Phospholipid Flippases in B cells and Platelets

Abstract

The asymmetric distribution of phospholipids in the plasma membrane is generated and maintained through the action of phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Exposure of PS is indispensable for the clearance of apoptotic cells and promotion of blood coagulation. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being re-internalised. In addition to the plasma membrane, flippases also function in generating phospholipid asymmetry in intracellular membranes and play critical roles in vesicle-mediated protein trafficking. In my PhD I investigated the biochemical properties and physiological roles of the flippases ATP11C and ATP8A1. Using ATP11C-deficient mice, I demonstrated that: 1) ATP11C mediated significant flippase activity in murine B cell subsets. Loss of ATP11C resulted in a defective internalization of PS and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. These findings identified ATP11C as an aminophospholipid translocase in B cell lineages and suggested that the temporary increased PS accumulation on the surface of pro-B cells caused by impaired flippase activity contributed to the B cell lymphopenia observed in ATP11C-deficient mice. 2) Loss of ATP11C resulted in reduced life span and increased size of platelets but did not induce thrombocytopenia or affect platelet function. Interestingly, I found that ATP11C did not mediate the flippase activity in platelets as evident by the normal flippase activity in mutant platelets and absence of protein expression of ATP11C in wild type platelets. These results suggested that ATP11C was not a contributing flippase in murine platelets and ATP11C likely regulated cell extrinsic factors that were required for the survival of platelets in the peripheral blood. Together, these findings highlighted a fundamental difference between the action of ATP11C in leukocytes and platelets. Moreover, I identified that flippase ATP8A1was highly expressed in both murine and human platelets but was not present in the plasma membrane. ATP8A1 was cleaved by the cysteine protease calpain during apoptosis, and the cleavage was prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remained intact. These data revealed a novel mechanism of flippase cleavage and suggested that flippase activity in intracellular membranes differed between platelets undergoing apoptosis and activation. Collectively, these findings extended our understanding on the role of flippases in B cell development and important mechanisms of platelet survival and function. Show more