Endocytosed factor V is trafficked to CD42b⁺ proplatelet extensions during differentiation of human umbilical cord blood‐derived megakaryocytes

Abstract

Plasma- and platelet-derived factor Va are essential for thrombin generation catalyzed by the prothrombinase complex; however, several observations demonstrate that the platelet-derived cofactor, which is formed following megakaryocyte endocytosis and modification of the plasma procofactor, factor V, is more hemostatically relevant. Factor V endocytosis, as a function of megakaryocyte differentiation and proplatelet formation, was assessed by flow cytometry and microscopy in CD34 hematopoietic progenitor cells isolated from human umbilical cord blood and cultured for 12 days in the presence of cytokines to induce ex vivo differentiation into megakaryocytes. Expression of an early marker of megakaryocyte differentiation, CD41, endocytosis of factor V, and the percentage of CD41 cells that endocytosed factor V increased from days 6 to 12 of differentiation. In contrast, statistically significant decreases in expression of the stem cell marker, CD34, and in the percentage of CD34 cells that endocytosed factor V were observed. A statistically significant increase in the expression of CD42b, a late marker of megakaryocyte differentiation, was also observed over time, such that by Day 12, all CD42b cells endocytosed factor V and expressed CD41. This endocytosed factor V was trafficked to proplatelet extensions and was localized in a punctate pattern in the cytoplasm consistent with its storage in α-granules. In conclusion, loss of CD34 and expression of CD42b define cells capable of factor V endocytosis and trafficking to proplatelet extensions during differentiation of megakaryocytes ex vivo from progenitor cells isolated from umbilical cord blood.

Baffled‐flow culture system enables the mass production of megakaryocytes from human embryonic stem cells by enhancing mitochondrial function

“…For Factor V (FV) uptake assays, differentiated cells were cultured in BEL medium supplemented with 30 nM FV-Atto-488 for 18 h at 37 C. 25,26 FV-treated cells were collected, washed, and then stained with CD41a and CD42b antibodies for 30 min at 4 C. Images were captured using a microscope (Nikon). Stained cells were analysed using flow cytometry.…”

Developments in the production of platelets from stem cells (Review)

“…HSCs, also known as CD34 + cells, are the simplest stem cell source for generating platelets. At present, HSCs are mainly obtained from the BM, umbilical cord blood (UCB) and peripheral blood (27,28).…”