vascular integrity during inflammation. These studies will provide key mechanistic insights into how platelet CLEC-2 limits vascular permeability during inflammation. Our results could offer exciting translational applications, for example, S1P receptor agonists or related drugs to restore endothelial barrier function in inflammatory diseases such as sepsis.

PROJECT SUMMARY/ABSTRACT Our COBRE has conducted a small but successful pilot project program. The program has been highly selective, funding only applicants with outstanding training, innovative ideas, and institutional support. In other words, we leveraged pilot funds to intramural resources to sustain junior investigators during the vulnerable period before extramural funding is achieved. Each of our pilot grant recipients became competitive for a full project in an Oklahoma COBRE, was awarded grants from state and private agencies, and is close to obtaining R01 funding. The momentum generated by the productivity of COBRE investigators (full and pilot projects) in Phases I and II provides the opportunity to widen the scope of the pilot program in Phase III. Oklahoma has many outstanding junior investigators with novel ideas in cardiovascular biology. Some have faculty-level appointments with institutional support. Many do not, but data generated from pilot grants increases their competitiveness for internal promotion. Furthermore, we continue to recruit promising new faculty members from outside Oklahoma. We will build on our success with two specific aims: Aim 1, identify and fund innovative pilot projects from junior investigators, and Aim 2, evaluate progress of projects and effectiveness of mentors to ensure that pilot grants foster career development of investigators and strengthen the scientific infrastructure of the Center. Coupled to our strong mentoring program, the pilot projects will help outstanding junior investigators transition to independent research careers.

PROJECT SUMMARY / ABSTRACT Studying biologically relevant interactions at the level of individual molecules is increasingly important in all fields of medical research. For example, characterizing precisely how antibodies interact with antigens provides insights into immune responses during vaccination or during autoimmune responses in systemic lupus erythematosus (SLE) and other diseases. Measuring the interactions of recombinant wild-type and mutant proteins that participate in coagulation and inflammation helps design small molecule inhibitors as drug candidates. Studying bacterial surface components that contribute to drug resistance leads to improved antibiotics or to new approaches to make bacteria less virulent. The objective of this proposal is to equip biomedical researchers in Oklahoma with a state-of-the-art instrument that measures the interaction of two label-free biomolecules using minute quantities of reagents. We request a Biacore T200 instrument that uses surface plasmon resonance (SPR) to measure, with high precision and sensitivity, biomolecular interactions in real time, yielding high quality affinity and kinetic data at temperatures from 4-45°C. To optimize the instrument, we also request dedicated analysis software for epitope mapping, immunogenicity, concentration measurements, thermodynamics, and single cycle injections. The instrument will be vital for supporting numerous ongoing projects that have a major impact on human health and disease.