Targeting Protease Activated Receptor 2 in Human Breast Cancer

Michael E. Bromberg, MD, PhD [PI]
U.S. Army Medical Research-DOD Breast Cancer Research Program. The major goal of this project to prepare retroviral vectors that will deliver small inhibitory RNA (siRNA) against PAR2 to silence PAR2 expression by human breast cancer cells and determine the effects of knocked down PAR2 expression on cell proliferation, apoptosis, TF-FVIIa complex-induced cellular signaling, cell migration, and angiogenesis.

Kininogen-Urokinase Receptor Reaction in Tumor Angiogenesis (NIH) 

Robert W. Colman, MD [PI]
This grant will explore the anti-angiogenic effect of peptides from monoclonal antibodies to kininogens on the growth of tumors in animal models and cell culture.

Innate Immunity in Experimental Arthritis:  Role of Kininogen (NIH/NIAMS)

Robert W. Colman, MD [PI]
The role of the kallikrein-kinin system in experimental arthritis will be studied using monoclonal antibodies and peptides related to kininogen as well as murine knockouts of bradykinin receptors.

Kallikrein-Kinin System in Inflammatory Bowel Disease 

Robert W. Colman, MD [PI]
Broad Foundation Medical Research Program. The major goal of this project is to study the influence of kininogen deficiency and "knockouts" of B2R and B1 R kinin receptors and tissue kallikrein in inflammatory bowel disease.

Radiolabeled Peptides for Imaging Thrombi and Emboli (NIH/NHLBI)

Linda Knight, PhD [PI]
The major goals of this project are to produce recombinant bitistatin and perform preclinical testing of Tc-99m-labeled r-bitistatin; to conduct a Phase I/II clinical trial of Tc-99m-recombinant bitistatin to assess the safety and efficacy of this tracer to image deep venous thrombosis in human subjects; and to continue studies on smaller peptide analogues of bitistatin for imaging deep venous thrombi and pulmonary emboli.

Radiolabeled Peptides for Imaging Tumor Angiogenesis (NIH/NCI)

Linda Knight, PhD [PI]
The overall goal of this project is to develop a radiopharmaceutical for imaging solid tumors by targeting receptors in tumor vasculature. The aims are to measure the relative affinities of bitistatin and other disintegrins for alpha (v )beta (3 ), alpha (IIb )beta (3) and alpha (5 )beta (1 ) integrins in vitro; to determine whether modification by radiolabeling affects the relative affinity for the various receptors; to assess the tumor uptake and whole body biodistribution of radiolabeled bitistatin and other disintegrins in vivo; and to determine if a radiolabeled disintegrin can be used to monitor the effectiveness of anti-angiogenic therapy by imaging.

Molecular Pharmacology and Physiology of Nucleotide Receptors in Cardiovascular System 

Satya P. Kunapuli, PhD
The focus of research in my laboratory is on the molecular pharmacology and physiology of nucleotide receptors in cardiovascular system. We have active interest in the molecular mechanisms of ADP induced platelet activation.

Targeting Alpha1-Beta 1 Integrin in Cancer Development (NIH)

Cezary Marcinkiewicz, PhD [PI]

Obtustatin, A Potent and Selective Inhibitor of Alpha1-Beta 1 Integrin (American Heart Association - National)

Cezary Marcinkiewicz, PhD [PI]

Mechanism of JC Virus Activation by Suppression of Integrin Expressed in Immune Cells (NMSS Pilot Research Project)

Cezary Marcinkiewicz, PhD [PI]

Interactions of the Plasma Kallikrein Kinin System Proteins

Robin A. Pixley, PhD
The main focus of my research is examining the interactions of the plasma kallikrein kinin system proteins. We are currently examining the role of these proteins in in-vivo animal models and in-vitro systems. The kallikrein kinin system proteins have been demonstrated in our laboratory to play a role in inflammation and angiogenesis through the plasma enzyme and inhibitor reactions and interaction with cellular receptors.

Molecular Mechanisms in Inherited Disorders of Platelet Signal Transduction (NIH and March of Dimes)

A. Koneti Rao, MD [PI]
Patients with inherited platelet bleeding disorders are not uncommonly encountered in clinical practice. However, in the vast majority of these patients, the underlying molecular mechanisms leading to the platelet dysfunction are unknown. Our studies have focused on platelet signaling processes in these patients and have delineated hitherto undescribed abnormalities in key signaling proteins, including phospholipase C-beta-2, GTP binding protein G-alpha-q and protein kinase-C?-theta. The insights from these studies will lead to better understanding of the normal platelet mechanisms and the identification of novel targets to develop newer antiplatelet agents.

Hyperglycemia, Hyperinsulinemia, and Tissue Factor Pathway in Blood Coagulation (NIH)

A. Koneti Rao, MD[Co-PI; G. Boden, PI]
Diabetes mellitus is well-recognized risk factor for cardiovascular disease. These patients have a high incidence of acute events including heart attacks and strokes. The impact of hyperglycemia (high blood glucose) and hyperinsulinemia (high blood insulin) on the activation of blood coagulation mechanisms has not been fully clarified. These studies, performed in collaboration with Dr. Guenther Boden, focus on the activation of the tissue factor pathway induced by hyperglycemia and hyperinsulinemia in healthy subjects and patients with diabetes mellitus. Studies to date reveal a strong evidence for the activation of tissue factor pathway by both hyperglycemia and hyperinsulinemia, with the highest levels being observed with the combination of both. Further studies will lead to an understanding of the effect of antithrombotic agents on the expression of tissue factor in diabetes mellitus.

Impact of Antiplatelet Drugs Aspirin, Clopidogrel and Cilostazol on Platelet Function and Blood Coagulation in Patients with Peripheral Arterial Disease (PAD)

A. Koneti Rao, MD [PI].
Antiplatelet drugs aspirin, clopidogrel and cilostazol are widely used in the management of patients with PAD, but the effects of these agents when used in combination on platelet function and the blood coagulation system has not been clarified. Supported by a grant from the pharmaceutical industry, these studies seek to define these effects, including on the tissue factor pathway of blood coagulation. Our studies suggest that antiplatelet agents inhibit circulating levels of tissue factor, a mechanism hitherto not recognized, and which is likely to contribute to the antithrombotic effects of these agents.

Platelet Receptor-Mediated Factor-X Activation (NIH/NHLBI) 

Peter N. Walsh, MD, PhD [PI], Syed S. Ahmad, MD, PhD and Fredda London, PhD [Co-PIs].
The aims of our studies include: 1. To refine the three-receptor model for platelet-mediated, FIXa-catalyzed FX activation. a. To complete coordinate binding studies examining the interactions of FIX(IXa), FVIII(VIIIa), and FX(FII) with activated platelet receptors. b. To determine the functional consequences on the kinetics of FX activation of platelet receptor occupancy with FIX, FIXa, FVIII, FVIIIa, and FX. c. To understand the mechanisms of platelet activation that are necessary and sufficient for the binding of FIXa, FVIII(a) and FX to platelet receptors and the assembly of the FX-activating complex and to define the relationship between exposure of amino-phospholipids (Annexin V binding sites) and the binding of FIX, FIXa, FVIII(a) and FX to platelets activated by thrombin. 2. To define the structural domains of FIX, FIXa, and FVIII(a) that interact with components of the FX-activating complex. a. To define the molecular domains within FIXa that bind to platelets and promote assembly of the F–X activating complex. b. To define the molecular domains within FVIII(a) that interact with components on activated platelets.

Molecular Interactions of Factor XI (NIH/NHLBI)

Peter N. Walsh, MD, PhD [PI], Frank Baglia, PhD [Co-PI], and Dipali Sinha, PhD [Co-PI].
The aims are as follows: 1) To define the mechanism of homodimer formation mediated by the Apple 4 (A4) domain of FXI by carrying out a detailed physicochemical characterization of dimer formation using the rA4 domain. 2) To determine the morphology of the interface mediating dimer formation between the A4 domains of FXI. 3) To ascertain the structural determinants of dimer formation in full-length FXI using loss of function and gain of function chimeric rFXI/PK hybrids and A4 domain   and FXI mutants using homology scanning and alanine scanning mutagenesis. 4) To determine the functional significance of dimer formation using conformationally constrained synthetic peptides and rFXI mutants designed to produce a monomeric FXI molecule. 5) To determine the most physiologically relevant surface(s), enzyme(s), cofactor(s), and FXI subdomains required for FXI activation.

The Chemistry and Biology of Coagulation

Peter N. Walsh, MD, PhD [Project Leader, Project 3, and Frank Baglia, PhD and Dipali Sihna, PhD [Co-PIs].
(Program Director, S. Krishnaswamy, The Children’s Hospital of Philadelphia, University of Pennsylvania).
Project 3: Exosite Function in the Catalytic Domain of Coagulation Factor XIa .
To prepare constitutively inactive (zymogen-like), constitutively active  (enzyme-like) and active-site-inhibited forms of the FXIa catalytic domain and characterize them with respect to their functional interactions with the substrate, FIX; and to determine the structure and functional characteristics of the FXIa catalytic domain extended macromolecular substrate recognition exosite(s) that mediate(s) the interaction of FXIa with FIX. To determine the structure and functional characteristics of the FXIa catalytic domain exosite(s) that mediate(s) its interaction with the Kunitz protease inhibitor (KPI) domain of PN2. To determine the structural and functional characteristics of the FXIa catalytic domain exosite(s) that mediate(s) its interaction with heparin. To determine the structural and functional characteristics of the FXIa catalytic domain exosite(s) that mediate(s) its interaction with activated platelets.

Kininogen Regulation of Endothelial Cell Activation in Angiogenesis  

Wu Yi, MD, PhD
My research includes the investigation of kininogen regulation of endothelial cell activation in angiogenesis, such as adhesion, proliferation, migration and tube formation.