Targeting phosphatidylserine exposure in pro-coagulant platelets.

Abstract

Pro-coagulant platelets are a subpopulation of activated platelets that are central to blood clot formation in both thrombosis and haemostasis. They expose the anionic phospholipid, phosphatidylserine (PS), in their outer leaflet enabling coagulation complex assembly on the negatively charged surface. Assembly of the tenase and prothrombinase complexes enhances their activity, increasing thrombin generation. PS is normally confined to the inner leaflet of the plasma membrane by flippase activity, preventing its exposure on the outer surface of unstimulated and pro-aggregatory platelets. In pro-coagulant platelets, a high cytosolic calcium signal results in the activation of the scramblase protein, TMEM16F, causing PS to move into the outer leaflet. Flippase activity is also inhibited, removing the route for re-entry of PS into the inner leaflet. It is a combination of these two events that results in sustained PS exposure. Targeting either the scramblase or flippase proteins could be a viable strategy to reduce PS exposure and therefore pro-coagulant platelet activity in the prevention of thrombosis. As platelet scramblase activity has previously been attributed to TMEM16F, a range of reported TMEM16F inhibitors were tested for their efficacy at blocking PS exposure in platelets. Ivermectin, clofazimine and benzbromarone had no effect on PS exposure downstream of either thrombin and CRP-XL or A23187 activation. Abamectin and niclosamide treatment caused a modest reduction in the percentage of platelets that exposed PS in response to thrombin and CRP-XL but this inhibition was attributed to off target effects. Additional experiments in human red blood cells also suggested that the effects of these drugs were non-specific. An alternative approach would be to maintain platelet flippase activity, as previously demonstrated using the drug R5421. However, the protein responsible for platelet flippase activity remains unidentified. A novel approach was taken to identify the protein(s) responsible for this activity. Candidate flippase proteins (P4 ATPases: ATP11A and ATP11C) were mutated using CRISPR-Cas9 in two human induced pluripotent stem cell lines (iPSC) to produce genetic knockouts. These iPSCs were forward programmed to mature megakaryocytes where flippase activity could be assessed. ATP11A-/- and ATP11C-/- single knockouts had similar flippase activity to wild-type megakaryocytes, remaining sensitive to A23187 and NEM. ATP11A-/- ATP11C-/- double knockouts had inhibited flippase activity compared to wild-type, with no remaining NEM sensitive inward PS movement. This therefore identifies ATP11A and ATP11C as flippase proteins at the plasma membrane of in vitro megakaryocytes, acting together to translocate PS to the inner leaflet. Maintaining the activity of these proteins in pro-coagulant platelets could be a novel antithrombotic strategy.