Research Interests and Techniques

Mechanisms of platelet activation in thrombosis and haemostasis

We are particularly interested in the role of ion channels and calcium-dependent proteins. For many experiments we use the megakaryocyte as a surrogate for studies of signalling in the tiny, anuclear platelet. Techniques:  patch clamp, Ca2+-sensitive fluorescent indicators, quantitative RT-PCR, interference RNA, transgenic murine models, various assays of platelet function eg. aggregometry, adhesion, secretion, shape change

Development of platelet-producing megakaryocytes from stem cells 

We are investigating the mechanisms by which platelets develop within their precursor cell, the megakaryocyte. Techniques: single and multi-photon confocal microscopy, patch clamp, interference RNA, single cell RT-PCR

Interactions between ion channels and GPCRs

We are investigating how ion channels can modulate signalling through GPCRs. In particular, we have identified a marked ability for certain Gq-coupled receptors (eg P2Y1) to be directly modulated by the cell membrane potential. Techniques: Patch clamp, Ca2+-sensitive fluorescent indicators, GFP fusion proteins, FRET

Figure 1. The major contribution of P2X1 receptors to early collagen-evoked Ca2+ signalling in the platelet. ATP-gated P2X1 receptors are the only Ca2+-permeable ligand-gated ion channel reported to date on the platelet plasma membrane; it therefore represents one of the fastest means whereby vascular injury can activate a Ca2+ increase during haemostasis and thrombosis. For further details see Fung et al. 2007. J.Thromb.Haemost 5: 910-7.

Figure 2. Confocal fluorescence imaging of the demarcation membrane system of the megakaryocyte. The confocal images show the staining pattern of membranes continuous with the extracellular space (dye: 10 mM di-8-ANEPPS). The complex demarcation membrane system, which provides a reservoir of membranes for future platelet production, can be seen throughout the extranuclear space of the large megakaryocyte. Small marrow cells lack this membrane system and stain as halos. For further details see Mahaut-Smith et al. 2003, Biophys. J. 84: 2646-54

Research Group and Funding

Main sources of funding

British Heart Foundation
Medical Research Council
BBSRC

Group members

Alex Bye

Sangar Osman

Kirk Taylor

Dr Joy Wright

Main collaborators

Professor Richard Evans, University of Leicester
Professor Alison Goodall, University of Leicester

Professor Jon Gibbins, University of Reading
Professor Richard Farndale, University of Cambridge
Dr Koji Eto, University of Tokyo

Dr Stefan Amisten, University of Oxford
Dr Juan Martinez-Pinna, University of Alicante

Recent Publications

Fung, C.Y.E, Jones, S., Ntrakwah, A., Naseem, K.M. Farndale, R.W. & Mahaut-Smith,M.P. (2012).  Platelet Ca2+ responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors. (Blood, published ahead of print January 6, 2012, doi:10.1182/blood-2011-10-386052).

Markovic, D., Holdich, J., Al-Sabah, S., Mistry, R., Krasel, C. Mahaut-Smith,M.P.*, Challiss, R.A.J.*  (*co-authors of correspondence). (2012). FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists. PloS One, 2012;7(1):e29946. Epub 2012 Jan 17.

Jones, S., Evans, R.J. & Mahaut-Smith, M.P. (2011). Extracellular Ca2+ modulates ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation. British Journal of Haematology,153, 83-91.

McCloskey, C., Jones, S., Amisten, S., Snowden, R.T., Kaczmarek, L.K., Erlinge, D., Goodall, A.H., Forsythe, I.D. &  Mahaut-Smith, M.P. (2010). Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes : roles in membrane potential, Ca2+ signalling and platelet count. Journal of Physiology, 588:1399-406

Lalo, U., Allsop, R., Mahaut-Smith, M.P. & Evans, R.J. (2010). P2X1 receptor mobility and trafficking; regulation by receptor insertion and activation. Journal of Neurochemistry, 113:1177-87

Martinez-Pinna, J., Gurung, I.S., Mahaut-Smith, M.P. & Morales, A. (2010). Direct voltage control of endogenous lysophosphatidic acid G-protein-coupled receptors in Xenopus oocytes. Journal of Physiology, 588:1683-93.

Qiu, M.R., Jiang, L., Matthaei, K.I., Schoenwaelder, S., Kuffner, T., Mangin, P., Joseph, J.E., Low, J., Connor, D., Valenzuela, S.M., Curmi, P.M., Brown, L.J., Mahaut-Smith, M.,  Jackson, S. & Breit, S.N. (2010). Generation and characterisation of mice with null mutation of the chloride intracellular channel 1 gene. Genesis 2010 48:127-36.

Fung, C.Y.E, Marcus, A.J., Broekman, M.J. & Mahaut-Smith, M.P. (2009). P2X1 receptor inhibition and soluble CD39 administration as novel mechanisms to widen the cardiovascular therapeutic window.  Trends in Cardiovascular Medicine, 19, 1-5.

Mahaut-Smith MP, Martinez-Pinna J and Gurung IS. (2008). A role for membrane potential in regulating GPCRs? Trends in Pharmacological Sciences 29: 421-9

Gurung IS, Martinez-Pinna J and Mahaut-Smith MP. (2008). Novel consequences of voltage-dependence to a G-protein-coupled receptor. British Journal of Pharmacology. 154: 882-9

Tolhurst G, Carter RN, Amisten S, Holdich JP, Erlinge D and Mahaut-Smith MP. (2008). Expression profiling and electrophysiological studies suggest a major role for Orai1 in the store-operated Ca2+ influx pathway of platelets and megakaryocytes. Platelets 19: 308-13

Fung CYE, Cendana C, Farndale RW and Mahaut-Smith MP. (2007). Primary and secondary agonists can use P2X1 receptors as a major pathway to increase intracellular Ca2+ in the human platelet. Journal of Thrombosis & Haemostasis. 5: 910-7

Vial C, Fung CYE, Goodall AG, Mahaut-Smith MP and Evans RJ. (2006). Differential sensitivity of human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts. Biochem Biophys Res Commun 343, 415-9.

Carter RN, Tolhurst G, Walmsley G, Vizuete-Forster M, Miller N and Mahaut-Smith MP. Molecular and electrophysiological characterisation of transient receptor potential ion channels in the primary murine megakaryocyte. Journal of Physiology, 576: 151-62.

Fung CYE, Brearley CA, Farndale RW and Mahaut-Smith MP. (2005). A major role for P2X1 receptors in the early collagen-evoked Ca2+ responses of human platelets. Thrombosis & Haemostasis, 94,37-40.

Horner S, Menke K, Hildebrandt C, Kassack MU, Nickel P, Ullmann H, Mahaut-Smith MP and Lambrecht G. (2005). The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range. Naunyn Schmiedbergs Archives Pharmacology, 372, 1-13.

Tolhurst G, Vial C, Leon C, Gachet C, Evans RJ and Mahaut-Smith MP. (2005). Interplay between P2Y1, P2Y12 and P2X1 receptors in the activation of megakaryocyte cation influx currents by ADP; evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling. Blood, 106, 1644-51.

Martinez-Pinna MP, Gurung IS, Vial C, Leon C, Gachet C, Evans RJ and Mahaut-Smith MP. (2005). Direct voltage control of signaling via P2Y1 and other Gaq-coupled receptors. J.Biol.Chem. 280, 1490-1498.

Pitt SJ, Martinez-Pinna J, Barnard EA and Mahaut-Smith MP. (2005). Potentiation of P2Y receptors by physiological elevations of extracellular K+ via a mechanism independent of Ca2+ influx. Molecular Pharmacology, 67, 1705-13

Mahaut-Smith MP, Tolhurst G and Evans RJ. (2004). Emerging roles for P2X1 receptors in platelet function. Platelets, 15, 131-144.

Martinez-Pinna MP, Tolhurst G, Gurung IS, Vandenberg JI and Mahaut-Smith MP. (2004). Sensitivity limits for voltage control of P2Y receptor-evoked Ca2+ mobilisation in the rat megakaryocyte. Journal of Physiology, 555, 61-70. 

Mahaut-Smith MP. (2004). Patch clamp recordings of electrophysiological events in the platelet and megakaryocyte. In Platelets and Megakaryocytes, Vol 1. Editors: JM Gibbins and MP Mahaut-Smith: Methods in Molecular Biology, Vol 272, Humana Press Inc. Totawa, NJ. 2004. pp 277-299.

Mason MJ and Mahaut-Smith MP. (2004). Measurement and manipulation of intracellular Ca2+ in single platelets and megakaryocytes, In Platelets and Megakaryocytes, Vol 2. Editors: JM Gibbins and MP Mahaut-Smith: Methods in Molecular Biology, Vol 273, Humana Press Inc. Totawa, NJ. 2004. pp 251-275.

Vial C, Pitt SJ, Roberts J, Rolf MG, Mahaut Smith MP and Evans RJ. (2003). Lack of evidence for functional ADP-activated human P2X1 receptors supports a role for ATP during hemostasis. Blood 102, 3646-3651.

Mahaut-Smith MP, Thomas D, Higham AB, Usher-Smith JA, Hussain JF, Martinez-Pinna J, Skepper JN and Mason MJ. (2003). Properties of the megakaryocyte demarcation membrane system in living rat megakaryocytes. Biophysical Journal, 84, 2646-2654.

Books

Platelets and Megakaryocytes, Vol 1. Functional Assays. Editors: JM Gibbins and MP Mahaut-Smith: Methods in Molecular Biology, Vol 272, Humana Press Inc. Totawa, NJ. 2004. pp. 373

Platelets and Megakaryocytes, Vol 2. Perspectives and Techniques. Editors: JM Gibbins and MP Mahaut-Smith: Methods in Molecular Biology, Vol 273, Humana Press Inc. Totawa, NJ. 2004. pp 475.

Platelets and Megakaryocytes, Vol 3. Additional Protocols and Perspectives. Editors: J.M. Gibbins & M.P. Mahaut-Smith.: Methods in Molecular Biology, Vol 788, Springer Science+Business Media, LLC 2011 pp ≈400.

Platelets and Megakaryocytes - Functional Assays