The incredible journey: From megakaryocyte development to platelet formation

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

Evidence for a cytoplasmic proplatelet promoting factor that triggers platelet production

Not available

α-Adducin dissociates from F-actin and spectrin during platelet activation

Aspectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. α-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites where actin filaments attach to the ends of spectrin molecules. Platelets activated through protease-activated receptor 1, FcγRIIA, or by treatment with PMA phosphorylate adducin at Ser726. Phosphoadducin releases from the membrane skeleton concomitant with its dissociation from spectrin and actin. Inhibition of PKC blunts adducin phosphorylation and release from spectrin and actin, preventing the centralization of spectrin that normally follows cell activation. We conclude that adducin targets actin filament ends to spectrin to complete the assembly of the resting membrane skeleton. Dissociation of phosphoadducin releases spectrin from actin, facilitating centralization of spectrin, and leads to the exposure of barbed actin filament ends that may then participate in converting the resting platelet's disc shape into its active form

Cytoskeletal mechanics of proplatelet maturation and platelet release

New steps in the process of conversion of proplatelet extensions from megakaryocytes into mature platelets are defined

T granules in human platelets function in TLR9 organization and signaling

Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. In this paper, we show that the TLR9 transcript is specifically up-regulated during pro-PLT production and is distributed to a novel electron-dense tubular system-related compartment we have named the T granule. TLR9 colocalizes with protein disulfide isomerase and is associated with either VAMP 7 or VAMP 8, which regulates its distribution in PLTs on contact activation (spreading). Preincubation of PLTs with type IV collagen specifically increased TLR9 and CD62P surface expression and augmented oligodeoxynucleotide (ODN) sequestration and PLT clumping upon addition of bacterial/viral ODNs. Collectively, this paper (a) tracks TLR9 to a new intracellular compartment in PLTs and (b) describes a novel mechanism of TLR9 organization and signaling in human PLTs

PKCε expression is required during proplatelet formation in murine model

Megakaryocytes (MK) remodel their cytoplasm into long proplatelet extensions to generate platelets [1]. We have previously demonstrated that PKCepsilon expression is strictly regulated during megakaryocytopoiesis (MKpoiesis), and its forced expression in the late phases of MK differentiation impairs platelet production [2,3]. However, our preliminary data suggest that PKCepsilon positive platelets may be released around the acute event of myocardial infarction, affecting their aggregation potential. Primary fetal liver (FL) cells isolated from CD1 pregnant mice are the preferential model to study the platelet formation mechanism [4]. Therefore, here we focused on the mouse PKCepsilon positive model to elucidate the role of PKCepsilon in MK maturation. Our data show that not only PKCepsilon expression increases during mouse MK differentiation, but also its forced down-regulation strongly reduces pro-platelet formation. Therefore, PKCepsilon is strongly required for murine proplatelet production. On the basis of these results and other known model systems, we show that PKCepsilon has a relevant role in the completion of platelet release

Dynamic actin/septin network in megakaryocytes coordinates proplatelet elaboration

Megakaryocytes (MK) undergo extensive cytoskeletal rearrangements as they give rise to platelets. While cortical microtubule sliding has been implicated in proplatelet formation, the role of the actin cytoskeleton in proplatelet elongation is less understood. It is assumed that actin filament reorganization is important for platelet generation given that mouse models with mutations in actin-associated proteins exhibit thrombocytopenia. However, due to the essential role of the actin network during MK development, a differential understanding of the contribution of the actin cytoskeleton on proplatelet release is lacking. Here, we reveal that inhibition of actin polymerization impairs the formation of elaborate proplatelets by hampering proplatelet extension and bead formation along the proplatelet shaft, which was mostly independent of changes in cortical microtubule sliding. We identify Cdc42 and its downstream effectors, septins, as critical regulators of intracellular actin dynamics in MK, inhibition of which, similarly to inhibition of actin polymerization, impairs proplatelet movement and beading. Super-resolution microscopy revealed a differential association of distinctive septins with the actin and microtubule cytoskeleton, respectively, which was disrupted upon septin inhibition and diminished intracellular filamentous actin dynamics. In vivo, septins, similarly to F-actin, were subject to changes in expression upon enforcing proplatelet formation through prior platelet depletion. In summary, we demonstrate that a Cdc42/septin axis is not only important for MK maturation and polarization, but is further required for intracellular actin dynamics during proplatelet formation

Anti-apoptotic BCL2L2 increases megakaryocyte proplatelet formation in cultures of human cord blood

Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigorously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34-positive (+) cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41aHigh CD42aHigh phosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The SHG cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic morphology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakaryocyte apoptosis, increased CD41a+ LLG cells, and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing BCL2L2. BCL2L2 also induced small, but significant increases in thrombin-induced platelet-like particle αIIbβ3 activation and P-selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems

Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells