ATP11A and ATP11C are plasma membrane phosphatidylserine flippases in in vitro human megakaryocytes.

Thrombotic diseases are the major worldwide cause of cardiovascular death. Platelets prevent blood loss following injury (haemostasis), but inappropriate and excessive platelet activation can lead to thrombosis. Platelet activation must be tightly controlled. Pro-coagulant platelets expose phosphatidylserine (PS), enabling coagulation complex assembly, enhancing thrombin generation and thrombosis. PS is normally restricted to the inner leaflet of the plasma membrane by flippase (aminophospholipid translocase) activity. However, the flippase protein(s) responsible for this crucial activity in platelets remains unidentified. The P4 ATPases ATP11A and ATP11C, regulated by their obligatory partner CDC50A, flip PS at the plasma membrane in a range of different cell types. To investigate platelet flippases, human induced pluripotent stem cells (hiPSCs) were forward-programmed into CD41+/CD42+ megakaryocytes, the platelet precursor. Wildtype (WT) forward-programmed megakaryocytes showed similar flippase activity to human platelets with internalisation of NBD-PS that could be inhibited by high cytosolic Ca2+ or N-ethylmaleimide (NEM). We then generated CDC50A, ATP11A or ATP11C single knockout and ATP11A/11C double knockout (DKO) hiPSCs using CRISPR-Cas9. CDC50A-KO, ATP11A-KO, ATP11C-KO and DKO hiPSC clones successfully formed CD41a+/CD42a+ mature megakaryocytes. CDC50A-KO megakaryocytes bound Annexin V when unstimulated and had no remaining NEM-sensitive flippase activity indicating the involvement of a P4-ATPase. Although ATP11A-KO and ATP11C-KO megakaryocytes had similar flippase activity to WT clones, DKO clones had inhibited NBD-PS internalisation compared to WT and had no remaining NEM-sensitive flippase activity. This indicates that the CDC50A-regulated P4-ATPases ATP11A and ATP11C act together at the megakaryocyte plasma membrane and are responsible for PS flippase activity and therefore likely responsible in human platelets.