The incredible journey: From megakaryocyte development to platelet formation

Abstract

Circulating blood platelets are specialized cells that prevent bleeding and minimize blood vessel injury. Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets. Given the need for platelets in many pathological situations, understanding how this process occurs is an active area of research with important clinical applications.

Figure 1.

Schematic of platelet production. (1) HSCs in the bone marrow differentiate into MKs in a TPO-dependent manner. (2) MKs undergo endomitosis and develop nuclei ranging in DNA content from 2n to 128n. (3) As MKs mature, they develop a highly invaginated membrane throughout their cytoplasm, which is continuous with the external plasma membrane. This membrane serves as a reservoir for proplatelet formation. (4) MKs migrate to the vascular niche, where they extend proplatelets and release them into vascular sinusoids. The entire MK is converted into pre/proplatelets, and its nucleus is exuded and phagocytosed. (5) Once in the bloodstream, proplatelets interconvert into preplatelets. (6) A fission event creates two platelets from a barbell proplatelet.

Figure 2.

Transmission electron micrographs of murine MKs, preplatelets, proplatelets, and platelets. MK cultures generated from murine fetal liver cells were fixed with 1.25% paraformaldehyde, 0.03% picric acid, and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 1 h, postfixed with 1% osmium tetroxide, dehydrated through a series of alcohols, infiltrated with propylene oxide, and embedded in epoxy resin. Ultrathin sections were stained and examined with an electron microscope (Tecnai G2 Spirit BioTWIN; FEI Company) at an accelerating voltage of 80 kV. Images were recorded with a charge-coupled device camera (2K; Advanced Microscopy Techniques) using digital acquisition and analysis software. (A) Overview of one MK showing multilobulated nucleus and IMS. (B) MK with a highly developed IMS. (C) Released preplatelets (#), proplatelets (*), and platelets (^). (D) Detailed view of platelets (bottom right) and an MK, highlighting its contents. N, nucleus; IMS, invaginated membrane system; G, granule; M, mitochondria; V, multivesicular body.

Figure 3.

Microtubules in proplatelets and platelets. Microtubules composed of β1-tubulin line the shafts of proplatelet extensions and form a microtubule coil in nascent platelet tips and released preplatelets/platelets. To delineate the microtubule cytoskeleton, murine fetal liver MK-generated samples were incubated with a rabbit polyclonal primary antibody for β1-tubulin, washed, and probed with a secondary Alexa Fluor 488 nm–conjugated antibody. MKs were imaged on a microscope (Eclipse TE2000-E; Nikon) equipped with a 63× objective, NA 1.4, and 1.5× optivar. Images were acquired with a charge-coupled device camera (ORCA-II-ER; Hamamatsu Photonics). Image acquisition was under the control of MetaMorph software (Molecular Devices). (A–C) Images highlight the branching of proplatelets (A), heterogeneous mix of platelets, pre- and proplatelets released from MKs (B), and the figure 8 structure seen in preplatelet to proplatelet interconversion (C).

Figure 4.

Trafficking of α-granules. Granules are packaged in MKs, trafficked along microtubules lining proplatelet (Proplt) shafts, and captured in nascent platelet tips. To visualize α-granules, murine fetal liver–derived MKs were incubated overnight with 150 µg/ml Oregon green 488 human fibrinogen conjugate, which they take up and package into their α-granules. MKs were then washed by albumin gradient sedimentation, and the resuspended pellet was placed in a video chamber. MKs were imaged on a microscope (Nikon) equipped with a 100× objective, NA 1.4, and were acquired with a charge-coupled device camera (ORCA-II-ER). Image acquisition was under the control of MetaMorph software. (A and B) Images visualize MKs actively releasing proplatelets (A) and released proplatelets (B).