ABSTRACT
Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.
Subject(s)
Blood Platelets/cytology , Bone Marrow Cells/immunology , Factor V/genetics , Megakaryocyte Progenitor Cells/cytology , Megakaryocytes/cytology , Animals , Apoptosis/drug effects , Arterioles/drug effects , Arterioles/immunology , Arterioles/injuries , Biomarkers/blood , Blood Platelets/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cell Differentiation , Cell Lineage/immunology , Endocytosis , Factor V/immunology , Factor V/pharmacology , Flow Cytometry , Gene Expression , Humans , Immunophenotyping , Lasers , Megakaryocyte Progenitor Cells/immunology , Megakaryocytes/immunology , Mice , Mice, SCID , Platelet Glycoprotein GPIb-IX Complex/genetics , Platelet Glycoprotein GPIb-IX Complex/immunologyABSTRACT
Repeated surgical exposure to topical bovine thrombin is known to be associated with the development of antibodies to bovine and human thrombin and factor V. This is demonstrated by abnormalities of in vitro coagulation assays and, rarely, postoperative bleeding. We describe a 4-year-old child in whom an antibody to bovine factor X developed after cardiac surgery; this antibody interfered with the heparin anti-Xa assay, thereby complicating the monitoring of heparin therapy.