The Cardeza Center pursues a broad range of basic research that can be divided into three focus areas: basic research, clinical/translational and core laboratories. We also host a seminar series during the academic year.
Lawrence Goldfinger, PhD
We are seeking to understand the molecular cell biology regulating how circulating blood platelets contribute to hemostasis and thrombosis, and to apply this understanding to modulate platelet function in various contexts. Our research program spans molecular studies of control of platelet gene expression, the cell biological outcomes of this regulation, physiological studies of platelet function, and development of tools for pre-clinical applications in health and disease.
Current Research Projects
Platelet microRNAs as modulators of hemostasis and thrombosis
Hemostasis (prevention of blood leak after vascular injury by formation of a clot) and thrombosis (clot formation generally not in response to injury, which can cause pathological complications) are considered the primary functions of platelets, and platelets require many genes to carry out these functions. In addition to those genes, platelets are enriched in small non-coding RNAs known as microRNAs (miRNAs) that are generally understood to function to dampen the expression levels of protein-coding genes, and thereby suppress gene activity. We have found that miRNAs are important regulators of platelet function. We are seeking to understand how miRNAs regulate platelet function in hemostasis and thrombosis, to identify the specific miRNAs and gene targets involved, and to develop strategies to manipulate miRNA function and modulate platelet reactivity to support hemostasis or suppress thrombotic potential.
Molecular control of mRNA translation in platelets
Platelets are anucleate cell fragments that circulate in blood for 7-10 days. Hence, platelets lack genomic DNA but they do contain protein-coding message RNAs (mRNAs), un-spliced pre-mRNAs and mRNA splicing machinery, as well as all the necessary molecular and cellular components to support translation of mRNA into protein, and to degrade existing proteins. Platelet reactivity, hemostatic capacity and thrombotic potential vary widely in humans, and we are investigating how control of protein translation contributes to this variation. Although mRNA translation in platelets (despite an inability to generate new mRNAs) was recognized many years ago, very little remains known about how platelets translate new protein necessary for their ongoing metabolism and reactivity while in circulation. We are exploring the molecular signaling controlling mRNA translation in circulating platelets, including the roles of platelet miRNAs, and the cellular and physiological outcomes and effects. Based on these mechanistic studies, we are developing translational approaches to control platelet reactivity via miRNA manipulation and other approaches in multiple contexts.
Current Lab Members
Peisong Ma, PhD
Dr. Ma’s laboratory is involved with investigating thrombosis and hemostasis, with a special emphasis on understanding GPCR (G-protein coupled receptor) and G-protein mediated platelet activation. Many antiplatelet drugs target GPCRs and G protein signaling pathways. Our current studies provide novel insights into the regulatory mechanisms that allow platelets to produce an optimal response to vascular injury. Using well-established vascular injury models, CRISRP-Cas9 genome-editing, and biochemical approaches, we have provided strong evidence that defects in GPCR and G-protein signaling pathways translate into in vivo phenotypes. The following is a brief summary of major ongoing projects in the lab.
Project 1: RGS-insensitive Gq (G188S) as probes of G protein functions
We have recently developed a mutant mouse line with a mutation (G188S) in the Gq subunit that renders the G protein resistant to interaction with RGS (regulator of G protein signaling) proteins as a class. In contrast to enhanced Gi2 signaling in Gi2(G184S) mutant platelets, we have observed decreased platelet activation in Gq(G188S) mutant mice, suggesting that the negative feedback mechanism of Gq regulation is different from that of Gi2. An ongoing study is to fully characterize the effect of G188S mutation on platelet function and thrombus formation.
Project 2: The signaling machinery that provides negative feedback regulation to G protein-dependent signaling during platelet activation
We have recently provided strong evidence that GPCR kinases (GRKs) are critical negative regulators during platelet activation. SNPs (single nucleotide polymorphisms) of GRKs are associated with the risk of stroke, hypertension, cardiac failure, and venous thromboembolism. Our goals are to investigate how GRKs regulate GPCRs and G proteins during platelet activation and thrombus formation, and to understand how dysfunctional regulation of GRKs may lead to thrombotic events and cardiovascular disease.
Project 3: The regulatory networks that regulate platelet activation downstream of G protein signaling using genome-wide screening
We have established that αIIbβ3 activation as a readout for genome-wide pooled CRISPR-Cas9 screen in primary megakaryocytes. We will identify novel positive regulators and negative regulators that control integrin activation in response to GPCR-coupled agonists.
Current Lab Members
Peisong Ma, PhD
Steven McKenzie, MD, PhD
Dr. McKenzie is a Hematology physician-scientist with clinical expertise in adult and pediatric non-malignant hematologic disorders and a scientific expertise in translational research. He works in the Cardeza Hemophilia and Thrombosis Center and also in the Hereditary Anemias Center. He is a member of the Sidney Kimmel Cancer Center (SKCC), in the Molecular Biology and Genetics Program. He is a member of two Graduate Programs:
Genetics, Genomics and Cancer Biology
Immunology and Microbial Pathogenesis
He is a physician mentor for Thomas Jefferson University MD/PhD program.
Dr. McKenzie directs two major laboratory research projects. The first project has a focus on immune-mediated thrombocytopenia and thrombosis syndromes (see McKenzie and Sachais, Current Opinion in Hematology, September 2014). This work led to the first and only mouse model of heparin-induced thrombocytopenia and thrombosis (HIT). The current work explores Novel Therapeutics in HIT, in an NIH-supported Program Project grant with Drs. Poncz and Rauova at Children’s Hospital of Philadelphia, Drs. Cines, Sachais and Cuker at University of Pennsylvania, and Dr. Arepally at Duke. In another subproject, Dr. McKenzie is co-funded on an NIH R01 with Dr. Bergmeier at UNC Chapel Hill and Dr. Woulfe at University of Delaware to explore platelet signaling mechanisms.
The second major McKenzie lab project focuses on the Genomics and Molecular Genetics of inter-individual variation in human platelet activation via FcgammaRIIa. This molecule has dual functions, as a receptor for IgG immune complexes and as a transmembrane adapter in integrin “outside-in” signaling. In collaboration with Dr. Paul Bray, who led the PRAX1 study, our team has identified differentially expressed mRNAs and miRNAs as well as genomic variants that regulate platelet reactivity. Team members of our longstanding Platelet Interest Group are Drs. Bray, McKenzie, Holinstat, Edelstein and Naik at Thomas Jefferson University, Drs. Rigoutsos and Londin at Thomas Jefferson University Computational Medicine Center, Dr. Fortina of SKCC Genomics, Drs. Shaw and Simon at Baylor, and Dr. Kunapuli at Temple. Dr. McKenzie is also co-funded, with Dr. Holinstat as PI, on work that involves 12-LOX and diabetes vascular biology. Our foci in the McKenzie lab moving forward are novel molecular genetic pathways for determination of receptor levels, protein tyrosine phosphatase activity and oligo-ubiquitylation in platelet FcgammaRIIa functions.
The protease-activated receptor 4 Ala120Thr variant alters platelet responsiveness to low-dose thrombin and protease-activated receptor 4 desensitization, and is blocked by non-competitive P2Y
Erratum: TULA-2 protein phosphatase suppresses activation of Syk through the GPVI platelet receptor for collagen by dephosphorylating Tyr(P)346, a regulatory site of Syk (The Journal of Biological Chemistry (2016) 291 (22427-22441) DOI: 10.1074/jbc.M116.743732)
Identification of a developmental gene expression signature, including hox genes, for the normal human colonic crypt stem cell niche: Overexpression of the signature parallels stem cell overpopulation during colon tumorigenesis
Steven E. McKenzie, MD, PhD
Shaji Abraham, PhD
Robert Kilker, MS
Ulhas P. Naik, PhD
The Naik laboratory is focused on developing therapeutic strategies to interrupt the progress of cardiovascular diseases and cancer, which are the leading causes of death in the western world. In this regard, the team has identified several novel gene products that play key regulatory roles in the progression of these diseases, with an emphasis on how these genes affect platelet functions (since platelet activity often potentiates these diseases). Using cell and molecular biological approaches, the team has characterized the potential role of calcium- and integrin-binding (CIB) protein family and junctional adhesion molecule (JAM) family members in physiological and pathological settings. Cutting edge technologies, such as the yeast two-hybrid system, siRNA, transgenic mouse models, CRISPR/Cas9, and in vivo disease models are routinely employed in the laboratory. Dr. Naik is also the Director of the Cardeza Center for Vascular Biology, Director of Integrative Physiology Graduate Program, and a member of the following Graduate Programs:
Genetics, Genomics and Cancer Biology
Cell Biology and Regenerative Medicine
Biochemistry and Molecular Pharmacology
Our three major ongoing projects are:
Positive and negative regulatory mechanisms of platelet activation during thrombosis.
Our team has identified numerous novel regulators of platelet activity genetic ablation of which has shown protection from thrombosis (e.g. Cib1 [Naik MU, et al., 2009] and Ask1 [Naik MU, et al., 2017]) or amplification of platelet function (e.g. JAM-A [Naik MU, et al., 2012]). For example, the adjacent image from Naik MU, et al., 2017 shows how in a model of pulmonary thromboembolism, whereby clots are induced to form in the tail vein and subsequently lodge in the lungs leading to breathing cessation, Ask1 knockout mice have a much higher survival rate than their wildtype counterparts; hence deletion of Ask1 protects from thrombosis. Currently, several other genes products are being investigated through exciting in vitro, ex vivo, and in vivo methods, including carotid artery injury, cremaster injury, stroke, and Deep Vein Thrombosis models.
Regulation of new blood vessel formation (angiogenesis) by Junctional Adhesion Molecule A (JAM-A).
We have cloned and characterized a novel junctional adhesion molecule, JAM-A (Naik, U.P., et al., Biochem. J. 1995; Naik, U.P., et al., J. Cell Sci, 2001; Naik, U.P. and Eckfeld, K. J. Biol. Regul. Homeost. Agents, 2003). JAM-A is expressed in endothelial and epithelial cells and resides at the tight junctions. We were the first to demonstrate that JAM-A regulates bFGF-induced angiogenesis through its interaction with integrin alphavbeta3 (Naik, M., et. al., Blood, 2003; Naik, M. and Naik, U.P., J. Cell Sci. 2006). Using siRNA and Jam-A knockout mouse, we have shown that JAM-A is essential for bFGF-induced angiogenesis (Naik, M., et al., Arterioscler Thromb. Vasc. Biol, 2003,; Cooke, et al., Arterioscler Thromb. Vasc. Biol, 2006). We extended this work further to demonstrate that JAM-A suppresses VEGF/VEGFR2 expression on endothelial cells, thus regulating vascular permeability and angiogenesis.
Mechanism of breast and prostate cancer cell metastasis.
Recently, we have found that JAM-A expression is inversely related to the metastatic ability of breast cancer cells (Naik, M., et al., Cancer Res. 2008). Overexpression of JAM-A in highly metastatic cells reduced their invasiveness; conversely, the knock-down of JAM-A in low metastatic cells increased their invasiveness. Studies are now ongoing to elucidate the molecular mechanism of this regulation. Furthermore, in collaboration with Justin Lathia of Cleveland Clinic, it has been shown that JAM-A regulates cancer stem cell function (Lathia, et al., Cell Rep, 2014).
Fraunhofer-UD Research Grant
Ulhas P. Naik, PhD
Kalyan Golla, PhD
Meghna U. Naik, MSc
Research Lab Manager
Pravin Patel, BS
Noor Shaik, BS
Latoya Watkins, BS