Theme 3: Proinflammatory roles for coagulation proteins and their receptors

Supervisors:  A/Professor Peter Tipping
Telephone: 9594 5547
Email: Peter.Tipping@med.monash.edu.au

Interactions between coagulation and inflammation

Involvement of inflammation and coagulation is a feature of a number of important human diseases such as atherosclerosis, endotoxaemia, severe (crescentic) forms of glomerulonephritis and inflammatory arthritis. In the initiation phase of coagulation, tissue factor (TF) orchestrates the assembly of Factor VIIa and Factor X, forming a ternary complex in which Factor X is generated. The resulting complex is an efficient activator of protease activated receptors PAR-1 and PAR-2 (see figure) as well as activation of thrombin and fibrin clot generation.

Coagulation receptors expressed on the surface of leukocytes and endothelial cells have potent effects on cellular functions, including their capacity to produce inflammatory mediators. These interactions between coagulation and inflammation suggest that therapies directed at inhibiting coagulation receptors in these diseases may have beneficial anti-inflammatory effects.

Projects in my group are directed at unravelling the basic cellular mechanisms by which key coagulation receptors -TF and PARs activate macrophages and other leukocytes. In addition, using experimental models of disease, we are exploring the importance of these interactions on development of inflammatory diseases in vivo, and the potential of new therapeutic agents to reduce injury. Examples of projects on coagulation and inflammation available in my group are included below.

1. Tissue factor and PAR-2 signalling in macrophage activation.
A/Prof Peter Tipping, Dr Jim Apostolopoulos

Tissue factor is a cell membrane protein which is essential for the initiation of coagulation. TF initiation of the coagulation cascade is intimately linked to inflammatory cell signalling. Our recent studies suggest that this protein may act as a signalling receptor which has the capacity to augment macrophage pro-inflammatory responses and that leukocyte expression of the cytoplasmic domain of TF contributes to antigen specific cellular adaptive immune responses via effects on leukocyte recruitment. The mechanisms of this signalling function have not been defined, but in other systems co-operation with the PAR-2 is required. We have also determined that the cytoplasmic domain of the TF molecule may play an important role in the inflammation response and we hypothesise that PAR-2 signalling specifically phosphorylates TF. Therefore, we will study the mechanisms of TF receptor function and we will investigate the cell signalling pathways that are triggered by these two molecules using transfected monocytic cell lines and in peritoneal macrophages isolated from normal mice, mice lacking the cytoplasmic domain of TF (TFδCTδCT mice) and with mice that lack PAR receptors. This project will involve training in techniques such cell culture, molecular biology and protein biochemistry.

2. The role of protease activated receptors in atherosclerosis.
A/Prof Peter Tipping, Ms Anh Cao

Atherosclerosis is a chronic inflammatory disease of the large arteries and the major cause of heart attack and stroke in humans. Coagulation proteins play an important role in thrombotic events after rupture of atherosclerotic plaques but they may also be involved in earlier stages of plaque development, via their capacity to activate protease activated receptors (PAR’s) on macrophages and smooth muscle cells in lesions. Apolipoprotein-E deficient mice (Apo-E-/-) develop severe hypercholesterolemia and atherosclerotic lesions similar to those observed in man and allow the pathological mechanisms underlying the development of atherosclerosis to be dissected. By breeding these mice with protease activated receptor (PAR-1 and PAR-2) deficient mice, the specific contribution of these 2 receptors to the development of atherosclerosis can be determined. In this project, development of atherosclerosis will be mice studied in Apo-E deficient mice which also lack either PAR-1 or PAR-1. The effects on macrophage recruitment, smooth muscle proliferation and production of inflammatory mediated will be determined. This project will develop skills animal models of atherosclerosis, basic surgical procedures, histological techniques, PCR genotyping, ELISA, real-time PCR and flow cytometry.

3. The role of PKR in atherosclerosis
A/Prof Peter Tipping, Dr Tong Sadler

The protein kinase R (PKR) is a cell signalling kinase involved in immune responses to infection. This protein has been shown to play a role in inflammation and recent studies suggest a role for PKR in atherosclerosis. In this project, the contribution of PKR in the development of atherosclerosis will be studied by breeding PKR deficient mice onto the Apo-E deficient background. The effect of PKR deficiency on the development of atherosclerosis will be determined as described in the project above.