Senior Lecturer University of Westminster London, England, United Kingdom
Background: CD148 is an R3 receptor-type protein tyrosine phosphatase (R3 PTP) expressed on the surface of platelets. It has a vital double regulatory role in platelet signalling pathways. Studies to date suggest CD148 as a potential antithrombotic drug target. A lot is known about the phosphatase domain of the CD148 protein. However, little is known about its extracellular regions.
Aims: This research aims to study the structure of the CD148 extracellular domain experimentally and through a bioinformatic artificial intelligence (AI) program called AlphaFold. Another aim is to analyze the function of CD148 in response to an inhibitor, Razuprotafib (AKB-9778) and its binding interactions.
Methods: 1. An E. coli bacterial expression system was used to develop constructs and analysed using Small-Angle X-ray scattering (SAXS). 2. AlphaFold software was installed to generate the predicted structures. 3. Flow cytometry was used to check the expression of CD148 upon applying different agonists. 4. A microchip-based flow chamber system called the Total-thrombus formation analysis system (T-TAS) was used to mimic in vivo conditions for evaluating whole-blood thrombogenicity when applying different Razuprotafib concentrations. 5. DiFMUP phosphatase assay to measure the selectivity of the inhibitor 6. In-silico molecular docking of CD148 and the inhibitor to identify the specificity and its binding interactions.
Results: Six constructs for the extracellular domain of the CD148 protein have been developed through the E. coli bacterial protein expression system. The construct having the first five exons was used for the SAXS analysis. A high level of CD148 expression was found in whole blood when being activated by CRP and PAR4. A population of CD148 still remains inactivated even when applying high concentrations. T-TAS and DiFMUP results show that 10uM of Razuprotafib attenuates CD148 activity.
Conclusion(s): Interestingly a loop was found on CD148 extracellar domain. Razuprotafib attenuates CD148 activity however it doesn't fully inhibit its function.
