An investigation of the structure and function of the receptor-type protein tyrosine phosphatase CD148
CD148 is an R3 receptor-type protein tyrosine phosphatase (RPTP) found on platelet surfaces, where it has a vital dual role in regulating platelet signalling and thrombosis. Inhibition of CD148 has been suggested as a novel anti-thrombotic strategy. Loss-of-function polymorphisms located in the extracellular domain (ECD) have been identified; however, there is limited information on the structure and function of this large region of the protein. The aims of this thesis were 1- to investigate the structure of CD148 ECD, gain insight into how specific missense polymorphisms affect CD148 function and 2- to test an inhibitor of CD148 on thrombosis and platelet activation. To investigate the structure, I analysed the AlphaFold predicted structure of CD148 ECD and also used small-angle X-ray scattering (SAXS). My findings reveal that CD148 adopts an elongated non-globular structure resembling a cylinder. Moreover, it has fewer fibronectin type III (FNIII) domains than sequence analysis predicts and features an interesting, unstructured flexible loop. The AlphaFold prediction correlated well with the experimental SAXS data. With regards to the second aim, I tested an RPTP inhibitor, AKB-9778 (Razuprotafib), on CD148 phosphatase activity, whole blood in vitro thrombogenicity and platelet activation. The inhibitor showed weak inhibition of overall thrombosis, and molecular docking was used to predict a binding model. Additional studies examined whether CD148-specific sub-populations of platelets exist, as has been noted for megakaryocytes. Flow cytometry (FC) experiments indicated that CD148 is uniformly expressed on all platelets; however, a population of CD148-positive unresponsive platelets was detected. My conclusions are that the ECD of CD148 shows a previously uncharacterized unstructured loop, AKB-9778 is a weak inhibitor of CD148 that has anti-thrombotic activity, and that there was higher receptor expression in high-RNA platelets compared to low-RNA platelets. However, this was significantly dependent on the size of platelets.
Networked Digital Library of Theses and Dissertations