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.

Simultaneous flow cytometric analysis of megakaryocyte polyploidy and a labile intracellular protein using zinc-based fixation.

Differentiating megakaryocytes undergo a unique endomitotic cell cycle leading to large polyploidal cells, which fragment to generate platelets, blood cells important for normal hemostasis. Simultaneous assessment of DNA content and cellular proteins by flow cytometry is a useful tool to study megakaryocyte differentiation and to define expression of proteins important for megakaryocyte development and platelet formation. The usefulness of zinc salt-based fixation (ZBF), a non-crosslinking method of cell fixation that permits downstream analysis of nucleic acids (Jensen et al.

Mechanisms Regulating Acquisition of Platelet-Derived Factor V/Va by Megakaryocytes.

Factor Va serves as the nonenzymatic protein cofactor for the prothrombinase complex, which converts prothrombin to thrombin in the events leading to formation of a hemostatic plug. Several observations support the concept that platelet-derived factor V/Va is physically and functionally distinct and plays a more important role in thrombin generation at sites of vascular injury as compared to its plasma counterpart. Platelet-derived factor V/Va is generated following endocytosis of the plasma-derived molecule by the platelet precursor cells, megakaryocytes, via a two receptor system consisting of low density lipoprotein (LDL) receptor-related protein-1 (LRP-1) and an unidentified specific "binding site".

Platelet shape change and spreading.

Hemostasis is dependent upon the successful recruitment and activation of blood platelets to the site of a breach in the vasculature. Platelet activation stimulates the rapid reorganization of the cortical actin cytoskeleton, resulting in the transformation of platelets from biconcave disks to fully spread cells. During this process, platelets extend filopodia and generate lamellipodia, resulting in a dramatic increase in the platelet surface area.

Platelets and megakaryocytes contain functional nuclear factor-kappaB.

Objective: To investigate the presence and role of NF-kappaB proteins in megakaryocytes and platelets. The nuclear factor-kappaB (NF-kappaB) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-kappaB family members, including the regulatory inhibitor-kappaB (I-kappaB) and I-kappa kinase (IKK) molecules.

Identification of coagulation factor XI as a ligand for platelet apolipoprotein E receptor 2 (ApoER2).

Objective: Factor XI (FXI) promotes hemostasis and thrombosis through enhancement of thrombin generation and has been shown to play a critical role in the formation of occlusive thrombi in arterial injury models. The aim of this study was to investigate the mechanisms governing interactions between FXI and platelets.

Methods And Results: Platelet adhesion to immobilized FXI was abrogated in the presence of the low-density lipoprotein (LDL) receptor antagonist, receptor-associated protein (RAP), soluble recombinant apolipoprotein E receptor 2 (ApoER2), or the LDL-binding domain 1 or 2 of ApoER2.

15-deoxy-delta12,14-PGJ2 enhances platelet production from megakaryocytes.

Thrombocytopenia is a critical problem that occurs in many hematologic diseases, as well as after cancer therapy and radiation exposure. Platelet transfusion is the most commonly used therapy but has limitations of alloimmunization, availability, and expense. Thus, the development of safe, small, molecules to enhance platelet production would be advantageous for the treatment of thrombocytopenia.