Fibrinolysis and Gene Regulation Laboratory
ISFP 2004 Congress
Members of Staff
Head: Dr Robert Medcalf
Post-doctoral Research Scientists
Dr Hong Yu
Dr Stan Stasinopoulos
Mr Andre Samson
tel: +61 3 9895 0317
Ms Mythily Sachchithananathan
tel: +61 3 9895 0317
tel: +61 3 9895 0317
B.Sc. (Hons). Student
Research activities undertaken in the Fibrinolysis and Gene Regulation Laboratory
Background: The Plasminogen Activating system
The removal of blood clots from the circulation and the turnover of extracellular matrix proteins is facilitated by specialized enzymes. One of the most important enzymes in this setting is plasmin. Plasmin performs many functions, but it is generally accepted that its primary role is to degrade fibrin, the structural scaffold of a blood clot. The generation of plasmin from its inactive precursor plasminogen is mediated by serine enzymes known as tissue-type plasminogen activator (t-PA) and urokinase (u-PA). The proteolytic activity of t-PA and u-PA is in turn regulated by specific protease inhibitors, plasminogen activator inhibitor (PAI)-1 and PAI-2. A specific cell surface receptor for u-PA also exists which not only provides a means of generating localised proteolytic activity in the pericellular environment, but, with the help of adjacent transmembrane proteins, can transmit signals to the cell nucleus and influence the expression pattern of other genes. The plasminogen activating system also actively participates in cell movement, wound healing and the metastatic spread of cancer. Finally, in addition there is now clear evidence that the plasminogen activating system contributes to the turnover of the extracellular matrix in the central nervous system. For example, t-PA has been shown to play a role in cognitive memory, can mediate reverse occlusion plasticity of the visual cortex, and promotes neurodegeneration. Therefore, our research impacts directly into these areas of cell and neurobiology and pathophysiology.
Figure 1 provides a schematic overview of the plasminogen activating system
Our laboratory is interested in the molecular and cellular biology of this system. Most of our efforts are focused on the regulation of expression of its individual components at the levels of transcription, mRNA accumulation, and protein production.
We have a number of projects on-going in the laboratory, most of which are focused on the molecular and cellular biology of plasminogen activation both in vivo and in vitro. We have also initiated a separate project to determine the functional consequences of the G20210A polymorphism in the human prothrombin gene.
Current research projects in the Medcalf Laboratory:
- Post-transcriptional regulation of PAI-2 gene expression: Our laboratory has identified two regions within the PAI-2 transcript that influence PAI-2 mRNA decay. One of these regions is located within the coding region, and the other is an AU-rich element within the 3’-UTR. For the latter, we have identified HuR as a PAI-2 mRNA binding protein and also cloned a protein that recognises the AU-rich element (Tristetraprolin; [TTP]) and we are currently evaluating the effect of TTP in the post-transcriptional regulation of the PAI-2 gene. We are also undertaking a proteomics-based approach to identify other functionally relevant PAI-2 mRNA binding proteins.
- Functional consequences of the prothrombin G20210A polymorphism: G20210A occurs at the last residue of the prothrombin mRNA and has a bifunctional effect: it slows down the rate of prothrombin mRNA and also increases the efficiency of prothrombin mRNA processing. We have shown that the G20210A polymorphism alters the binding pattern of proteins to this region of the prothrombin transcript. A proteomic approach is being used to clone and characterise the prothrombin mRNA binding protein.
- Transcriptional regulation of t-PA expression: The t-PA gene is regulated very differently in different cells. Part of this effect is due to the differential expression of key transcription factors that associate with critical cis-acting elements in the t-PA gene promoter. We are presently manipulating expression of these factors to determine whether the pattern of the t-PA expression can be selectively altered in different cells.
- Regulation of t-PA expression in vivo: Transgenic mouse lines that express different lengths of the human t-PA promoter fused to the LacZ reporter gene are being used to study t-PA promoter-directed regulation in vivo during constitutive conditions and during administration of agents that modify t-PA expression. We are also using these mice to study changes in the regulation of the t-PA gene during excitotoxic injury and during cerebral ischaemia (middle cerebral artery occlusion model.
- The role of t-PA and novel thrombolytic agents during neurodegeneration: We are exploring the role of t-PA during neurodegeneration using both in vitro and in vivo approaches. Our laboratory is also comparing the effects of t-PA and the plasminogen activator derived from the salivary gland of Desmodus rotundus (common vampire bat; DSPA/desmoteplase) during excitotoxic and ischaemic injury.
- Development of novel anticancer agents. We are exploring the role of novel hydroxamic acid derivatives on the regulation of genes of the plasminogen activating system and the matrix metalloprotease system. These compounds are being tested using in vitro invasion assays and also in animal models of cancer metastasis.
Dr Medcalf also serves on the Editorial Boards of two International Journals (European Journal of Biochemistry and The Journal of Thrombosis and Haemostasis) and is also President of the forthcoming Congress of the International Society for Fibrinolysis and Proteolysis, Melbourne, 2004 (http://www.icms.com.au/isfp2004). Dr Medcalf is also president-elect of the ISFP.
American Heart association:
Recent publications from the Medcalf Laboratory since 1998
- Costa, M., Shen, Y., Maurer, F., and Medcalf, R.L. (1998) Transcriptional regulation of the tissue-type plasminogen activator (t-PA) gene in human endothelial cells: identification of inducible nuclear factors which recognise functional elements in the t-PA gene promoter. Eur. J. Biochem. 258: 123-131.
- Dear A.E and Medcalf R.L (1998) The Urokinase Type Plasminogen Activator Receptor (CD-87) is a Pleiotropic Molecule. Eur. J. Biochem. 252:185-193.
- Maurer, F., Tierney, M., and Medcalf, R.L. (1999). An AU-rich sequence in the 3’-UTR of plasminogen activator inhibitor type 2 (PAI-2) mRNA promotes PAI-2 mRNA decay and provides a binding site for nuclear HuR. Nucleic Acids Res. 27: 1664-1673.(PDF file)
- Dear A.E and Medcalf R.L. (2000) The novel anti-tumour agent oxamflatin differentially regulates urokinase and plasminogen activator inhibitor type 2 expression and inhibits urokinase-mediated proteolytic activity. Biochim. Biophys. Acta. 1492:15-22.
- Katsikis, J., Yu, H., Maurer, F., and Medcalf R.L. (2000) The molecular basis for the aberrant expression of plasminogen activator inhibitor type 2 in THP-1 monocytes. Thromb. Haemost. 84:468-473.
- Iannello, R.C., Gould, J. A., Young, J.C., Giudice, A., Medcalf, R.L and Kola, I. (2000) Methylation-dependent silencing of the testis-specific Pdha-2 basal promoter occurs through selective targeting of an ATF/CRE binding site. J. Biol. Chem. 275:19603-19608.
- Costa, M., Shen, Y., and Medcalf R.L (2000). Overexpression of a dominant negative CREB protein in HT-1080 cells selectively disrupts plasminogen activator inhibitor type 2 but not tissue-type plasminogen activator gene expression. FEBS Lett. 482:75-80.
- Costa, M and Medcalf, R.L. (2001) Ectopic expression of the cAMP-responsive element binding protein inhibits phorbol ester-mediated induction of tissue-type plasminogen activator gene expression. Eur. J. Biochem. 268:987-996.
- Tierney, M., and Medcalf, R.L. (2001) Plasminogen activator inhibitor-type 2 contains mRNA instability elements within exon 4 of the coding region: Sequence homology to coding region instability determinants in other mRNAs. J. Biol. Chem. 276:13675-13684. (PDF file)
- Costa, M., Grant, P.J., Rice, G.I., Futers, T.S., and Medcalf, R.L. (2001). Human endothelial cell-derived nuclear proteins that recognise polymorphic DNA elements in the von Willebrand Factor gene promoter include YY1. Thromb. Haemost. 86:672-679.
- Black, S., Yu, H., Lee, J., Sachchithananthan, M., and Medcalf, R.L. (2001) Physiologic Concentrations of Magnesium and Placental Apoptosis: Prevention by Antioxidants. Obstet. Gynecol. 98:319-324.
- Yu, H., Schleuning, W.-D., Michl, M., Liberatore, G., Tan, S.S., and Medcalf, R.L (2001). Control elements between –9.5 and –3.0 kb in the human tissue-type plasminogen activator gene promoter direct spatial and inducible expression to the murine brain. Eur. J. Neurosci. 14:799-808. (PDF file)
- Yu, H., Maurer, F., and Medcalf R. L. (2002) Plasminogen activator inhibitor-2: a regulator of monocyte proliferation and differentiation. Blood 99:2810-2818. (PDF file)
- Carter, A.M., Sachchithananthan, M., Stasinopoulos, S., Maurer, F., and Medcalf, R.L (2002). Prothrombin G20210A is a bifunctional gene polymorphism Thromb. Haemost. 87:846-853.(PDF file)
- Liberatore, G.T., Samson, A., Bladin, C., Schleuning, W.D., and Medcalf, R.L. (2003). Vampire bat salivary plasminogen activator (desmoteplase) - a unique fibrinolytic enzyme that does not promote neurodegeneration. Stroke 34:537-543. (see recent press releases) (PDF file)
- Yu, H., Stasinopoulos, S., Leedman, P, and Medcalf, R.L. (2003). Inherent instability of plasminogen activator inhibitor type 2 mRNA is regulated by tristetraprolin. J. Biol. Chem. (in press).
- Plambeck, C.A., Kwan, A.Y., Adams, D., Westman, B.J., Weyden, L, Medcalf, R.L., Morris, B., and Mackay, J. P. (2003). The zinc finger domain from human splicing factor ZNF265 forms a novel fold. J. Biol. Chem. (in press)