Platelet and Megakaryocyte Group

Group Leader: Professor Beng Chong

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Overview of Research

The focus of Professor Beng Chong's research is megakaryocyte / platelet pathobiology. It can be broadly divided into two areas: (1) transcription regulation of megakaryopoiesis, and (2) platelet pathobiology.

In health, blood platelet concentrations are kept within a constant range. A decrease in platelets can cause bleeding and an increase in platelets, such as in myeloproliferative disorders, can lead to thrombosis and cardiovascular diseases.

Research Projects

1. Transcriptional regulation of megakaryopoiesis

To maintain a constant blood platelet level, the production of platelets in the bone marrow needs to be tightly controlled. This is achieved by a complex mechanism that involves thrombopoietin (TPO) and key transcription factors such as FOG-1 and GATA-1. However, some of the transcription factors are still unknown. The interaction of these proteins with one and other and their binding to DNA, have not yet been fully characterized. We have recently cloned human FOG-2, a new member of FOG family of transcription factors. FOG-2 can effectively replace FOG-1 in its interactions with the GATA proteins. To identify transcription factors involved in the control of platelet production, three experimental strategies were employed:

i. Identification of important regulatory domains in the promoter region of megakaryocyte-specific genes
We found that the proximal Ets binding site in the promoter region of the gene was critical for GP IX expression. The Ets factor was subsequently identified as Fli-1. Work on other megakaryocyte-specific genes is proceeding.

ii. Identification of TPO-inducible genes
This may lead to identification of genes that are involved in the regulation of megakaryopoiesis. We used the technique, representational difference analysis (RDA) for this and have identified some known TPO-induced genes such as GP IX, PU.1/Spi-1, Fli-1, EV11 and Pim-1 and several novel genes. Characterization of the novel genes is proceeding.

iii. Identification of the transcriptional partners of FOG-2 and Fli-1
We have used the yeast two hybrid technique to screen the K562 cDNA library using FOG-2 or Fli-1 as a bait. A number of novel clones have been identified with this approach. In addition, known transcription factors, not previously reported to interact with these two proteins (for example, GATA-1 not previously known to interact with Fli-1) were uncovered. Characterization of the protein-protein interaction between Fli-1 and GATA-1 revealed that this interaction synergistically promotes transcription of megakaryocyte-specific genes such as GP IX. Our data suggests the interaction of these two transcription factors is critical for the expression of megakaryocyte-specific proteins and, probably also for differentiation of megakaryocytes.

The studies of gene regulation of megakaryocyte differentiation will result in a better understanding of the mechanisms involved in platelet production and may also lead to improved diagnosis and management of platelet disorders, including post-chemotherapy and radiotherapy-induced thrombocytopenia which is a serious complication of cancer treatment.

2. Platelet Pathobiology

Professor Chong has a longstanding research interest in the pathogenesis of platelet disorders, particularly immune platelet disorders.

a. Drug-induced immune thrombocytopenia
This is a common adverse effect of drug therapy. It is caused by binding of a drug-dependent antibodies to platelets resulting in their premature clearance. In quinine-induced thrombocytopenia, we had previously observed that all patients had antibodies against GP IX. Using human/mouse GP IX chimeric proteins, we have now mapped the major binding site to the C-terminus extracellular region of GP IX. This is also the binding site of the monoclonal antibody SZ1 which cross-blocks the binding of the drug-dependent antibodies in quinidine-, ranitidine- and rifampicin-induced thrombocytopenia, suggesting the binding sites of these antibodies are also located in this region of GPIX.

b. Bernard-Soulier syndrome (BSS)
We have identified a novel mutation GPIb???Leu®Pro in a patient with BSS co-existing with HPA 5b polymorphism (GPIb???5Thr® Met). Unlike the mutations in other BSS patients, these mutations do not significantly affect the cell surface expression of GPIb-IX but lead to decreased vWF binding, and consequently to a dysfunctional receptor.

Group Members

Key Publications

Chong BH, Pitney WR, Castaldi PA. Heparin-induced thrombocytopenia: Association of thrombotic complication with a heparin-dependent IgG antibody which induced platelet aggregation, release and thromboxane synthesis. Lancet ii:1246-1248, 1982.

Brighton T, Evans S, Castaldi PA, Chesterman CH, Chong BH. Prospective evaluation of the clinical usefulness of M.A.I.P.A. assay in Idiopathic Thrombocytopenic Purpura and other immune thrombocytopenias. Blood 88:194-201, 1996.

Sungaran R, Markovic B, Chong BH. Localisation of cells that express thrombopoietin mRNA in the kidney and liver using in situ hybridization. Blood 89:101-107, 1997.

Burgess JK, Lopez JA, Berndt MC, Gaudry LE, Dawes I, Chesterman CN, Chong BH. Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex characterization of the epitopes. Blood 92:2366-2373, 1998.

Tao H, Gaudry L, Rice A, Chong BH. Ex vivo expansion of megakaryocytic progenitor cells derived from normal human bone marrow cord blood. Exp Hematol 27: 293-301, 1999.

Holmes M, Turner J, Fox A, Chisholm O, Crossley M, Chong BH. hFOG-2: a novel zinc finger protein binds the co-repressor mCtBP2 and modulates GATA-mediated activation. J Biol Chem 274: 23491-8, 1999.

Holmes M, Turner J, Fox A, Chisholm O, Crossley M, Chong BH. hFOG-2: a novel zinc finger protein binds the co-repressor mCtBP2 and modulates GATA-mediated activation. J Biol Chem 274: 23491-8, 1999.

Sungaran R, Chisholm O, Markovic B, Chong BH. The role of platelet alpha-granular proteins in the regulation of thrombopoietin mRNA. Blood 95: 3094-3101, 2000.

Newman P, Chong BH. Heparin-induced thrombocytopenia: New Evidence for the Dynamic Binding of Purtied anti-PF4-heparin Antibodies to Platelets and the Resulting Activation. Blood 96: 182-187, 2000.

Eisbacher M, Khachigian LM, Khin TH, Holmes ML, Chong BH. Inducible expression of megakarocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase. Cell Growth Differ, 12, 435-445, 2001.

Tait AS, Cranmer SL, Jackson SP, Dawes IW, Chong BH. Phenotype changes resulting in high-affinity binding of von Willebrand factor to recombinant glycoprotein Ib-IX: analysis of the platelet-type von Willebrand disease mutations Blood, 98, No. 6, 1812-1818, 2001.

Funding Sources

NHMRC Program Grant: Vascular Biology in Thrombosis 1998-2002.
CN Chesterman, BH Chong, PJ Hogg, DA Owensby, LM Khachigian.

NSW State Government Research & Development Infrastructure Grant 2000-2002.
CN Chesterman, BH Chong, ND Dudman, PJ Hogg, LM Khachigian, DEL Wilcken.

National Heart Foundation of Australia, 1999-2001. Generation of recombinant human antiplatelet antibodies for the treatment of coronary artery disease.
BH Chong, RL Ward, OT Chisholm.

AusAid-sponsored Australia-China Institutional Link Program Grant, 1997-2000. Drug development from Chinese Medicinal Herbs for the treatment of blood diseases.
BH Chong, R-L Gao.

Singapore National Medical Research Council, 2001-2004. Development of Platelet Inhibitors for the Treatment of Coronary Heart Disease.
BH Chong.

Aventis Pharma Pty Ltd, 2002-2004. IMPROVE - International medical prevention registry on venous thromboembolism.
BH Chong.

Aventis Pharma Pty Ltd, 2002. EXCLAIM - A double-blind , placebo-controlled, parallel, multicenter study on extended VTE prophylaxis in acutely ill medical patients with prolonged immobilisation.
T Brighton, BH Chong.

Organon Australia Pty Ltd, 2002. ARTEMIS - A multinational, randomised, double-blind comparison of once daily subcuteneous fondaparinux sodium with placebo for the prevention of VTE in acutely ill medical patients.
T Brighton, BH Chong.

Sanofi Synthelabo Australia Pty Ltd, 2002. PEGASUS - Study to compare the efficacy and safety of fondaparinux sodium with dalteparin (Fragmin) in the prevention of venous thromboembolic events in high risk abdominal.
T Brighton, BH Chong.