Fibrinolysis and Gene Regulation Laboratory

Staff Members

Laboratory Head

•  Associate Professor Robert Medcalf
  robert.medcalf@med.monash.edu.au 

Post-doctoral Research Scientists

• Dr Stan Stasinopoulos
  stanley.stasinopoulos@med.monash.edu.au
• Dr Andre Samson
  andre.samson@med.monash.edu.au

Research assistants

• Mr Be’Eri Niego
  beeri.niego@med.monash.edu.au
• Ms Roxann Freeman
  roxann.freeman@med.monash.edu.au

Ph.D student

• Ms Rachael Borg
  rachael.borg@med.monash.edu.au

Honours student

• Ms Amanda Au

Introduction

While it is important to avoid blood clots in arteries leading to the heart and brain, once they are formed, it is vital that the blood clot is removed so blood flow can be restored thereby minimising the damage to the affected organ. The body has a natural clot dissolving enzyme system (the “fibrinolytic” or “plasminogen activating” cascade) that targets fibrin, the scaffolding of a blood clot. Clinically, proteases such as tissue type plasminogen activator (t-PA) have been used to treat patients following an ischaemic stroke or heart attack by restoring blood flow to the oxygen deplete tissue. However, while blood flow is often successfully restored, t-PA can cause serious complications. For example, it is now known that t-PA has a direct action on neurons and can lead to neuronal death in ischaemic stroke.

Our laboratory studies the cell and molecular biology and regulation of the plasminogen activating cascade. We are particularly interested in understanding how t-PA and related proteases damage normal cells in the brain.  We are also interested in understanding how gene expression of t-PA and other components of the plasminogen activating system (notably plasminogen activator inhibitor type 2) are controlled and regulated. Our previous work has shown that a vampire bat saliva derivative (desmoteplase) has the potential to be clot busting drug like t-PA but without the side effects. This research has been featured on CNN, in Scientific American and Stroke and was considered by the American Heart Association as one of the top ten advances in 2003. 

Selected Projects:

1. Comparison of the neurotoxic potential of variants of tissue-type plasminogen activator.

2. Post transcriptional regulation of plasminogen activator inhibitor type 2 gene expression.

3. The role of mRNA stability in the regulation of tissue-type plasminogen activator gene expression in endothelial cells and neurons.

4. The role of t-PA in models of traumatic brain injury

5. The role of thrombin on neuronal cells and astrocytes.

Background

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. In addition to these function of this system, there is now clear evidence that the plasminogen activating system also plays an important role 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 promote 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. Some of our efforts are focused on the regulation of expression of its individual components at the levels of transcription, mRNA accumulation, and protein production, while other projects address functional consequences of this system using animal models of brain injury.

Current research projects in the Medcalf Laboratory:

1. 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 (Tristetraprolin; [TTP] ) that recognises the AU-rich element and we are currently evaluating the effect of TTP and other mRNA binding proteins 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.
   
   
2. 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. We are also undertaking experiments to explore the role and contribution of the 3’-UTR of the t-PA transcript in t-PA gene regulation.
   
   
3. 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.
   
   
4. The role of t-PA and novel thrombolytic agents during neurodegeneration and traumatic brain injury: We are exploring the role of t-PA during neurodegeneration and traumatic brain injury using 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.
   
   
5. To determine the means by which thrombin stimulates neurons. Akin to t-PA, thrombin is well  associated with the coagulation cascade. However, thrombin has additional effects in vivo, including its ability to protect neurons at low concentrations but can be deleterious at higher concentrations. We are exploring this effect of thrombin in our in vitro and in vivo models of neuronal injury and also determining the influence of thrombomodulin in this process. Thrombomodulin has the ability to alter the substrate specificity of thrombin and we are determining whether it can attenuate thrombin-induced neurotoxicity.

Associate Professor Medcalf also serves on the Editorial Advisory Boards of two International Journals (European Journal of Biochemistry and The Journal of Biological Chemistry), is Associate Editor of The Journal of Thrombosis and Haemostasis, and is also Chairman of the International Society for Fibrinolysis and Proteolysis (ISFP; 2008-2010) (http://www.fibrinolysis.org).