Creation of a molecular and structural model of mouse thrombos is

• Project/Area Number: 22790313
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Pathological medical chemistry
• Research Institution: Kyoto University
• Principal Investigator: NICOLAS Prevost 京都大学, 生命科学系キャリアパス形成ユニット, 助教 (60561967)
• Project Period (FY): 2010 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2011: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2010: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: 分子病態学 / 血栓症 / 分子腫瘍学

Research Abstract

Real-time labeling and analysis of vWF:
Initial plan : Labeling of vWF in CHO cells using fluorophore-labeled vWF-targeted peptide.
Outcome : unsuccessful.
Approach used : We have created an HEK293T cell line that stably expresses the human histamine receptor 1(HEK293T-HRH1). Transient expression of WT and and mutant vWF in HEK293T-HRH1 results in constitutive expression of vWF multimers in pod-like structures on the surface of the cell. The amount of vWF multimers secreted on the surface of the cell can be greatly enhanced by histamine treatment of HEK293T-HRH1 cells. This cell model has allowed us to successfully express all 12 vWF constructs and label them by immunostaining and monitor vWF string release, proteolysis and/or platelet binding in real-time microscopy flow chamber assays.
Conclusion : we have created the first cellular model that allows the simultaneous detection of intact or cleaved vWF strings and platelets in live cells in the absence or presence of flow. This assay has allowed us to perform a thorough testing of our candidate vWF mutants and establish that some of them were utterly resistant to cleavage by ADAMTS13 even at very high shear rates. As expected, these proteolysis-resistant mutants were capable of promoting rapid platelet accumulation under flow.
In vivo studies:
Initial plan : Creation of a knock-in construct for the expression of our most-proteolysis resilient and pro-thrombotic vWF A2 mutant in live mice.
Outcome : grossly inadequate funding has rendered this approach unfeasible.
Approach used : we have started breeding vWF-null mice in our facility and we are currently in the process of performing plasma vWF reconstitution experiments in these animals. We have subcloned all our vWF constructs into pLIVE vectors. Hydrodynamic injection of these constructs into vWF animals allows us to stably induce hepatocyte-driven expression of WT or mutant vWF in live animals for up to a month. Ongoing experiments will demonstrate whether expression of our cleavage-resistant mutants is indeed sufficient to induce thrombotic thrombocytopenic purpura in these animals.
Crystallography
Initial plan : purification and X-ray crystallography analysis of WT and mutant vWF.
Outcome : we have been extremely successful in purifying large amounts of uncontaminated recombinant vWF A2 proteins. Crystal preparation is currently underway.

Research Products

• [Journal Article] What is vinculin needed for in platelets? (2010)
  • Author(s): Mitsios JV, Prevost N, Kasirer-Friede A, Gutierrez E, Groisman A, Abrams CS, Wang Y, Litvinov RI, Zemljic-Harpf A, Ross RS, Shattil SJ.
  • Journal Title: Journal of Thombosis and Haemostasis Volume: 8 Pages: 2294-304
  • Peer Reviewed
• [Remarks]
  • URL: http://www.cp.kyoto-u.ac.jp/Prevost/IndexJ.html
• [Remarks]
  • URL: http://www.cp.kyoto-u.ac.jp/Prevost/PublicationsJ.html
• [Remarks]
  • URL: http://www.cp.kyoto-u.ac.jp/Prevost/IndexJ.htm