Research report 2002–2004 - Monash University · 2017. 7. 18. · 4 Highlights 2002–2004 2004, Rudolph Virchow Medal for Outstanding Contributions to Thrombosis Research In October

1www.acbd.monash.org

The Australian Centre for Blood Diseases(ACBD) brings together the skills andfacilities of separate yet complementaryorganisations in order to enhanceunderstanding of blood and its diseases.

Level 6, Burnet Tower89 Commercial RdMelbourne Victoria 3004

Telephone +61 3 9903 0122

Research report 2002–2004

2 www.acbd.monash.org

Foreword – Rod Carnegie 3

Highlights 2002–2004 4

Directors report 5

Relocation to AMREP 7

Research reports 9Serpin Biology Group 9

The Fibrinolysis and Gene Regulation Laboratory 11

Platelet and leukocyte biology and Platelet signalling laboratories 15

Clinical Heamatology and Bone Marrow Transplantation 21

Myeloma Research Group 23

ECRU 24Clinical research – Trials division 24

Biotechnology – Research division 26

State-of-the-art facilities 28

Academic news 30ACBD prizes and awards 30

Arrivals to the ACBD 31

Departures 32

Postgraduate education 33PhD program 34

Honours Degree Program 35

ACBD Summer Scholarship Program 36

Grant funding 2002–2004 37

ACBD personnel 38

Contents


Approximately 80 per cent of Australians will suffer from a blood-related diseaseat some stage of their lives and over 75 per cent of people will require a bloodtransfusion. It is a little known fact that more lives are lost each year becauseof a blood clot to the lung than from breast cancer.

It is also not widely recognised that the development of blood clots in vital organs,such as the heart and brain, is responsible for the majority of heart attacks andstrokes. Blood cancers such as lymphomas, leukemias and myeloma representup to 15 per cent of all cancers. The incidence of these problems is on the rise.Collectively blood-related diseases are responsible for significant morbidity, lossof productivity and mortality in our society.

The ACBD is a fully-integrated blood centre with strong clinical, research andeducational capabilities. This has been realised through the co-location of manyof Box Hill Hospital’s leading haematology personnel to the Alfred campus.The combined Alfred and Box Hill haematology expertise will provide the mostextensive network of haematology services in the State.

The ACBD research and medical educational services are on a par with the worldsleading blood centers. The centre provides a seamless integration of acute healthcare, research and educational activities. The centre has an international focus.The objectives of the ACBD are to remain a leading international centre forresearch, treatment and education in blood and its diseases.

The fusion of acute health care, academic endeavors and a broad range ofresearch talents including biotechnology expertise is a best international practiceconcept. This concept is both solid in its foundation and practical in the executionof its plans. The ACBD pays its way in the cost-effective delivery of the bestalternative solutions to medical problems by its focus on issues that areimmediately relevant to patient care.

It is exciting and rewarding to be part of such a proactive and cutting edgeresearch centre that aims to improve the health of all Australians.

Sir Roderick Carnegie

Foreword


Highlights 2002–2004

2004, Rudolph Virchow Medal for Outstanding Contributionsto Thrombosis Research

In October of 2004, Assoc Professor Shaun Jackson was awarded theRudolph Virchow Medal for Outstanding Contributions to Thrombosis Research.This presentation was made at a formal ceremony during the InauguralInternational Haemostasis Symposium, in Würzburg, Germany.

2003, Monash University Silver Jubilee Research Prize

In 2003, Dr Medcalf was awarded the Monash University Silver Jubilee ResearchPrize. This was awarded by the board of the Faculty of Medicine, Nursing andHealth Sciences for his contributions to the field of fibrinolysis and plasminogenactivator research.

2002 Victoria Fellowship

Dr Cindy Yap was awarded one of six Victoria Fellowships by the VictorianGovernment, presented by the Governor of Victoria at a gala ceremony atGovernment House. The Victoria Fellowships, which consist of six awardsin the form of travel grants of up to $15,000, recognise emerging innovators inscience, technology and engineering early in their careers. Dr Yap received heraward for her role in identifying an enzyme that is largely responsible for theabnormal blood clotting that underlies heart attack and stroke.

Associate Professor Shaun Jackson

Dr Robert Medcalf

Dr Cindy Yap


Director’s report

This report covers the period of 2002-2004. Unfortunately we were not able to puttogether a report last year due to many changes and happenings. One of themajor changes has been the relocation of our basic research laboratories fromBox Hill Hospital to the Alfred Medical Research and Education Precinct (AMREP).The move was in response to the Victorian Government decision to clusterbiomedical research in the State. We had a couple of options to choose from,but felt that a move to the AMREP would be more consistent with our researchand clinical direction, and that we stood a much better chance of success byrelocating to this site. Our new home is located on level 6 of a new tower builton the AMREP site.

Considerable amount of time was spent planning the fit-out of the 1800 squaremeters of space allocated to our group. In this regard I would like to acknowledgeall the hard work of Robert Medcalf and other members of the centre who workedclosely with the architects to complete this awesome task. The space we occupyis state-of-the-art in every respect. We have comfortable well lit and equippedfacilities that are admired by all who visit. The project of relocating us was acooperative effort of the Baker Medical Research Institute, Monash University,The Alfred Hospital, and The Victorian Government. The Baker Medical ResearchInstitute used considerable funds to realise the project and for that we are verygrateful. We hope that our close proximity to the Baker will allow a closer workingrelation. Our focus in the area of blood clotting blends very well with the focus atthe Baker Institute and other partners on site and we look forward to developingjoint programs that make use of all the cardiovascular skills on site.

The concept of collocating research and education on a precinct is most attractiveand clever. It gives us all the opportunity to effectively work together and bringsthe best out of our staff. Our tower houses many diverse yet complementary andsynergistic activities. The Burnett Institute with their focus on the biology andepidemiology of infectious disease occupies several floors, The MonashDepartment of Epidemiology and Preventive Medicine has many experts that wecan call on and is a couple of floors below us. The new facility for clinical trials(Clinical Trials Victoria) and the National Trauma Institute are also our neighbours.The scope of working with all these groups in a productive manner is enormous,exciting and in the long run likely to yield substantial dividends.

Members of the blood centre have settled well in their new home and we are backto business as usual, writing papers, grants and carrying out our research from thenew facilities. My appointment as head of clinical hematology at the Alfred Hospitalhas brought with it a closer working relation with all the clinical haematologists atthe hospital that we hope to build on. The Alfred has a wealth of clinicalhaematologists and patients and we all stand to benefit from our new workingrelationship. I anticipate that we will develop new research directions and activitiesas our relationship further develops and cements.

One aspect of the ACBD that we hope to further develop over the next few yearsis our educational programs. We are committed to develop a basic educationprogram in blood diseases that will assist patients and their families as a well asprovide the latest information to clinicians caring for patients with these conditions.

Biotechnology and commercialisation/patenting of new ideas or discoveries arealways at the forefront of the minds of our scientists. The biotechnology companyKinacia developed by members of the ACBD has continued to grow and develop.The company was taken over in 2004 by Cerylid Pty, Ltd and now carries outits development program from the Cerylid laboratories in Richmond, Victoria.

Professor Hatem Salem

Our vision:

Is to be a leadingnational and

international blooddiseases centrewith recognized

research, treatmentand educational

programs forblood diseases.


Members of the ACBD are now working on setting up a new company tocapitalise on new intellectual property and commercial activities in the centre.Robert Medcalf has an active collaboration with the German biotechnologycompany PAION. The work has enabled PAION to progress its new thrombolyticagent to a clinical program. If successful we will soon have a new, more effectiveand potentially safer thrombolytic agent to use in the context of thromboticcerebrovascular accidents.

The Eastern Clinical Trial Unit (ECRU) has maintained its standing as the largestcooperative clinical trial unit in the country. ECRU has 35 research coordinatorsand more than 125 active clinical trials. Cheryl Gillzan, the manager of ECRU,continues to do a wonderful job marketing ECRU to the pharmaceutical industry,a very demanding and competitive area of clinical research. ECRU now has threeclinical sites, Box Hill, Maroondah and the Alfred Hospitals.

Two years gap in reporting is a long period of time. We saw many staff membersleave and have welcomed many others. I will not attempt to list all who have leftor those who arrived, this information will appear elsewhere in the report. I wouldhowever like to mention two of our CJ Martin Fellows who are currently studyingin the United Kingdom. Both Suhasini Kulkarni and Sascha Hughan are currentlyworking at Cambridge and Birmingham respectively as a part of their fellowshipprogram. We wish them the very best and look forward to their return in the nearfuture. I also want to mention Cindy Yap who was one of our shining stars.Cindy decided that a change was in order in her life, and joined her husbandin a new commercial venture. We wish them both success and happiness.

August 2005 will see the largest gathering of experts in Haemostasis andThrombosis in Sydney. They will be attending the 20th congress of theInternational Society of Haemostasis and Thrombosis. This biennial event iseagerly awaited by all. The preparation for the meeting took several years andinvolved numerous individuals. Many members of the ACBD feature prominentlyon the organising committee and contributed to what should be a wonderfulscientific exchange.

Finally I would like to thank all my colleagues, members of the centre.Their self belief and strong commitment has allowed us to progress and achieveour current standing. I am particularly grateful to Simone Schoenwaelder whois a mastermind in organisation. Without Simone’s hard work and dedication wewould be a long way behind. I look forward to writing my next report in twelvemonths from now when I hope to report much further progress.

Hatem Salem


Relocation to AMREP

The story behind our relocation to AMREP

2004 proved to be a very exciting year for all concerned at the ACBD. Apart fromour continuing success in the area of blood biology and diseases, we were allparticularly excited with the completion of our brand new state-of-the-art facilityat AMREP. This was quite some undertaking and it is worthwhile to cast somethoughts back a few years to highlight the key events that shaped our decisionto choose AMREP as our eventual relocation.

Most of you will already be aware of the fact that the ACBD was initiated in thelate 1990’s as a centre within the Department of Medicine at Box Hill Hospital.The department and the ACBD steadily grew in stature and became synonymouswith cutting-edge science focusing on blood diseases. In addition, our clinicalresearch unit (ECRU) was established and this was also continuing to expand.Collectively, this resulted in a greater intake of honours and PhD students,research assistants, nursing staff and other senior scientists, not to mentionthe need to purchase new equipment. With this demand on space, it becameapparent that we had to relocate to a bigger facility. We were also conscious ofthe fact that our long-term research success would be maximised if our newfacility was to be positioned close to other research facilities with a commonfocus. Location in real estate circles is often used to signify asset value and tosome extent this applies to science. With this in mind, it soon became apparentthat the most appropriate decision for us was to move our facility to the AlfredMedical Research and Education Precinct (AMREP) at the Alfred Hospital. This site provided proximity of the Baker Heart Research Institute, the BurnetInstitute, and MonashUniversity Department of Immunology, and Epidemiologyand Department of Community Medicine, and also provided the necessary clinicalinterface with the Alfred Hospital. It was a perfect location.

We could also look forward to fostering new collaborations with colleagues withinAMREP, and this location would provide an enviable opportunity for new studentsand in fact for entire research teams. The other immensely important componentbehind this decision was the development of the Burnet Building that was beingbuilt adjacent to the Baker Heart Research Institute. This building was to consistof seven levels and we had the opportunity of developing the top level for theACBD. This level contained approximately 1800 square meters of open floorspace (about double our space at Box Hill Hospital) that included at the north enda magnificent view of the Melbourne skyline, a view of the Dandenongs to theEast, and the southern suburbs and Port Philip Bay to the south.

I remember this time very well and also the challenge to come up with a floor planthat would not only suite our needs, but I have to admit, make our colleagueselsewhere in Melbourne drool with envy. In early 2002, we sat down with ourarchitects (S2F) to draft the plans for our new home. We decided on an open floorplan with all laboratories having natural light and window views, integrated tissueculture rooms, and a centralised core facility where heavy duty and communalequipment could be easily accessed. Then of course we needed to provideattractive office accommodation for our staff, and enough office space to allow forexpansion to accommodate new research teams. It was also equally importantthat we give considerable thought to the needs of our students and technical staff,provide for an adequate number of meeting rooms, reception area and finally astaff tea room that would be comfortable, attractive and contain the necessaryfacilities. This was not a trivial exercise particularly when this whole operation hadto be funded within a tight and largely inflexible budget.

The ACBD, 6th floor Burnet Tower pre renovation

The Burnet Tower

Ms Jennie Saravanamuttu, personalassistant to Professor Salem.


After many drafts and discussions with all stakeholders, we eventually agreedon a floor plan that addressed all of our needs. Although our floor was availablefor fit out in early 2002, it took almost two years before we could commenceour relocation. We boxed-up our laboratories and offices at Box Hill Hospitalin late March 2004 and the complete relocation occurred over the weekendof April 3 to 4.

After some predictable teething problems, we have now settled in and are fullyoperational. We have also added flavour to our facility by including pieces oforiginal art and other visual aids that are both attractive and related to ourresearch interests. Looking back, it is still hard to imagine that we transformed1800 square meters of wall-less space into a facility that we are all immenselyproud of, especially me. Our facility, now officially recognised as the ACBD, andhas been described as the most attractive and functional in Melbourne and ourInternational visiting scientists have all been as equally impressed. We look backon our previous chapter at Box Hill Hospital with pride and fully acknowledge thesupport provided to us by the Hospital and of course Monash University, thatgave us that initial opportunity to develop our ideas and to be recognisedinternationally for research into blood diseases. With an eye on our past, we nowlook forward to the future of the ACBD with an air of confidence and anticipation.

Rob Medcalf

View of Melbourne City Skyline across Faulkner Park from the ACBD.

Packing up Box Hill


From Left: Melinda Missen, Anita Horvath,Corinne Hitchen and Trifina Sofian.

Ms Melinda Missen and Dr Anita Horvath

Research report

Serpin Biology Group

The evolutionary success of serpins marks them as a very special group ofproteins. Their name is an acronym derived from the best known feature of thefamily, serine protease inhibitor. Over 800 serpin genes are present in sequencedatabases from prokaryotes, plants, simple multicellular animals and mammals.The human genome alone contains 35 serpin genes.

Serpin gene duplication and functional diversity

The focus of research in our laboratory has been on the biological role of membersof the serpin super family of proteins. Much of this research has involved the ‘A’clade cluster of serpins on human chromosome 14q32.1 and the syntenic regionon mouse chromosome 12F1. The murine locus contains an orthologous clusterof serpin genes in which two members, corresponding to antitrypsin andantichymotrypsin, have undergone dramatic expansion. We have examined theorganisation of the human and murine ‘A’ clade clusters making use of theirrecently completed genomes sequences. One of the most surprising outcomesof this analysis was that human antichymotrypsin is represented by 14 genes inthe mouse. While these genes appear to have conserved elements indicating thatwill make functional proteins, they are divergent within the specificity-determiningreactive loop. Using gene-specific PCR we have studied the expression pattern ofthe murine antichymotrypsin-like proteins. We have also cloned several membersof this group and analysed their biochemical and biophysical properties. Recently,we have crystallized one of the serpins, EB22.4 and obtained a high-resolutionstructure which gives important insights into its inhibitory mechanism.

One of the murine antichymotrypsin-like group, serpin2A, is primarily expressedin primitive haemopoietic stem cells and activated T-cells. We have produced arecombinant version of serpin2A and examined its biochemical properties with aview to defining its role of serpins in regulating the growth and differentiation ofhaemopoietic progenitors. We have also investigated the expression patterns ofother members of the serpin family and their cognate proteases in human bonemarrow and peripheral blood cells. The long-term aim of this work is to give newinsights into the control of blood cell development.

Centerin: a serpin expressed in lymphoma

Another member of the ‘A’ clade serpin cluster is the germinal centre cell-specificprotein, centerin. Preliminary investigations by other investigators have shown thatthis is not expressed in any other tissues but it is markedly upregulated in B-celllymphomas. We have cloned the gene for this protein and have produced arecombinant protein. Ongoing studies are evaluating the role of centerin in normalB-cell development and investigating its usefulness in the diagnosis andmanagement of lymphomas.

New members of the serpin ‘A’ clade cluster

In addition to the known members of the ‘A’ clade cluster we noticed twomembers of the family which had not previously been studied. One of these(SERPINA12) has been described in rat adipose tissue and is upregulated indiabetes. The other new gene is SERPINA11 and is expressed in fetal liver.We are currently developing studies on these proteins to define their normal rolesand investigate whether they are involved in human disease.

Laboratory members

Head: Dr Paul Coughlin

Dr Anita Horvath, PhD, Post-Doctoral Fellow

Ms Corinne Hitchen, Research Assistant

Ms Melinda Missen, PhD student

Mr Lukas Stolarski, Honours student

Ms Trifina Sofian, Honours student


Antiplasmin: a key regulator of clot dissolution

We are currently developing new research in the area of fibrinolysis (clotdissolution). A major problem in the management of patients with deep veinthrombosis is long term pain, swelling and ulceration in the legs. The standardapproach to treatment of these disorders is the use of anticoagulants becauseclot dissolving agents often cause an unacceptable risk of serious bleeding.

There is a need for therapeutic agents which promote clot dissolution withoutsignificantly increasing the risk of haemorrhage. A member of the serpin family,antiplasmin is the major inhibitor of the clot dissolving enzyme, plasmin. Althoughthis protein has been known for a long time remarkably little is known about itsprecise mechanism of action or regulation. We are therefore investigatingrecombinant antiplasmin using X-ray crystallography and domain-specificantibodies to facilitate production of new therapeutic agents which will allowmodulation of the fibrinolytic system.

Research in the Serpin Biology Group is complemented by active collaborationswith members of Monash University Department of Biochemistry and MolecularBiology and the Stem Cell Group at Peter MacCallum Cancer Institute.

Current areas of research focus during 2004

• Mechanisms of ligand dependent activation of murine antichymotrypsin

• Centerin: a serpin expressed in lymphoma

• New members of the serpin ‘A’ clade cluster – involvement in humandisease states

• α2-antiplasmin: a key regulator of clot dissolution

Recent publications

1 Hamerman JA, Hayashi F, Schroeder LA, Gygi SP, Haas AL, Hampson L,Coughlin P, Aebersold R, Aderem A. Serpin 2a is induced in activatedmacrophages and conjugates to a ubiquitin homolog. J Immunol2002;168(5):2415-23.

2 Morris EC, Dafforn TR, Forsyth SL, Missen MA, Horvath AJ, Hampson L,Hampson IN, Currie G, Carrell RW, Coughlin PB. Murine serpin 2A is aredox sensitive intracellular protein. Biochem J 2003; 371(1):165-73.

3 Sharon Forsyth, Anita Horvath and Paul Coughlin. The murine ±1-antitrypsinand ±1-antichymotrypsin multi-gene clusters: a review and comparison withthe human clade A serpins. Genomics 2003; 81(3):336-45.

4 Noelene S. Quinsey, Hazel L. Fitton, Paul Coughlin, James C. Whisstock,Timothy Dafforn, Robin W. Carrell, Stephen P. Bottomley and Robert N. Pike.The introduction of a mutation in the shutter region of antithrombin(Phe77-Leu) increases affinity for heparin and decreases thermal stability.Biochemistry 2003 Sep 2;42(34):10169-73.

5 Horvath, A. and Coughlin, P.B. Expression patterns of the murineantichymotrypsin-like genes reflect evolutionary divergence at the serpina3locus. J Mol Evol 2004 Oct;59(4):488-97.

6 Horvath A. J., Forsyth S. L., and Coughlin P. B. (2004). Expression patternsof murine antichymotrypsin-like genes reflect evolutionary divergence at theSerpina3 locus. J Mol Evol 59: 488-97.

7 Baker, R., Coughlin, P., Gallus, A., Harper, P., Salem, H. and Wood, E.Australia and New Zealand Consensus Guidelines for Warfarin Reversal.Med J Aust. 2004 Nov1;181(9):492-7.

Dr Paul Coughlin

Dr Anita Horvath


Research report

The Fibrinolysis and Gene Regulation Laboratory

The Plasminogen Activating system

The removal of blood clots from the circulation and the turnover of extracellularmatrix proteins is facilitated by specialised enzymes. One of the mostimportant enzymes in this setting is plasmin. Plasmin performs many functions,but it is generally accepted that its primary role is to degrade fibrin, thestructural scaffold of a blood clot.

The generation of plasmin from its inactive precursor plasminogen is mediated byserine 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 specificprotease inhibitors: plasminogen activator inhibitor (PAI)-1 and PAI-2. A specificcell surface receptor for u-PA also exists which not only provides a means ofgenerating localised proteolytic activity in the pericellular environment, but, with thehelp of adjacent transmembrane proteins, can transmit signals to the cell nucleusand influence the expression pattern of other genes. The plasminogen activatingsystem also actively participates in cell movement, wound healing and themetastatic spread of cancer.

Finally, there is now clear evidence that the plasminogen activating systemcontributes to the turnover of the extracellular matrix in the central nervoussystem. For example, t-PA has been shown to play a role in cognitive memory,visual processing, and can promote neurodegeneration. Therefore, our researchimpacts directly into the areas of neurobiology and neuro-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 individualcomponents at the levels of transcription, mRNA accumulation, and proteinproduction. We have also initiated a study into the role of the plasminogenactivating system (particularly t-PA) in the central nervous system and alsowe are evaluating novel thrombolytic agents that lack the neurotoxic featurescharacteristic of t-PA. One such agent in the highly fibrin-specific plasminogenactivator found in the saliva of the common vampire bat.

Summary of on-going projects

1 Regulation of tissue-type plasminogen activator gene expression in vitro:

The activation of plasminogen by t-PA is the principle means by which plasminis generated in the circulation. Many agents influence the transcriptional controlof the t-PA gene, most notably cytokines and growth factors. Indeed, oneparticular agent (Phorbol ester [PMA]) has been shown to either induce orrepress t-PA depending on the cell type. We are interested in determining themolecular basis for this opposite regulation as this might shed light on how thet-PA gene is regulated. We are also interested in determining how cytokinesregulate t-PA gene expression. One particularly important cytokine in thissetting is the inflammatory mediator, tumour necrosis factor (TNF).High levels of TNF in the blood are associated with the development ofthrombosis and in this context, addition of TNF to endothelial cells results ina marked suppression of t-PA production. Hence it follows that the means bywhich TNF can promote thrombosis occurs, at least in part, by switching offthe t-PA gene, thereby reducing the ability of the host to remove blood clots.One project we are undertaking addresses the molecular mechanism behindTNF-mediated suppression of t-PA transcription in endothelial cells. We areexamining the regulatory domains within the t-PA promoter to identify thecontrol sequences that are needed to convey TNF-mediated suppression to

Laboratory members

Head: Dr Rob Medcalf

Dr Stan Stasinopoulos, PhD

Dr Phil Daniel, PhD

Dr Thomas Weiss, MD

Dr Hong Yu, MBBS, PhD

Ms Mythily Sachchithananthan,PhD student, Assistant

Mr Andre Samson, PhD student

Mr Courtney Reddrop, Research assistant

Lovisa Dousha, BSc (Hons) student

Emily Chen, BSc (Biomed) student

Visiting scientists

Ms Anna Tjarnlund, PhD student

Goteborg, Sweden

Associate Professor Christina Jern,

Goteborg, Sweden

Dr Robert Medcalf

Figure 1


the t-PA gene promoter. In addition to these approaches, we are exploringpost-transcriptional regulation of the t-PA gene by TNF and other agents,including PMA.

2 t-PA gene regulation in vivo

Transgenic mice provide a means to assess the in vivo expression patterndirected by defined sequences of gene promoters. Transgenic mice and ratsexpressing 9.5 kb of the human t-PA promoter fused to a reporter gene(9.5 tPALacZ) have been used to visualise t-PA promoter directed expressionin vivo. We previously showed that the 9.5 kb human t-PA promoter directshigh level reporter gene expression in discrete areas of the brain includingthe limbic region, hippocampus, the superior colliculis and cerebellum.The expression pattern in the brain was reproducible and consistent withthe expression pattern of the endogenous mouse (and human) t-PA gene.We are now using these mice to look more closely at how the t-PA geneis regulated in neuronal cells following treatment with agents thatpromote neurodegeneration.

Figure 2 shows the pattern of expression directed by the human t-PA genepromoter in the mouse brain. Panels i and ii show Dorsal ventral views,respectively of transgenic mice expressing the LacZ reporter gene controlledby 9.5 kb of the human t-PA gene promoter. Blue-green colouration indicatesregions of reporter gene expression on the brain surface. For comparativepurposes, panel iii shows a dorsal view of a transgenic line expressing theLacZ reporter gene driven by only 1.4 kb of the human t-PA promoter thatdoes not show reporter gene expression. Coronal sections of the brains oftransgenic mice expressing 9.5 kb of the t-PA promoter were cut and stainedfor reporter gene expression (Figure 2, panel iii). As shown, LacZ staining isdirected to discrete regions of the brain. Panel iv shows a magnified regionof the area outlined in panel iv showing more clearly the areas expressing thereporter gene: H (hippocampus); DG (dentate gyrus); MH (medial habenula).Scale bar in panel i = 5 mm; Scale bar in panel v = 2.5 mm

3 Regulation of the plasminogen activator inhibitor type 2 gene

3.1 Post transcriptional regulation of PAI-2 gene expression

We have devoted much effort to elucidate the molecular mechanismsunderlying the regulation of the PAI-2 gene. We have been particularlyinterested in the biology and regulation of PAI-2 for a number of reasons:

1. PAI-2 is an unusual protease inhibitor since it resides intracellularly, yetits targets are located extracellularly. This suggests that PAI-2 may havean intracellular function independent of the inhibition of u-PA. In fact, twolaboratories have provided evidence for a role for PAI-2 as an inhibitor ofapoptosis.

2. The PAI-2 gene is one of the most responsive genes known: for example,the PAI-2 gene is the most prominently induced gene in fibroblasts treatedwith the inflammatory mediator, tumour necrosis factor (TNF).

Our laboratory recently demonstrated that the PAI-2 gene is impressivelyregulated at the level of PAI-2 mRNA stability. We have identified a numberof sequences within the PAI-2 3’-UTR as well as in the coding region thatprovide binding sites for cellular factors that appear to influence the decayrate of PAI-2 mRNA. Two of these mRNA binding proteins have beenidentified (“HuR” a known mRNA stabilising protein; and tristetraprolin[TTP] an mRNA destabilizing protein) and work is underway to definethe role of these factors in the control of PAI-2 mRNA metabolism.We are also using a proteomics approach to identify other novel mRNAbinding proteins.

3.2 To assess the role of PAI-2 using THP-1 monocytes

Not all cells express PAI-2, but those which do exhibit a similar PAI-2expression profile. There is however, one notable exception: in THP-1monocytes, the PAI-2 gene is mutated and PAI-2 protein cannot beproduced. The availability of the THP-1 cell line has allowed us to exploreaspects of PAI-2 biology which were previously difficult to assess.

Mythily Sachchithananthan

Dr Phil Daniel


We have recently stably introduced the fully functional PAI-2 into THP-1cells and we are using these cells to explore the influence of PAI-2 onmonocyte biology. Results to date have shown that the presence of PAI-2in these cells alters the rate of cell proliferation. Microarray data havefurther shown that the presence of PAI-2 alters the expression pattern of anumber of other genes. Therefore, PAI-2 can also modulate cell behaviourby influencing or reprogramming gene expression patterns. We are lookingmore closely at the PAI-2 responsive target genes to determine how PAI-2alters their pattern of expression. We are also interested in determiningwhether PAI-2 alters the gene expression pattern on other cell systems.

4 The regulation of metastatic disease by novel compounds

The metastatic ability of tumour cells is dependent to a large extent on theirability to generate localised proteolytic activity to facilitate cell migration.Much evidence has indicated that u-PA is critical for this activity. Indeed, manyclinical studies have shown that tumours with high levels of u-PA are moreaggressive and display a greater degree of metastasis than tumour with lowlevels of u-PA. Furthermore, animal studies have demonstrated that alterationin u-PA activity either by decreasing expression of u-PA itself, or alternativelyby increasing expression of u-PA inhibitory agents (e.g. PAI-2) can alter themetastatic potential of certain cancers.

We have described a novel compound that has the ability to suppress u-PAactivity by two mechanisms: directly suppressing u-PA expression andsimultaneously increasing expression of its natural inhibitor, PAI-2. We are nowusing animal models of metastatic cancer to explore the therapeutic potentialof this agent.

5 Functional effects of the prothrombin G20210A gene polymorphism

We have been investigating the functional consequences of a G to Apolymorphism at position 20210 in the prothrombin gene. Prothrombin is acritical enzyme required for the generation of fibrin from fibrinogen. Individualscarrying this polymorphism have increased levels of prothrombin in the bloodand an increase in the incidence of venous thrombosis. We have evidence tosuggest that the increase in plasma prothrombin levels in individuals carryingthe A allele prothrombin variant promotes a pro-thrombotic state whichprecipitates the increased incidence of venous thrombosis.

The mechanism(s) whereby G20210A polymorphism leads to elevatedprothrombin is unknown but its location in the 3’-untranslated region (3’-UTR)immediately preceding the poly (A) tail of PT mRNA suggested that it mayinfluence PT mRNA stability, processing or protein translation. Our preliminaryin vitro experiments have indicated that cDNA expression vectors containing Aallele variant of prothrombin produce at least three-fold more prothrombin instably transfected cells compared to cells expressing the G allele counterpartand this certainly involves changes in the post-transcriptional expressionpattern of prothrombin. We are presently using a proteomics approach toidentify the cellular factors that influence prothrombin mRNA expression.

6 The Role of t-PA in the central nervous system

In addition to its well known role as a fibrinolytic agent, t-PA also plays apreviously unsuspected role within the central nervous system (CNS). In thiscompartment, t-PA plays a positive role under normal conditions as itparticipates in neuronal plasticity and memory formation and also plays a rolein stress-induced anxiety. Under conditions of neuronal injury, the presence oft-PA intensifies the degree of cell death indicating a negative effect of t-PAunder these conditions. We are presently attempting to determine the meansby which t-PA is neurotoxic. This has important implications in patients withischaemic stroke. In this context, we have been evaluating the neurotoxiceffects of a plasminogen activator derived from the saliva of the commonvampire bat (Desmodus rotundus). This substance, known as DSPA(Desmodus rotundus salivary plasminogen activator) is far more fibrin selectivethan t-PA (hence more clot specific). We have also shown that DSPA does notdisplay the neurotoxic effects seen with t-PA. DSPA therefore holds muchpromise as a new plasminogen activator for stroke patients.

Andre Samson

Be'Eri Niego

Dr Thomas Weiss


Current areas of research focus during 2004

• Regulation of tissue-type plasminogen activator gene expression in vitro:

• t-PA gene regulation in vivo

• Regulation of the plasminogen activator inhibitor type 2 gene (PAI-2)

– Post transcriptional regulation of PAI-2 gene expression

– To assess the role of PAI-2 using THP-1 monocytes

• The regulation of metastatic disease by novel compounds

• Functional effects of the prothrombin G20210A gene polymorphism

• The Role of t-PA in the central nervous system

Recent publications from Dr Medcalfs Laboratory (2002-2004)

1 Yu, H., Maurer, F., and Medcalf R. L. (2002) Plasminogen activator inhibitor-2:a regulator of monocyte proliferation and differentiation. Blood 99:2810-2818.

2 Carter, A.M., Sachchithananthan, M., Stasinopoulos, S., Maurer, F., andMedcalf, R.L (2002). Prothrombin G20210A is a bifunctional genepolymorphism Thromb. Haemost. 87:846-853.

3 Liberatore, G.T., Samson, A., Bladin, C., Schleuning, W.D., and Medcalf, R.L.(2003). Vampire bat salivary plasminogen activator (desmoteplase) – a uniquefibrinolytic enzyme that does not promote neurodegeneration. Stroke 34:537-543.

4 Yu, H., Stasinopoulos, S., Leedman, P, and Medcalf, R.L. (2003). Inherentinstability of plasminogen activator inhibitor type 2 mRNA is regulated bytristetraprolin. J. Biol. Chem. 278:13912-13918.

5 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 humansplicing factor ZNF265 forms a novel fold. J. Biol. Chem. 278:22805-22811.

6 Cakarovski, K, Leung, J., Restall, C., Carin-Carlson, A., Yang, E, Perlmutter,P., Anderson, R., Medcalf, R.L. and Dear, AE. (2004) Novel inhibitors ofurokinase type plasminogen activator and matrix metalloproteinase expressionin metastatic cancer cell lines. Int. J. Cancer 110: 610-616.

Invitations and talks given by Dr Medcalf 2002-2004

1 Invited Speaker, Lorne Genome Conference, Lorne, February 17-21, 2002

2 Invited Speaker and Session Chairman, Combined Biochemistry andMolecular Biology Conference (“ComBio”) 2002, Sydney, September 29- October 3.

3 Invited Speaker, “t-PA: the other side of the coin” Australian Society forNeuroscience, Stroke satellite meeting, Adelaide, February 2003.

Press releases related to research results from Medcalf lab

Monash University www-pso.adm.monash.edu.au/news/Story.asp?ID=856&SortType=1

Scientific American:www.sciam.com/article.cfm?articleID=000293D5-D911-1E1D-8B3B809EC588EEDF

American Heart associationwww.americanheart.org/presenter.jhtml?identifier=3007500www.cnn.com/2003/HEALTH/conditions/01/09/stroke.bat.treatment

Dr Stan Stasinopoulos

Desmodus Rotundus

Desmodus Rotundus


Research report

Platelet and leukocyte biology and Platelet signalling laboratories

Introduction

The organs and tissues that make up the human body rely on a continualsupply of blood, which is circulated throughout the body via a complex seriesof arteries, veins, and capillaries. This blood not only carries vital oxygen andnutrients to the tissues, as well as removing carbon dioxide and other wasteproducts, but it is also a vehicle for various blood cells, including platelets andleukocytes, which play key roles in the maintenance of blood vessel integrity.The main focus of the Platelet and Leukocyte Biology Laboratories is to identifythe mechanisms regulating blood vessel injury and repair, with particularemphasis on the role of blood platelets and leukocytes in these processes.

Platelets are small blood cells produced by megakaryocytes. In the event of bloodvessel injury, these tiny blood cells are rapidly recruited to the area of damage,where they spread and form a stable thrombus (blood clot).

This response must be tightly regulated to ensure that the formation of a bloodclot is of sufficient size to seal off the damaged area, preventing blood loss, whilstnot disrupting blood flow to vital organs by causing vessel occlusion.

Unfortunately, the consequences of abnormal platelet regulation are seen all toofrequently in the clinical setting, with the incidence of cardiovascular relateddiseases such as heart attack and stroke, remaining some of the major causesof death in the western world today.

Therefore, it is important to gain a comprehensive understanding of the processesperformed by platelets that participate in vessel wall maintenance, as thisknowledge may have important implications for the future development of novelantithrombotic strategies.

Leukocytes are nucleated white blood cells (WBCs) that are formed from stemcells in the bone marrow, and defend the body against infecting organisms andforeign agents. The interaction of these WBCs with platelets at sites of vascularinjury has been found to play a critical role in the inflammatory responsesassociated with vascular diseases such as atherosclerosis.

The recruitment of leukocytes (in particular monocytes) to vessels is a key step inthe initiation of atherosclerosis, and circulating activated platelets and platelet-monocyte aggregates have also been shown to promote the formation of theatherosclerotic lesion. Monocyte-platelet interaction has also been associated withvascular lesions that re-develop as a consequence of mechanical injury fromballoon angioplasty and stenting, which are both currently used for the treatmentof vascular disease and vessel occlusion. Therefore, a better understanding of theleukocyte-platelet interaction, and the consequence of this interaction to vesselmaintenance, is fundamental in order to better understand the progression ofvascular disease.

Laboratory members

Head: Associate Professor Shaun Jackson

Platelet and Leukocyte Biology

Associate Professor Shaun Jackson(Laboratory Director, NHMRC

Principal Research Fellow)

Dr Yuping Yuan

Dr Warwick Nesbitt

Dr Sue Cranmer

Dr Varuni Kanagasundarum

Dr Pierre Mangin

Dr Suhasini Kulkarni

Dr Cindy Yap

Dr Kevin Woollard

Ms Teresa Domagala

Ms Inna Pikovski

Mr Isaac Goncalves

Ms Mhairi Maxwell

Ms Thanae Georgakopoulos

Mr Mark Frazzetto

Mr Denison Chang

Ms Kate Fernandez

MS Hilary Hoare

Platelet Signalling

Dr Simone M Schoenwaelder(Head, NHMRC RD Wright and

Monash University Logan Fellow)

Ms Akiko Ono

Ms Joanna Lim

Mr Shannon Turnbull

Ms Karen Boniface

Ms Megha Mulchandani

Ms Carolina Cavez

Mr Swaroop Manjunath

Dr Karen Anderson(Head, NHMRC RD Wright Fellow)

Dr Steve McMaugh

Ms Penelope Ball


Areas of research focus during 2002–2004

Signal transduction and platelet function

Calcium Flux and Platelet Activation

A major focus of the group is the investigation of calcium signalling mechanismsgoverning platelet adhesion and activation at the surface of thrombogenic proteinmatrices. We have developed a number of microscopy/imaging based assays thatenable us to quantitatively measure changes in platelet calcium concentration withinflowing blood. This advance in platelet calcium imaging has enabled us to elucidatedistinct signalling mechanisms responsible for the activation of the major plateletintegrin αIIbβ3, during platelet adhesion. These studies have demonstrated that theefficiency by which calcium signals are propagated within platelet aggregates playsan important role in dictating the rate and extent of thrombus growth.

Nesbitt WS, et al, J Biol Chem. 2002; 277(4):2965-72 Nesbitt, W.S., et al. 2003; J. Cell Biol. 16 0(7): 1151-1161.Commentary: LeBrasseur N, 2003 ; J. Cell Biol., 160:980Commentary: Schubert C, 2003; Nature Med., News & Views, 9:511

Regulation of platelet-fibrinogen interactions

Fibrinogen is an important adhesive protein involved in platelet function.Platelets bind to fibrinogen during blood clot formation, via the major plateletintegrin αIIbβ3. Studies in our laboratory have investigated the signallingmechanisms that regulate platelet-fibrinogen interactions. In recent work, usingmice deficient in the enzyme phospholipase Cγ2 (PLCγ2), we have identifiedan important role for this enzyme in integrin αIIbβ3-dependent calcium flux,necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore,our studies establish an important cooperative signalling role for PLCγ2 and thenucleotide ADP in regulating platelet adhesion efficiency on fibrinogen.

Goncalves I and Hughan SC, et al. 2003; J. Biol. Chem., 278(37):34812-34822.

PI 3-kinase signalling and platelet function

The ability of platelets to adhere to injured vessels under conditions of blood flowis dependent on two major platelet surface receptors, however the mechanismsby which they trigger platelet activation remain poorly defined. Using state-of-the-art technology developed in the laboratory, we have examined the role ofa ubiquitous enzyme, known as phosphoinositide 3-kinase (PI 3-kinase), inregulating platelet activation. Our studies have uncovered a novel role forPI 3-kinase in regulating platelet activation under flowing conditions, anddemonstrated that inhibiting the action of this enzyme prevents platelets frombecoming activated normally. This finding has formed the basis for thedevelopment of a new class of anti-clotting drugs which are currentlyunder development in Victoria.

Yap CL, et al, 2002; Blood, 99(1):151-8.Anderson and Jackson SP, 2003; Int J Biochem Cell Biol. 35(7):1028-1033.Jackson SP and Schoenwaelder SM, 2003; Nat Rev Drug Disc, 2(10):775-89.Jackson SP, Yap CL, Anderson KE. 2004; Biochem Soc Trans. 32(Pt 2):387-92.

Ras Family small GTPases and platelet function

Members of the Ras family of small G-proteins (GTPases), including RhoA andRap1b have been implicated in regulating the adhesion of many cell types,through their ability to modulate integrin receptors. A major area of researchfocus for the platelet signalling group has been to examine a potential role forthese enzymes in platelet integrin function. Our studies have demonstrated forthe first time that the activation of RhoA, downstream of integrin αIIbβ3, isresponsible for maintaining stable adhesion of platelets to vessel wall proteins,particularly under conditions of blood flow. These studies have found that RhoAachieves this regulation of adhesion through stabilising and sustaining theintegrin/ligand bonds, rather than regulating the ability of the platelets to stick tovessel wall proteins. In further ongoing studies, we are investigating preliminaryevidence demonstrating the existence of distinct yet cooperative roles for bothRhoA, and another Ras family member, Rap1b, in regulating integrin αIIbβ3adhesive function.

Schoenwaelder SM, 2002; J Biol Chem. 2002 277(17):14738-46. Chrzanowska-Wodnicka M et al, 2003; Blood, 102(11):773a [abstract].

Associate Professor Shaun Jackson

From left: Sacsha Hughan, Akiko Ono, JoannaLim, Cenk Suphioglu, Simone Schoenwaelder,

Sharelle Sturgeon, Siew Mei Chan.

Akiko Ono


Platelet morphology

Within the normal circulation, platelets exist in a resting flat, discoid morphology.However, in the event of blood vessel injury, platelets undergo a dramatic‘activation’ process that involves rapid morphological changes, becomingspherical and extending membrane projections called filopodia. These changesalter the dynamics of platelet behaviour, potentially influencing platelet-plateletinteractions, as well as platelet-vessel wall interactions. We are investigating thesignalling mechanisms involved in platelet morphological change using variouspharmalogical inhibitors in combination with physiological in vitro flow basedassays developed in our lab. We are also undertaking experiments involvingintravital microscopy techniques, which allow us to image thrombus formationoccurring in vivo in real-time. These studies will help to identify the functionalsignificance of platelet morphological changes during the formation of blood clots.

Dopheide SM, 2002; Blood, 99(1):159-67.

Soluble P-selectin in cell signalling

In human plasma, the levels of soluble P-selectin (sP-selectin - a soluble form ofthe adhesive protein P-selectin) is suggested to reflect the activation of bothplatelets and/or endothelial cells. In normal human plasma, sP-selectin is presentat relatively low levels (30-60 ng/ml). However, in patients with vascular disorders,such as peripheral vascular disease, unstable angina, postangioplasty restenosis,diabetes and those with hypertension and hyperlipidemia, levels of sP-selectin areincreased ranging from 120-200 ng/ml with extreme values of 1000 ng/ml. Whilethe significance of this increase in humans has not been determined, previouswork has shown that an increased level of sP-selectin in mice is associated with aprocoagulant state in these animals. Plasma from mice with elevated sP-selectinclots faster than plasma from wildtype mice and more fibrin is deposited onplatelet thrombi formed in a perfusion chamber. Whether the findings in mice canbe extrapolated to the human situation requires further investigation. We areinvestigating the consequences of sP-selectin on neutrophil activation andinteraction with platelet monolayers, during flow mediated adhesion. The overallrationale of the project being to determine the consequences of sP-selectin in vivoand ultimately determining whether modulating sP-selectin in vivo with novelcompounds would be beneficial in vascular disease.

Regulation of platelet surface reactivity

Following their initial adhesion to the injured vessel, activated platelets provide ahighly reactive surface for the recruitment of additional platelets, leading to plateletaggregation. While much is known regarding the factors promoting platelet surfacereactivity, the mechanism(s) by which platelet surface reactivity is downregulatedremain poorly understood. Using confocal-based assay techniques that allow thevisualisation of calcium dynamics in platelets adhering to vessel wall substratesunder physiologically relevant perfusion conditions, the laboratory has identified a

novel role for calcium and the calcium-dependent clotting factor, FXIII, in regulatingthe adhesive function of platelets. This novel mechanism appears to limit the sizeof thrombi at sites of vascular injury. This mechanism may be important in theprevention of vaso-occlusive clot formation upon serious vascular trauma.

Kulkarni S and Jackson SP. 2004; J Biol Chem. 279(29):30697-706, 2004.

Platelet adhesion receptors studies

A Role for the GPIb/V/IX -Filamin Interaction

The platelet glycoprotein (GP)Ib/V/IX receptor complex plays a major role inpromoting platelet adhesion and thrombosis, and as such, has become a majortarget for the development of novel anti-clotting drugs. The importance ofGPIb/V/IX in the clotting process is highlighted by the congenital bleeding disorder,Bernard-Soulier syndrome. Over the last several years, a major focus of ourlaboratory has been to investigate the association between GPIb/V/IX and theplatelet membrane scaffolding (cytoskeleton). Our most recent findings hasdemonstrated that the interaction between GPIb/V/IX and the cytoskeletal-associated protein, filamin-1, regulates the ability of platelets to maintain adhesionto blood vessels under high shear conditions. We believe this may be an importantmechanism that regulates platelet adhesion and normal blood clotting.

Mhairi Maxwell and Yu (Joy) Yao

Dr Suhasini Kulkarni


We are currently investigating the role of specific structural domains in theinteraction of this receptor with intracellular structural and signalling proteins.These studies will help to further define receptor interactions that regulate plateletadhesion and activation and contribute to our overall understanding of theprocesses that lead to normal (haemostasis) and pathological (thrombosis) bloodclot formation.

Williamson D, et al, 2002; J Biol Chem. 2002 Jan 18;277(3):2151-9.Perrault CP, et al, 2003; Blood, 101:3477-3484.Cranmer SL and Jackson SP, 2003; Blood [editorial], 102:1937.Cranmer SL, et al, 2004; Biochem J. Mangin P, et al, 2004; Blood. 104(2):420-7, 2004.

The importance of collagen and its receptors in supporting blood clot formation

The sudden rupture of atherosclerotic plaques can precipitate life-threateningdiseases such as heart attack and stroke. It is thought that the increasedconcentration of fibrillar collagens, in particular types I and III, in these advancedatherosclerotic lesions contribute significantly to the increased reactivity of theseplaques. Despite the potential importance of collagens in regulating the processof blood clot formation, there is still limited insight into their role in the body. Thereare a large number of potential receptors for collagens on the platelet surface,however the GPVI receptor was recently shown to play a major role in

supporting blood clot formation on collagen surfaces. This receptor is onlyexpressed in the presence of the FcR γchain, which forms the signalling_ elementof the GPVI/FcR γ complex. Using mice deficient in the expression of theGPVI/FcR γ complex (FcR γknockout mice), we are in the-chain process ofinvestigating the importance of the GPVI/FcR γ complex in regulating theformation of occlusive blood clots. To do this, we have established two in vivomouse techniques which allow the examination of the importance of platelet-collagen interactions in blood clot formation within large arteries. Our studies havefound that FcR ≥knockout mice are able to-chain form clots to the same rate andextent as normal mice in both in vivo models. This suggests that the collagenreceptor, GPVI, is not absolutely critical for the formation of occlusive blood clotsin the large vessels, and may not be the best target for development of anti-clotting compounds. These studies are ongoing.

Platelet-Leukocyte biology

Monocyte-platelet interactions

The recruitment of leukocytes (Monocytes) to vessels is a key step in the initiationof atherosclerosis. Studies in the laboratory are investigating the regulation of theprimary integrins and adhesion molecules that mediate the firm adhesion ofmonocytes under flow conditions. Thus far, our studies have identified a novel rolefor the αXβ2 integrin in monocyte interaction with platelets under flow. The role forthis integrin, in co-operation with others, to regulate the interaction of monocyteswith activated platelets under flow conditions may have important implications inthe regulation of inflammatory responses of monocytes in vascular injury.Examination of the signalling mechanisms involved in the regulation of αM andαXβ2 integrins during monocyte-platelet interactions under flow conditions is a keyaspect to understanding the regulation of monocyte interactions duringinflammatory response.

In vivo study of platelet biology

Models to examine arterial thrombosis

Despite great advances in treatment, cardiovascular disease continues to be theleading cause of death in the world today. Heart attack and stroke, caused byplaque rupture and occlusive thrombi (blood clots) in the major blood vesselssupplying the heart and brain, contribute to the majority of deaths. Thus researchinto the mechanisms by which thrombi form, and ultimately the development ofnovel treatments, is ongoing. The advent of genetically modified mice hascontributed greatly to such research in recent years. By being able to remove aprotein from the animal’s genome, we can then investigate how important thatprotein is in the formation of thrombi in disease states. To investigate the role ofsuch proteins, we must be able to mimic these diseases states in animals.

Akiko Ono and Joanna Lim

Dr Simone Schoenwaelder

Dr Sue Cranmer


To this end, we have successfully developed a number of models of arterialthrombosis in our laboratory.

The folts and electrolytic models are two of the most common and wellcharacterised animal models of arterial thrombosis in cardiovascular research.Histologically, the thrombi produced in each model contain fibrin, platelets andred blood cells. The thrombi formed in the Folts model are primarily composed ofplatelets whilst the thrombi formed in the electrolytic model are equally composedof fibrin and platelets. Thus, in addition to examining thrombus formation ingenetically modified mice, these models are ideal for the pre-clinical assessment ofnovel anti-thrombotic drugs. The nature of the thrombus formed in the Folts modelmeans that this model is best suited to the study of novel anti-platelet drugs,where as the electrolytic model is suited to the study of both anti-platelet andfibrinolytic drugs.

Key collaborations held by the platelet and leukocyte biologyand platelet signalling laboratories [2002-2004]

Our research groups continue to establish extensive collaborative researchnetworks, both nationally and internationally. Our collaborators include:

Professor Peter Shepherd, UCL, London UK Drs Len Stephens and Phillip Hawkins, Babraham Institute,Cambridge UKProfessor Keith Burridge, UNC, NC USADr M. Wodnicka, Dr Gil White, UNC USADr Francois Lanza, INSERM, Strasbourg, FranceProfessor Christian Gachet, INSERM, Strasbourg, FranceDr Zaverrio Ruggeri, Scripps, San Dieo, USA

Other selected publications from the platelet and leukocytebiology and platelet signalling laboratories [2002-2004]

1. Jackson SP, Nesbitt WS and Kulkarni S. (2003) Signalling events underlyingthrombus formation. J Thromb Haemost., 1(7): 1602-1612.

2. Guiliano S, Nesbitt WS, Rooney M and Jackson SP (2003) Bidirectionalintegrin alpha IIb beta 3 signalling regulating platelet adhesion under flow:contribution of protein kinase C. Biochem J, 372(Pt 1):163-172.

3. Kulkarni S, Nesbitt WS, Dopheide SM, Hughan SC, Harper IS, Jackson SP.Techniques to examine platelet adhesive interactions under flow. Methods MolBiol. 272:165-86, 2004.

4. Abbott BM and Thompson PE. (2004) PDE2 inhibition by the PI3-kinaseinhibitor LY294002 and analogues. Bioorg Med Chem lett, 14(11):2847-51.

5. Pimanda JE, Ganderton T, Maekawa A, Yap C, Lawler J, Kershaw G,Chesterman CN and Hogg PJ. (2204) Role of thrombospondin-1 in control ofvon Willebrand factor multimer size in mice. J Biol. Chem, 279(20):21439-48.

Invitations and talks presented by the platelet and leukocytebiology and platelet signalling laboratories [2002-2004]

Invited symposia chair

2002 S.P. Jackson – XIIth International Vascular Biology Meeting. Karuizawa, Japan

2003 S.M. Schoenwaelder – ISTH XIX Congress, Birmingham, UK

2004 S.M. Schoenwaelder, HAA (HSANZ/ANZSBT/ASTH) 2004 AnnualScientific Meeting, Melbourne, Victoria

2004 S.P. Jackson, XIXth European Platelet Meeting, Bad Bruckenau, Germany

Dr Pierre Mangin

Dr Sharelle Sturgeon and Mhairi Maxwell

Dr Yuping Yuan and Yu (Joy) Yao


Invited seminars/presentations

2002 S. Cranmer, International Society of Biorheology and International Society of Clinical Haemorheology Congress, Antalya, Turkey

2002 S.M. Schoenwaelder, Dept. Cell Biol. & Anat., University of NorthCarolina at Chapel Hill, USA

2002 S.P. Jackson, Ludwig Institute, London, UK

2002 S.P. Jackson, Plenary Lecture, VIIth Annual Haemostasis RheologyMeeting. Tokyo, Japan

2002 S.P. Jackson, XIIth International Vascular Biology Meeting. Karuizawa, Japan

2002 S.P. Jackson, Gordon Conference, Haemostasis, Maine, USA

2002 S.P. Jackson, The American Society of Hematology, 44th AnnualNational, Convention, Philadelphia, USA

2002 S.P. Jackson, Millenium Pharmaceuticals, Cambridge, Massachusetts,USA

2003 S.P. Jackson, State-of-the-Art Lecture, The International Society onThrombosis & Hemostasis, XIX Congress, Birmingham, England

2003 S.P. Jackson, Babraham Institute, Cambridge, UK

2003 S.M. Schoenwaelder, ISTH XIX Congress, Birmingham, UK

2003 S. Cranmer, Department of Physiology, University of Birmingham, UK

2004 S.M. Schoenwaelder, XIXth European Platelet Meeting, Bad Bruckenau,Germany.

2004 S.P. Jackson, Inaugural International Haemostasis Symposium,Wurzburg, Germany

2004 S.P. Jackson, Haematology Society of Australia, Annual ScientificMeeting, Melbourne

Erik Westein

Biorheology workstation

Mhairi Maxwell and Mark Frazetto


Research report

Clinical Haematology and Bone Marrow Transplantation

Bone marrow transplantation

The bone marrow transplant program has had a busy year with activity inclinical research and translational research. The primary goal of ourtranslational, laboratory-based research is the understanding of the graft-versus-leukaemia phenomenon and the development of laboratory techniquesthat attempt to harness this phenomenon to increase the chance of curingpatients with leukaemia and, at the same time, potentially decreasing theside-effects and risks of transplantation. Our clinical research activityincludes involvement in national and international trials of new treatmentsfor malignant diseases such as leukaemia, lymphoma and myeloma as wellconducting trials that we have designed ourselves.

The bone marrow transplant laboratory has three senior scientists (Gerri Bollard,Mirek Kapuscinski and Ann Stewart), one research scientist (Mingus Rose), twoPhD students (Kate Ward and Tamara Etto) and two BSc Honours students (OanhNguyen and Christina Ioannidis). The laboratory is headed by Associate ProfessorAnthony Schwarer. Gerri Bollard runs the stem cell processing andcryopreservation section of the laboratory, manipulating the peripheral blood stemcells and bone marrow stem cells from patients and donors. Mirek Kapuscinskiassists Gerri Bollard on the clinical side as well as conducting his own researchinto chimerism post transplant. He also supervised Christina Ioannidis. Christinainvestigated the association of vitamin D receptor polymorphisms with posttransplant complications such as graft-versus-host disease.

Ann Stewart has been collaborating with Joanna Paddle-Ledinek of the Burns Unitto generate large numbers of keratinocytes that may help develop techniques thatwill allow us to remove immune cells from the donor stem cells that are capableof causing graft-versus-host disease while retaining those immune cells that areimportant for the graft-versus-leukaemia phenomenon. In addition, Dr Stewartsupervised Oanh Nguyen who developed a technique using insect cells toproduce large amounts of a viral protein (CMV pp65) which can be used togenerate immune cells that can prevent infection with CMV post transplant.This technique can be adapted to allow us to develop specific immune cellsagainst many infectious organisms.

Mingus Rose has been working with natural killer (NK) cells. Recent work byothers suggested that NK cells play a crucial role in generating a graft-versus-leukaemia effect in a special transplant scenario termed haploidenticaltransplantation where the donor and patient are only half-matched for the tissuetyping antigens (HLA). This is an exciting new transplant technique that has thepotential to change the way transplantation is performed throughout the world.

Christina Ioannides and Oanh Nguyen, the BSc Honours students in the lab,both obtained first class (H1) honours for their work in 2004.

The clinical team of the bone marrow transplant program consists of AssociateProfessor Anthony P. Schwarer, Associate Professor Andrew Spencer andDr Sharon Avery. Associate Professor Spencer runs the myeloma researchprogram which is described in a separate section of this report. The clinical teamis involved in many clinical trials conducted by international groups (eg EuropeanOrganisation for the Research and Treatment of Cancer, Centre for InternationalBlood and Marrow Transplantation and Research), and by national groups(eg Australasian Leukaemia and Lymphoma Group, Australian Bone MarrowTransplant Recipient Registry).

Laboratory members

Head: Associate Proffessor Anthony Schwarer

Gerri Bollard,

Mirek Kapuscinski

Ann Stewart

Mingus Rose

Kate Ward, PhD student

Tamara Etto, PhD student

Oanh Nguyen, Honours student

Christina Ioannidis, Honours student


These cooperative trials have focused on new treatment of chronic myeloidleukaemia, non-Hodgkin’s lymphoma, acute myeloid leukaemia, Burkitt’sleukaemia/lymphoma and multiple myeloma. Such trials are important in helpingset a new standard of care for these diseases.

The bone marrow transplant program has also conducted a number of in-housetrials including a technique for reactivating the thymus to help improve immunerecovery after transplantation as well as a trial pioneering haploidenticaltransplantation in Australia. This latter trial is based on the work of a group ofItalian researchers who have developed a technique that has the promise to finddonors for patients who need transplantation but do not have tissue-typematched donors. This new technique uses family member donors who are onlyhalf matched to generate a graft with a very potent graft-versus-leukaemia effect.Preliminary results from the Italian researchers show a cure rate five times that ofstandard transplantation. We are the first group in Australia to set up such aprogram.

Our clinical trials would not be possible without Jenny Muirhead and RosemaryMcGinnes, our two tireless data managers.

Laboratory research projects

• Evaluation of peripheral blood stem cell engraftment in non-myeloablativetransplant recipients

• Development of sensitive molecular detection methods for haemopoieticchimerism measurement

• Vitamin D receptor gene polymorphisms as a predictive marker innon-myeloablative allogeneic stem cell transplantation

• Epigenetic regulation of alkylating drug sensitivity in malignant lymphoid cells

• Decreasing graft-versus-host disease: The use of dendritic cells andkeratinocytes to stimulate potential GVHD-causing T cells to allow depletionof these cells while preserving the T cells responsible for the graft-versus-leukaemia phenomenon and immune recovery after haematopoietic stemcell transplantation

• Characterisation of CD34+ derived dendritic cells for the use in the generationof CMV specific T cells

• Generation of the polymorphic minor histocompatibility antigen, HA-1, for usein immunotherapy

• Production and purification of human cytomegalovirus (HCMV) recombinantprotein IE1.pp65 using the baculovirus system to generate a multiple anti-HCMV T cell response

Database contributions

• Prospective study of allogeneic and autologous bone marrow transplantrecipients (Australian Bone Marrow Transplant Recipient Registry)

• Prospective study of allogeneic and autologous bone marrow transplant forpatients with life threatening haematological disorders (Centre for InternationalBlood and Marrow Transplant Research)

• Prospective study of matched unrelated donor transplants (Australian BoneMarrow Donor Registry)


Research report

Myeloma Research Group

Laboratory members

Head: Associate Proffessor Andrew Spencer

Tiffany Khong

Janelle Sharkey

Sung-Lin Yeh

Rita Broner

Karly Koutrouvelis

The MRG was established in 2001 and is an expanding translational andclinical research entity based at Bayside Health’s Alfred campus but withaffiliations to numerous other researchers both nationally and internationally.The MRG is responsible for the development, resourcing and implementationof numerous multicentre Phase II and Phase III trials evaluating noveltherapeutic approaches to multiple myeloma (MM), lymphoma andrelated haematological malignancies.

In 2004 the MRG data management team administered 16 clinical trials includingthe centralised data-management for 3 multicentre trials, the largest being theAustralasian Leukaemia and Lymphoma Group (ALLG) MM6 trial. MM6 is thelargest clinical trial in MM ever undertaken in Australia and is already attractinginternational attention with invited presentations of interim data planned for theupcoming 10th International Myeloma Workshop and the Annual ScientificMeeting of the European Haematology Association (EHA).

In parallel with clinical trial activity the MRG has developed the infrastructure toenable the rapid pre-clinical evaluation of novel compounds for the treatmentof MM. An essential element of this infrastructure was the establishment in 2004of 2 murine tumour implant models of MM including an entirely novel xenograftmodel using human KMS-11 MM tumour cells. This success attracted theattention of the Novartis pre-clinical/Phase I drug development team based at theNovartis Institute of Biomedical Research (NIBR), New Jersey, arguably the largestcancer drug discovery organisation in the world. Representatives of the NIBRvisited the MRG late 2004 and based on that meeting the MRG is now a‘preferred’ collaborating site for pre-clinical and early phase clinical anti-MM drugdevelopment. Collaborative pre-clinical work is planned to commence in March2005 with the first collaborative Phase I/II trials planned for third quarter 2005.Ongoing translational activities in 2004 included the further evaluation of severalnovel potential therapeutics including TRAIL, PKC412, flavpiridol and azacitiddine.

Laboratory projects

• Establishment of the 5T33 murine multiple myeloma model

• Establishment of a KMS11 NOD-SCID human-murine multiple myelomaxenograft

• Mechanisms of action and resistance to TRAIL in multiple myeloma

• In vitro and in vivo evaluation of the preclinical efficacy of azacitidine for thetreatment multiple myeloma

• Characterisation of a novel p53-dependent p21-mediated drug resistancemechanism in multiple myeloma

• In vitro and in vivo evaluation of the preclinical efficacy of PKC412 for thetreatment multiple myeloma

• In vitro and in vivo evaluation of the preclinical efficacy of flavopiridol forthe treatment multiple myeloma


Eastern Clinical Research Unit (ECRU)

Clinical research – Trials division

The Eastern Clinical Research Unit (ECRU) is an initiative of the Monash UniversityDepartment of Medicine and is based at Box Hill, Maroondah and more recentlythe Alfred Hospitals in Melbourne. ECRU has been involved in clinical trials since1996 and has consolidated its position as the premier clinical research facility inAustralia. In 2003, new trials in anaesthetics and surgery have been added to theother disciplines involved in clinical studies with ECRU. ECRU employs 35 nurses,and has more than 80 active clinical programs. A very large number of patientsare regularly screened for suitability to enter these studies. ECRU has enrolledmore patients in many of its studies than other Australasian sites. This reputationis rapidly growing and will ensure the ongoing success of the clinical researchprogram in our hospitals.

It is pleasing to note that several drugs that have been studied by ECRUresearchers have made their way to the clinic. One of these drugs is a new bloodthinner that was approved in 2003 by the Federal Government for use inprevention of blood clots in patients undergoing hip and knee surgery. Thediabetes research group continues to expand its clinical activities with severalstudies focusing on new forms of insulin and novel ways of delivering thisimportant drug. The people who have volunteered with ECRU have benefitedfrom advanced health care for themselves and the knowledge that theirinvolvement will help others.

It is now the largest multidisciplinary Clinical Trial Unit in Australia. We havecombined essential logistic resources with an enthusiastic team with the skills tomanage clinical trials in most disciplines. Ready access to the resources and vastnetwork of contacts of Monash University and Eastern Health provide invaluableinput for associates and interested parties. ECRU has particularly distinguisheditself in its ability to recruit, collect statistical data and retain trial participants,essential components of any successful clinical trial. We seek to deliver qualitytrial results and involve ourselves in projects that make a significant contributionto health care.

Dr Anthony Dear, Deputy Director of ECRU, has established a new activity knownas ECRU biotech. ECRU biotech aims to expand the capabilities of ECRU beyondclinical research to a better understanding of how the drugs work and exert theireffects. This in turn promises to deliver better treatment than currently available.Active contributors to ECRU Biotech include members of the endocrinology andneurology teams at Eastern Health.

Unit Manager, Cheryl Gillzan has worked hard to meet the ever changing needsof ECRU. The department has had major renovations at Box Hill with the creationof offices for each separate discipline, meeting rooms, monitoring space and alarge patient lounge with kitchen facilities. The new facilities are comfortable,attractive and easy to maintain.

ECRU Personnel

Principal Investigators

Box Hill

Professor Hatem Salem (Haem), Ecru Director

Dr Anthony Dear (Haem), Ecru Deputy Director

Dr Paul Coughlin (Haem)

Prof Chris Bladin (Neuro)

Dr Richard Simpson (Endo)

Dr Harvey Newnhan (Endo)

Dr Gary Gordon (Cardiol)

Dr Carol Tong (Endo)

Dr Sylvia Lim-Tio (Endo)

Associate Professor Graham Schmidt (Gasstro and Endo)

Prof Peter Gibson (Gastro)

Dr Mariko Howlett (Gastro)

Dr Sanjay Nandurkar(Gastro)

Associate Professor Joe Mckendrick (Oncol)

Dr Phillip Parente (Oncol)

Dr Paul Fogarty (Resp)

Dr David Beilby (Anaesth)

Dr Tony Chow (Anaesth)

Maroondah

Dr Murray Gerstman (Endo), Maroondah Director

Dr Kam Narayan (Oncol/Haem)

Dr Michael Leyden (Oncol/Haem)

Dr Afif Hadj (Surgical)

Dr Henry Voselis (Haem)

Dr Michael Digby (Haem)

Dr Martin Lew (Haem)

Dr Amanda Gilligan (Neuro)

Left: Hatem Salem, Cheryl Gillzan and Tony Dear


Below is a table showing the number of studies in the different clinical fieldsof ECRU and the number of patients recruited in each area for 2004:

Discipline studies in Patients recruited breakdown progress for 2004

Box Hill ECRUBH Haematology 11 112BH Endocrinology 17 50 BH Respiratory 2 0BH Neurology 15 61 BH Gastroenterology 20 59 BH Anaesthetics/surgery 2 23 BH Oncology 38 72

Maroondah ECRUM Haematology 8 55M Anaesthetics 0 21M Surgery 2M Endocrinology 3 32

Alfred ECRUA Haematology 3

Total 121 485

Study Coordinators

Box Hill Staff

Cheryl Gillzan, Ecru Manager

Joanne Phillips (Diab Ed)

Sue Varley (Endo)

Rowena Stewart (Diab Ed)

Vanessa Viola (Diab Ed)

Prue Robertson (Diab Ed)

Sue Dal Sasso (Endo)

Shanne Kast (Promotion/Diab Ed)

Todd Christina (Endo)

Miniver Siah (Gastro)

Gillian Syres (Gastro)

Vathy Nagalingam (Gastro)

Anna Harvey (Gastro)

Margaret Abbott (Gastro)

Lesley Poulton(Haem)

Belinda Patford (Haem)

Jacinta Macormack (Haem)

Anne Buckland (Neuro)

Zofia Ross(Neuro)

Melissa Wright (Neuro)

Alan Mcmanus (Oncol)

Carmela Corfield (Oncol)

Janelle Peppin (Oncol)

Helen Ficatas (Oncol)

Fiona Page (Oncol)

Pamela Badawy (Oncol)

Irina Shinkarsky (Resp)

Maria Di Staso (Resp)

Maroondah Hospital Staff

Debra Mccall

Andriana Chronopolous

Maree Farley

Alfred Hospital Staff

Prue Freeman (Haem)

Left: Hatem Salem, Paul Coughlin, LesleyPoulton and Gil Syres

Dr Richard Simpson and Joanne Phillips


Biotechnology Research Division

Laborartory members

Head: Dr Anthony Dear

Dr Hong Bin Liu

Dr Yunshan Hu

Dr Richard Simpson

ECRU Biotechnology (ECRU Biotech) was conceived in response to arecognized need to expand the activities of ECRU beyond the clinicalsphere and into the fields of basic and translational research.

Modern biotechnology programmes require a broad range of research activitiesspanning evaluation of existing agents with potential application to novel clinicalscenarios to design, development and testing of novel agents in in vitro and invivo models of disease.

ECRU Biotech has embraced the need for a wide range of biotechnologyresearch activities, reflected in its current research program. Currently ECRUBiotech is involved in the evaluation of compounds, in vitro and in vivo, currentlyused clinically in the treatment diabetes with a view to determining their potentialfor vascular protection. In addition ECRU biotech has established, with itscollaborators, several in vivo systems for assessment of novel and existingagents in the setting of disease states ranging from metastatic cancer tovascular disease.

ECRU Biotech is supported by Monash University, individual principleinvestigators from ECRU’s clinical operations, has strong links with thepharmaceutical industry, and is engaged in seeking funding from both thepublic and private sectors in order to further it research activities.

Current ECRU Biotechnology research program 2002–2004

Projects

1. Effects of Thiazolidinediones on Plasminogen Activator Inhibitor Type 1Expression in Models of the Metabolic Syndrome. Dr Richard Simpson (Glaxo Smith Kline/ Takeda Industries/ Novo Nordisk)

2. Novel Small Molecule Treatment for The Prevention of Abdominal AorticAneurism Formation and Progression. Associate Professor Rob Widdop (Dept Pharmacology, Monash University)

3. Protease Inhibition in Metastatic Malignancy and Bone Disease. Dr Robin Anderson (Peter Mac Callum Cancer Institute), Associate ProfessorMatthew Gillespie (St Vincents Institute of Medical Research), AssociateProfessor Andrew Spencer (Alfred Hospital)

4. Histone Deacetylase Inhibitors as Novel Treatments in Acute Leukaemia,Myelodysplastic Syndrome and Lymphoma. Dr Ricky Johnstone (Peter Mac Callum Cancer Institute), Dr Richard Lock(Children’s Medical Research Institute, Sydney Australia) and Professor GLeone (Gemmelli Hospital, Rome, Italy)

5. In vitro and In vivo Assessment of Novel Treatments for NeointimalHyperplasia in the Setting of Bypass Graft Failure and Stent Implantation.Associate Professor Rob Widdop (Dept Pharmacology Monash University),Professor Michael Grigg (Dept Surgery Box Hill Hospital)

6. Chemical Synthesis of Novel Inhibitors of Protease and Histone Deacetylase Activity. Associate Professor Patrick Perlmutter (Monash University, Dept of Chemistry)

7. Molecular Assessment of Gene Profiles Responsible For DiseasePredisposition and Drug Metabolism. Dr Keith Byron (Gribbles Molecular Sciences).

Dr Richard Simpson, Dr Yunshan Hu, Dr Anthony Dear and Dr Hong Bin Liu


Research Publications Arising from ECRU Biotechnology Division Projects

1. Yu Ming, Dear AE, and Grigg M.J. “Homocysteine: An Aetiological Factor inPeripheral Vascular Disease”. Australian and New Zealand Journal of Surgery72:668-671 (2002).

2. Peart MJ, Dear AE, Tainton KM, Ruefli AA, Sedelies K, Trapani JA, Smyth MJ,Johnstone RW. “Novel Mechanisms of Apoptosis Induced by HistoneDeacetylase Inhibitors”. Cancer Res. Aug 1;63(15):4460-71 (2003).

3. Cakarovski K, Leung J, Restall C, Carin-Carlsson A, Yang E, Perlmutter P,Anderson R, Medcalf RL and Dear AE. “Novel Inhibitors or Urokinase TypePlasminogen Activator and Matrix Metalloproteinase Expression and Activityin Metastatic Cancer Cell Lines”. Int.J.Cancer 110:610-616 (2004).

4. Liu HB, Dear AE, Medcalf RL and Simpson RW. “Rosiglitazone andPioglitazone-Mediated Inhibition of Plasmonogen Activator Inhibitor Type 1(PAI-1) Expression in C11-STH Human Vascular Endothelial Cells. A PossibleMechanism for Thiazolidinedione-Mediated Attentuation of Atherosclerosis”Cardiovascular Pathology, 13:3 Suppl 1:99-100 (2004).

Patents

1. “An agent for Plasminogen Activator and Matrix Metalloproteinase associatedconditions and method of use”. Australian Provisional Patent IRN 602223Inventors, Dear AE, and Medcalf RL. Refiled September 23rd 2003 on Behalfof Monash University.

2. “Compositions and Methods for Treating Aneurysm”. Australian ProvisionalPatent # IRN 679911 Inventor: Dear AE. refiled 24th December 2003 on Behalfof Monash University.


State-of-the-art facilities

Imaging at the ACBD

The ACBD under the auspices of Monash Micro-Imaging (MMI) has establisheda state-of-the-art micro-imaging and biorheology laboratory with the purpose ofdeveloping novel imaging modalities and applications aimed at investigating thecellular and molecular mechanisms of platelet thrombosis and cardiovasculardisease. The overarching aim of the laboratory is to develop an internationalreputation in the development and application of cutting edge imaging modalitiesapplied to both live cell and intra-vital techniques, with a particular focus oncardiovascular biology and related areas. The great strength of this facility is theintegration of basic biology with the development of new imaging techniques.This boutique approach to imaging development is unlike the traditional ‘Turnkey’centralised approach used by many imaging facilities, which tend to operate ata distance from the day-to-day requirements of the biological researcher.

The current ACBD imaging laboratory consists of:

• Three dedicated bio-rheology imaging workstations comprised of invertedwide-field instruments with a range of both long working distance and shortworking distance high numerical aperture objectives, MTI-DAGE analogueCCD cameras, analogue video acquisition and an integrated MCID digitalacquisition/analysis workstation.

• Confocal suite, consisting of a Leica TCS-SP and both invert(DM-IRBE) andupright dedicated microscopes.

• Two offline imaging workstation for analysis along with a range of softwareapplications.

• Analogue -MTI-Gen-II-Sys image intensifier

• Leica MPV-Combi

• A range of specialist incubation chambers, platforms and hoods for both livecell and intravital microscopy applications.

Dr Warwick Nesbitt


We are in the process of extending upon our existing micro-imaging facilities withinthe ACBD by developing a novel multi-mode imaging system that combines recentadvances in objective-type Total Internal Reflection Fluorescence Microscopy(TIRFM) and high speed epi-fluorescence techniques. Dr Warwick Nesbitt at theACBD, in association with Olympus Australia and SDR Clinical Services has madesignificant progress in the development of this imaging system, such that aworking system was established in July of 2004. The unique properties of theTIRF imaging technique will allow us to examine many aspects of plateletmembrane adhesion and signalling processes that would otherwise be beyondthe resolution of existing techniques. At present work is underway to develop asingle molecule detection capability using this unique device that will allow us todirectly image and monitor the adhesion dynamics of the platelet intergin αIIbβ3in live cells.

State-of-the-art technologies in the Fibrinolysis and Gene Regulation Laboratory

Stereotactic microinjection procedures have been developed to study the roleof fibrinolytic enzymes during neuronal degeneration in the murine brain.Transgenic models are available to allow us to study the expression pattern andregulation of these fibrinolytic enzymes under normal and pathological conditions.Cultures of primary neurons have also been established to complement these invivo approaches and we have developed imaging systems to study in real time,changes in free intracellular calcium levels in these primary neurons followingtreatment with excitoxic injury. We are also culturing human primary brainmicrovascular cells to understand the basis for the damaging role of theseenzymes under conditions of low oxygen (hypoxia).

Dr Erik Westein


Academic news

ACBD prizes and awards 2003 Inaugural Firkin Awards

The Firkin awards were implemented in 2003 by the ACBD in recognition ofProfessor Firkin’s many achievements. These awards were designed toencourage and inspire young Australian scientists and medical practitioners whowish to pursue a career in medical research. The Firkin Awards will be open toi) Medical undergraduates wishing to pursue a career in medical research(BMedSci); ii) Medical graduates wishing to enrol in an MD or PhD program, andiii) Science graduates (BSc, BBioMedSc) entering an Honours or PhD program.

In 2003, recipients of the Inaugural Firkin Awards were:

1. Dr Georgia, Baker Institute

2. Melanie Ivey, Baker Institute

3. Akiko Ono, ACBD

ACBD scholarships and fellowships

Several staff members have received prestigious promotions, including AssociateProfessor Jackson, who has now been promoted to NHMRC Principal ResearchFellow (PRF) and Dr Robert Medcalf, who was awarded a NH&MRC SeniorResearch Fellowship (SRF).

NH&MRC CJ Martin Overseas Training Fellowships

Dr Suhasini Kulkarni was awarded a Partnership CJ Martin overseas postdoctoralFellowship Award by the NH&MRC and NHF. Dr Kulkarni will be travelling to oneof the premier institutes (Babraham) in Cambridge, UK, for a period of two yearsto investigate the role of phosphatidylinositol 3-kinase in inflammation. She willthen return to the Australian Centre for Blood Diseases to continue her work inthe field of cardiovascular medicine examining the processes of thrombosisand inflammation using transgenic mouse models.

The prestigious CJ Martin Overseas postdoctoral fellowships were also awardedto Dr Cindy Yap and Dr Sascha Hughan. Dr Hughan plans to train as a post-doctoral scientist with Dr. Steve Watson in Birmingham, UK, where she hopesto gain further insight into the signal transduction pathways involved in plateletfunction and to better understand the processes of haemostasis and thrombosis.

NH&MRC BioMedical (Dora Lush) postgraduate scholarship

Melinda Missen was awarded an NHMRC Dora Lush Postgraduate ResearchScholarships, of which only 150 are awarded nationwide to PhD applicants. This scholarship will allow Ms Missen to commence PhD studies in the SerpinBiology Laboratory in 2004, under the supervision of Dr Paul Coughlin.

From the left Dr Suhasini Kulkarni and Dr Cindy Yap

Dr Sascha Hughan

Ms Melinda Missen


Arrivals to the ACBDPhil Daniel, PhD

Phil Daniel received his PhD from Lincoln University, New Zealand in 1995, andundertook post doctoral studies in the field of gene regulation in the labratoryof Professor Joel Habener at Massachusetts General Hospital, Boston, USA.He returned to New Zealand in 2000, conducting research into hypothalamicexpression of genes involved in obesity at the Auckland University School ofMedicine until 2003. He is currently investigating factors influencing the expressionof tissue-type plasminogen activator in neurons and endothelial cells, in theLaboratory Fibrinolysis and Gene Regulation Laboratory.

Pierre Mangin, PhD

Dr Mangin completed his PhD studies in June 2003 at the INSERM U311research laboratory, Strasbourg (France), under the guidance of Dr FrancoisLanza. This research group is mainly interested in the biology and pharmacologyof hemostasis and thrombosis. During his PhD studies, Dr Mangin providedinteresting insights into the signalling role of various adhesive receptors onplatelets, in particular the GP Ib-V-IX complex. In 2003, he was awarded theprestigious INSERM/NH&MRC exchange fellowship to perform a post-doctoralstudy in Dr. Shaun Jackson’s research group, where he endeavours to continuehis research in platelet receptor signalling and its regulation of platelet function.

Dr Kevin Woollard

Dr Woollard graduated from Aston University (Birmingham, UK) with a honorsdegree in Human Biology, he then stayed on to complete his PhD in the MolecularBiosciences department studying cellular and molecular mechanisms ofinflammation in disease. In 2003 Dr Woollard was awarded a 3 year internationalresearch fellowship from F.Hoffmann La Roche (Basel,Switzerland) to study at theBaker Heart Research Institute and the ACBD in the lab of Shaun Jackson,investigating P-selectin mediated platelet/leukocyte activation in both thrombosisand vascular inflammatory disease.

Thomas Weiss, MD

Thomas Weiss received his MD from University of Vienna, Austria in 2003.He started his research work in 1999 in the laboratory of Professor Johann Wojtaat the Department of Internal Medicine, Medical University of Vienna, Austria.This research group is mainly interested in atherosclerosis, vascular biology andmolecular cardiology. In 2004 Dr Weiss was rewarded a 2 year Erwin SchroedingerScholarship of the Austrian Science Fund to undertake his research at the ACBD,investigating the role and regulation of tissue-type plasminogen activator in stroke.

Phil Daniel, PhD

Pierre Mangin, PhD

Dr Kevin Woollard

Thomas Weiss, MD


Departures In 2002-2004, the Department said farewell to a number of staff members,including our Laboratory Manager Mr Ron Morris, and several recent doctoralgraduates – Dr Sascha Hughan, Dr Suhasini Kullarni, Dr Simon Guiliano andDr Nayna Mistry.

Mr Ron Morris has retired from his long standing position asDepartment/Laboratory manager.

Dr Sascha Hughan left the department to pursue postdoctoral studies withDr Steve Watson in Birmingham, UK. During this time, she hopes to gain furtherinsight into the signal transduction pathways involved in platelet function and tobetter understand the processes of haemostasis and thrombosis.

Dr Simon Guiliano has taken up a position as Marketing Manager for SigmaAldrich in the Victorian and South Australian regions.

Dr Mistry has taken a position at Mayne Pharma, in the Global office forRegulatory Affairs and Drug Safety, as a Regulatory Associate.

The department also said farewell to Two Senior Research Officers: KarenAnderson and Sharon Forsyth. Sharon is now an Assistant Editor on the‘Respirology’ journal and also a Science/Medical Editor for an online AcademicEnglish Editing and Writing Service called SCRIPTORIA.

We wish them all the best with their future endeavors.

Mr Ron Morris

Dr Sascha Hughan


Postgraduate education

Our Scientists of the Future

The ACBD provides encourages the development of science at both anundergraduate and postgraduate level. The centre hosts dynamic postgraduatePhD and honours programmes, as well as fostering undergraduate sciencetalent.

PhD ProgrammeThe ACBD is currently responsible for the supervision of 9 PhD students:

Isaac GoncalvesProject title: Adhesion-specific signalling mechanisms in platelets.Supervisors: Dr S. Jackson and Dr Y. Yuan

Mhairi MaxwellProject title: Investigation of signalling molecules involved in integrin-mediatedadhesion.Supervisors: Dr S. Jackson and Prof. H. Salem

Mark FrazzettoProject title: Selective inhibitors of PI3-kinase isoforms as reagents for identificationof platelet signalling mechanisms.Supervisors: Professor Hatem H. Salem and Dr P. Thompson.

Anita HorvathProject title: Molecular characterisation of the murine ±1-ACT-like serpins.Supervisor: Dr P. Coughlin

Mythily SachchithananthanProject title: Post-transcriptional regulation of prothrombin gene expression.Supervisors: Dr R. Medcalf and Dr S. Stasinopoulos

Andre SamsonProject title: Biology of tissue-type Plasminogen Activator (t-PA) in the centralnervous system.Supervisor: Dr R. Medcalf

Belinda AbbottProject title: Synthesis and structure activity studies of antiplatelet 2-morpholinochromones.Supervisors: Prof. H. Salem and Dr P. Thompson

Antony VinhProject title: Assessment of novel inhibitors of matrix metalloproteinases inabdominal aortic aneursyn formationSurervisors: Dr Anthony Dear and Associate Professor Robert Widdop

Penny MayesProject title: Development of novel compounds for the treatment of metastaticmalignancy and vascular seaseSupervisors: Dr Anthony Dear and Associate Professor Patrick Perlmutter


We congratulate the following students who were successfully awardedtheir doctorate during the 2002-2004.

Dr Suhasini.Kulkarni Title – Investigation of novel mechanisms regulating platelet aggregation under flow

Dr Cindy YapTitle – Investigation of Shear-Dependent Signalling between Platelet GlycoproteinIb/V/IX and Integrin IIb/IIIa.

Dr Nayna MistryTitle – Investigation of the role of the platelet cytoskeleton in regulating interactionbetween glycoprotein Ib/V/IX and von willebrand factor.

Dr Sascha Hughan Title – Investigation of Integrin αIIbβ3 activation during platelet adhesion

Dr Simon GuilianoTitle – Calcium signalling regulating platelet adhesion and thrombus growth

Masters awarded

Ms Inna PikovskiProject title: The structure-function relationship of platelet glycoprotein receptorIb/V/IX.

Firkin PhD Scholarship

Students that are interested in pursuing doctorate studies in cardiovasculardisciplines, and who have the appropriate graduate qualifications, are encouragedto apply for the Firkin PhD. Award to undertake a PhD. program at the ACBD oraffiliated institutes comprising AMREP, commencing February 2006.

Further information on these scholarships can be obtained by contactingDr Robert Medcalf (03 9903 0133). Up to four scholarships of three yearsduration will be awarded with an annual tax-free stipend of $22,500.

To apply for one of these prestigious awards, please send a copy of yourcurriculum vitae including a transcript of your academic record together to:

Dr Robert MedcalfAustralian Centre for Blood DiseasesMonash University6th Floor, Burnet Building, AMREPCommercial Road, Melbourne, Victoria 3004Australia

Closing date for applications is October 31 (of each year).Professor Barry Firkin


Honours degree program

Over the last few years, the honours programme at the ACBD has beenconducted under the umbrella of several Monash University Departments;Anatomy and Cell Biology, Biochemistry, Pathology and Immunology, andMicrobiology. The programme is heavily devoted to the student’s research project,and thereby emphasizes the importance of experimental design, data collectionand analysis, literature reviews and trouble shooting.

The ACBD has supported a large number of honours students in the past, and theperiod 2002-2004 was no exception. We congratulate the following students whosuccessfully completed their honours year with us.

2002

Megha MulchandaniProject title:Investigation of a role for the p110β Isoform of Phosphoinositide 3-kinase in platelets.Supervisors: Dr S. Schoenwaelder and Dr S.P Jackson

Ramona MuttucumuraProject title: Investigation of a role for the small GTPase Rac in platelet functionSupervisors: Dr Y. Yuan and Dr S Jackson

Denuja KarunakaranProject title: Signal Transduction in platelets: The role for phospholipase C in vWf-stimulated platelets.Supervisors: Dr S Jackson and Ms T. Domagala

Kristina CakarovskiProject title: In vitro assessment of novel inhibitors on the modulation of the matrixmetalloproteinase enzyme system.Supervisors: Dr R. Medcalf and Dr A. Dear

2003

Emily ChenProject title: The post-transcriptional regulation of Plasminogen Activator InhibitorType 2.Supervisors: Dr R. Medcalf and Dr S. Stasinopoulos

Lovisa Dousha Project title: Characterization of novel inhibitors of Matrix Metalloproteinaseexpression in vascular smooth muscle cells.Supervisors: Dr R. Medcalf and Dr A. Dear

Corinne HitchenProject title: Zebrafish serpins: Identification, characterization and patterns of expression.Supervisor: Dr P. Coughlin and Dr A. Perkins

Swaroop ManjunathProject title:Investigation of the relative roles of the p110β and p110γ isoforms of phosphoinositide 3-kinase in platelets.Supervisors: Dr S. Schoenwaelder and Dr S. Jackson

Denison ChangProject title: Investigation of diYF mouse platelets and the role of tyrosinephosphorylation in the activation of integrin αIIbβ3.Supervisors: Dr Y. Yuan and Dr S. Jackson

2004

David MartinProject title: Mechanisms Involved in Atherosclerosis and Neointimal Formation in ApoE-deficient MiceSupervisors: Dr Anthony Dear and Associate Professor Robert Widdop


ACBD Summer Scholarship Program

Each year the Department of Medicine invites currently enrolled Science,Biomedical Science and Medicine undergraduates to apply for a B.G FirkinSummer Scholarship. The scholarships recognise the enormous contribution ofthe late Prof. B.G Firkin to the fields of thrombosis and cardiovascular disease.The scholarships, both second and third year, are designed to encourage highachieving undergraduates who wish to pursue a career in biomedical researchby giving them the opportunity to participate in a research project supervisedby senior research staff for six to ten weeks during their university holidays.The ‘hands on’ involvement of the students in these projects provides themwith a unique chance to enhance their practical skills and to challenge them withthe intellectual rigors inherent in good research. In many cases, the summerscholarships also serve as a springboard for students to undertake an Honoursyear within the department.

Further information about the B. G Firkin Scholarship may be obtained bycontacting the ACBD or Simone Schoenwaelder directly.

Molecular Medicine and Biotechnology Research Laboratory Miniprojects

The ACBD was pleased to continue to take part in the Bachelor of BiomedicalSciences (BMS) – Molecular Medicine and Biotechnology Research LaboratoryMiniprojects scheme. This teaching initiative involves groups of third year studentsbeing assigned a miniproject within the department, to be undertaken as part oftheir university contact hours. Students visiting the laboratories were required toparticipate in laboratory work, prepare a formal presentation and critically reviewsome of the relevant literature. This scheme took the students beyond thebounds of the university environment and into some of the research Institutesaffiliated with Monash University.

Opportunities for Undergraduates to gain working experience at the ACBD

In addition to offering summer scholarships and opening up our researchlaboratories to host miniprojects for BMS undergraduates, the ACBD also offersthe opportunity for 2nd and third year undergraduates to apply for a part-timeposition to gain valuable working experience in our research laboratories. Thesepositions are designed to allow undergraduates to experience the research labenvironment, and gain a better understanding of what it means to be a ‘scientist’before they delve into their honours and PhD years. This programme has beenon offer for a number of years now, and has proven to be very effective inassisting undergraduates in their career decision making, and also providing themwith that all important “first experience” that is required for many job applications.For more information please contact:

Dr Simone SchoenwaelderAustralian Centre for Blood DiseasesMonash University, 6th Floor, Burnet Building, AMREPCommercial Road, Melbourne, Victoria 3004 Australia

Email: [email protected]

“Being awarded a SummerVacation Scholarship at the

ACBD at the end of my 2ndyear undergraduate studies

has not only given me anexcellent opportunity to

experience first hand theworkings of a medical

research laboratory, but ithas also provided me with a

solid foundation with which topursue honours studies. It

has also opened up a wholenew and interesting field ofblood clotting studies, one

that was never evident tous as students during

undergraduate lectures”

Ms Akiko Ono (2002/2003; 2003/2004

scholarship recipient)

From Left: Ms Rivka Lilian, Ms Akiko Ono,Ms Mei Chan and Ms Joanna Lim.


Grant funding 2002–2004

Scholarships/Fellowships

2001–current S.M. Schoenwaelder, Monash University Logan Fellowship

2001–2004 S.M. Schoenwaelder, NHMRC R Douglas Wright Fellowship

2002–2003 S.P. Jackson, NHMRC SFRB

1997–2002 K.E. Anderson, NHMRC CJ Martin Fellowship

2000–2004 Wellcome Senior Reseach Fellowship, P Coughlin $163,000/year

2003–2004 K.E. Anderson, NHMRC, R.D. Wright Fellowship

2003–2007 Medcalf, NH&MRC, Senior Research Fellowship $115,000/year

2004–current S.P. Jackson, NHMRC, Principal Research Fellowship, $135,000P. Coughlin, Wellcome Trust Senior Fellowship, $180,000/year

Project grants

1999-2003 NHMRC Project Grant, #9937630, S.P. Jackson ($772,000)

2000-2002 NHMRC Project Grant, #124312, S.P. Jackson & S.M. Dopheide ($210,000)

2000-2002 NHMRC Project Grant, #124424, H.H. Salem & Jackson S.P. ($300,000)

2000-2002 NHMRC #124316, R.L. Medcalf, ($79,000)

2000-2002 NHMRC #124315, R.L. Medcalf and A. Carter, ($70,000)

2001-2003 NHMRC Project Grant, #143569, H.H. Salem and S. Cranmer ($405,000)

2001-2004 NHMRC Project Grant, #143678, S.P. Jackson and Y. Yuan ($260,000)

2001-2002 NHF Grant-In-Aid, S.P. Jackson and M. Rooney ($68,000)

2001-2003 NHMRC#143615, R.L. Medcalf and H. Yu, (80,000)

2001-2002 NHF Grant-In-Aid, G 99M 0346 SM Schoenwaelder and HH Salem ($81,925)

2002 ECRU, A.E Dear, R. Simpson, R.L. Medcalf, ($50,000)

2002-2004 NHMRC Project Grant, No. 194210, S.P. Jackson ($220,000)

2002-2003 NHF Grant-In-Aid, G 01M 0330, SM Schoenwaelder ($92, 561)

2002-2004 NHMRC Project Grant, #194205, SM Schoenwaelder ($225,000)



2003-2005 NHMRC, Project grant, K.E. Anderson, H. Salem, Total $435,000

2003 NHMRC Equipment grant, KE. Anderson, S. M. Schoenwaelder, H.H. Salem, S.P Jackson, P. Coughlin, RL Medcalf, $66,000

2003-2005 ARC Linkage-Project, KE Anderson, P. Thompson, $71,100

2003 S. Cranmer, The Ian Potter Foundation Travel Grant

2003-2005 RL Medcalf, NH&MRC Project Grant , #236862, $155,000/year

2004-2006 RL Medcalf, H Yu, NH&MRC Project Grant, #284250, $105,000/year

2004-2006 NHMRC, Project Grant, No. 284266, S.P. Jackson ($172,500)

2004-2005 2004-2005 NHF Grant, S.M. Schoenwaelder, G 031M1131, $50,000/year.

Other Source of Funding (Commercial) 2004

2003-2004 RL Medcalf, PAION GmbH, $115,000/year


ACBD Personnel

Australian Centre for Blood Diseases

Professor Hatem Salem, Head of department

Ms Jennie Saravanamuttu, ]Personal assistant to Head of the Department

Mrs Anna Pugliese, Administative officer

Platelet and Leukocyte Biology lab

Dr Shaun Jackson (Head)

Dr Sue Cranmer

Dr Pierre Mangin

Dr Yuping Yuan

Dr Warwick Nesbitt

Dr Sharelle Sturgeon

Dr Sascha Hughan

Dr Justin Hamilton

Ms Mei Chan

Mr Mark Frazzetto

Ms Mhairi Maxwell

Mr Erik Westein

Ms Yu (Joy) Yao

Platelet Signalling lab

Dr Simone Schoenwaelder (Head)

Ms Akiko Ono

Ms Joanna Lim

Fibrinolysis lab

Dr Rob Medcalf (Head)

Dr Stan Stasinopoulos

Dr Phil Daniel

Dr Thomas Weiss

Mr Be’Eri Niego

Mr Andre Samson

Mr Christopher Gafforini

Ms Mythily Sachchithananthan

Serpin biology lab

Dr Paul Coughlin (Head)

Dr Anita Horvath

Ms Melinda Missen

Ms Corinne Hitchen

Ms Trifina Sofian

ECRU Biotech lab

Dr Anthony Dear (Head)

Dr Hong Bin Liu

Dr Yunshan Hu

Ms Prue Freeman (Study Coordinator)



Documents

Research report 2002–2004 - Monash University · 2017. 7. 18. · 4 Highlights 2002–2004 2004, Rudolph Virchow Medal for Outstanding Contributions to Thrombosis Research In October