CD148 : a positive regulator of GPVI and αIIbβ3 proximal signalling in platelets

Platelets are small anucleate blood cells that plug holes in damage blood vessels. They do so by adhering to exposed extracellular matrix proteins at sites of injury and aggregating together. Platelet responsiveness to injury is controlled by a diverse repertoire of surface receptors that can be divided into two broad categories based on how they signal; the tyrosine kinased-linked receptors and the G protein-coupled receptors (GPCRs). There has been much work on elucidating the functions of tyrosine kinases in platelets, whereas protein tyrosine phosphatases (PTPs) have been under-investigated. To date, six non-transmembrane PTPs (NTPTPs), PTP-1B, Shp1, Shp2, MEG2-PTP, LMW-PTP and HePTP and a single receptorlike PTP (RPTP), CD148, have been identified in platelets. The main objective of this thesis was to determine the functional role of CD148 in platelets, which had never been studied in platelets. Using a mouse model, I demonstrate that CD148 is a critical positive regulator of signalling from the main collagen activation receptor GPVI and the fibrinogen integrin αIIbβ3, and also plays a minor role in regulating thrombin and thromboxane A2 (TxA2) mediated aggregation and secretion via the PAR-4 and TP receptors, respectively. The molecular mechanism of how CD148 regulates signalling from so many receptors is by maintaining a pool of active Src family kinases (SFKs) in platelets, which it does by dephosphorylating a tyrosine residue in the C-terminal of all SFKs. In an attempt to identify other PTPs that perform a similar function to CD148 in platelets, I analyzed platelets from PTP-1Band TC-PTP-deficient mouse models for functional and phosphorylation defects. PTP-1B-deficient platelets exhibited minor aggregation/secretion and phosphorylation defects relative to CD148-deficient platelets; and TC-PTP, which I show to be expressed in human and mouse platelets for the first time, is involved in platelet development. My conclusion is that CD148, PTP-1B and TC-PTP have distinct functional roles in platelets. Acknowledgements I can remember clearly my first week in the Watson lab and how welcome and settled everybody made me feel from day one. I couldn’t have wished for a better environment to train and develop as a scientist and also as a person. The last four years have been some of the toughest and challenging years of my life, yet also some of the most enjoyable and rewarding. Throughout my PhD many people have provided me with help and support to achieve my goals. I would like to take this opportunity to thank them all for everything they have done. Firstly, I would like to thank my supervisor Yotis Senis for providing me with the opportunity to do this PhD. Yotis you have been a true inspiration to me and I would like to thank you for all your expert training, encouragement, belief in my ability and your continued support and guidance. Thank you for keeping focussed and pushing me to excel, you have been a truly fantastic supervisor! Thank you also for teaching me how not to behave at conferences. I would also like to thank my co-supervisor Steve Watson for all his support and advice, providing a great lab environment in which to work and for always testing me in lab meetings and making me think. A number of people provided me with technical expertise critical to the success of this thesis. I would like to thank Mike Tomlinson for all his help with molecular biology related problems, cloning and helping me setup the luciferase assays, but not for always beating me every year in the fantasy football league! Thanks to Ban Darwood for all things aggregometry and for being the mother of the group. Thanks to Simon and Stevie T for help with the microscope. Majd, thanks for all your computer help and funny talk about Legs Eleven. I am extremely grateful to all the other members of the Watson lab, past and present, for making my experience in the lab an enjoyable and enriching one. Andy, cheers for all your scientific advice and constructive comments in lab meetings, I am sorry for stealing your Mr. Potato head and sending you ransom notes for the body parts. Thanks to Jocelyn for helping me steal Mr. Potato head and always making me laugh in the office. Thanks to Alice for always being happy and smiley and cheering me up. Alex, thanks for our little chats as I made tea and coffee. Thanks to Jen for all your molecular biology help. Thanks to Sonya for your willingness to stay out late and come clubbing with me and Ian. Ying Jie, thanks for all your help in the lab and your funny darting runs in the touch rugby tournament. Thanks also to Jenson for taking some of the flack off me during pressurised times. Tarvinder, thanks for introducing me to the Birmingham nightlife and all our chats in the lab. I’d also like to thank the other students in the lab for all there help and banter. Thanks to Dario with help with NFAT experiments and ironman chat. Craig H, thanks for your help in the lab, taking blood for me and being a great mate. Thanks to Neil for your help with NFAT, the aliquotting job and also for the laughs and jokes in the lab, although I did not like it when you spiked my tea with half a bag of sugar. Dean, thanks for all the stories of your drunken antics. Thanks to Ian for our games of park the trolley, for being annoying and interrupting me during CPS (critical pipetting stage! ), always making me laugh and generally being a great friend. Craig N, thanks for all the SFK discussion and office banter. I’d also like to give a big thankyou to Gayle for helping out with admin things, ordering reagents, arranging Christmas parties and secret Santa and her funny laugh which made me smile. Thanks to everyone in the Bicknell lab for all their help. I would also like to acknowledge our collaborators in other labs, in particular Alistair Barr down in Oxford for his help with the phosphatase assays and also the Tremblay lab over in Montreal, Canada for allowing me to spend some time over there to utilise their PTP knockout mouse models. I would like to take this opportunity to tell my family and friends how much I love them and how grateful I am for all their continued support. Without the push from my Mum and Dad I wouldn’t be where I am today, thank you so much, I couldn’t have done it without you. And thank you Mum for asking me nearly every day for the past year if I have finished my thesis yet. I can now finally say I have! Finally I’d like to give a massive thank you and a big hug to my beautiful and loving girlfriend, Fiona who has supported me over the past years throughout my PhD, giving me continued support and encouragement, putting up with my bad moods when my experiments haven’t worked and my whinging whilst writing this thesis. I love you so much! Abbreviations AA arachidonic acid ADP adenosine diphosphate ATP adenosine triphosphate BCR B-cell receptor CD148 TM-KO CD148 transmembrane knockout mouse CRP collagen-related peptide CSK C-terminal Src kinase COX1 cyclooxygenase-1 DAG 1,2-diacylglycerol ECM extracellular matrix ER endoplasmic reticulum FNIII fibronectin type 3 GDP guanosine diphosphate GPCR G protein-coupled receptor GTP guanosine triphosphate HSC haematopoietic stem cell IP immunoprecipitate IP3 inositol 1,4,5-trisphosphate IRS-1 insulin receptor substrate-1 ITAM immunoreceptor tyrosine-based activation motif ITIM immunoreceptor tyrosine-based inhibition motif MAM meprin/A4/μ domain NO nitric oxide NTPTP non transmembrane PTP OCS open canalicular system PAS protein-A sepharose PGH2 prostaglandin H2 PGI2 prostaglandin I2 PGS protein-G sepharose PKA protein kinase A PS phosphatidylserine PTK protein tyrosine kinase PTP protein tyrosine phosphatase PTB phosphotyrosine binding RPTP receptor-like PTP SEM sub-endothelial matrix SFK src family kinase SH2 src homology 2 domain SH3 src homology 3 domain TCR T-cell receptor TKLR tyrosine kinase-linked receptor TM-KO transmembrane knockout TxA2 thromboxane A2 VASP vasodilator-stimulated phosphoprotein VWF Von Willebrand factor WCL whole cell lysates