Abstract
Factor V (FV) is an essential coagulation cofactor that circulates in distinct plasma and platelet pools. Unlike plasma FV, platelet FV is stored complexed to the megakaryocyte (MK) synthesized α-granule protein multimerin, which is not found in plasma. The origin of platelet FV has been controversial and recent studies have suggested human platelet factor V originates from plasma. To determine if human MK could endocytose FV and generate the FV-multimerin complexes, we investigated FV endocytosis and storage by human MK derived from cord blood CD34+ cells. Cells were cultured with or without 10% plasma in 15% BIT/ IMDM containing 5ng/ml SCF and 50ng/ml TPO. Human plasma or purified plasma FV was added on day 7 of culture. Cells were harvested to analyze α-granule proteins on day 11. Enzyme-linked immunoassays were used to evaluate MK content of FV and multimerin, and their complexes. Control α-granule proteins analyzed included albumin, IgG and fibrinogen, as they are known to originate from plasma. Multimerin was detected in all MK whereas FV, albumin, IgG, fibrinogen and FV-multimerin complexes were only detected in MK cultured with plasma. The amounts of plasma-derived proteins in MK were orders of magnitude lower than in platelets. MK cultured with purified FV had dose-dependent FV uptake. With SFLLRN stimulation, MK cultured with plasma released multimerin, FV, albumin, IgG and fibrinogen, consistent with their storage in secretion granules. Like platelet FV, MK FV showed a proteolyzed pattern. Double immunostaining showed partial colocalization of intracellular multimerin and FV in MK cultured with FV. Immunoelectron microscopy confirmed that like bone marrow MK, MK cultured with FV contained FV within demarcation membranes, small vesicles and α-granules. Analyses of FV expression by cultured MK, using semi-quantitative RT-PCR, indicated there were no significant differences in FV expression by MK cultured with or without plasma, at any of the time points evaluated. However, there was a trend to increased FV expression with MK maturation, and for MK cultured in plasma, FV expression was significantly higher at 15 compared to 7 days, raising the possibility that some FV was synthesized at late stages of megakaryopoiesis. Our study illustrates that cultured human MK can proteolyze and target endocytosed plasma FV into α-granules and that they can generate complexes of FV with multimerin.