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

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. Factor V is endocytosed from plasma by megakaryocytes to form the unique, physiologically‐relevant platelet‐derived cofactor pool. Endocytosis is developmentally regulated and defined by loss of the stem cell marker, CD34, and expression of the megakaryocyte/platelet specific cell surface protein, CD42b. Endocytosed factor V is trafficked to and localized in a punctate pattern in growing CD42b+ proplatelets.