Mechanisms of organelle transport and capture along proplatelets during platelet production

Megakaryocytes generate platelets by remodeling their cytoplasm into long proplatelet extensions, which serve as assembly lines for platelet production. Platelet packaging and release concludes at the tips of each proplatelet. Essential in this process is the distribution of organelles and platelet-specific granules into the nascent platelets. To investigate the mechanism of delivery of organelles into putative platelets, the distribution and dynamics of organelles/granules was monitored. Individual organelles are sent from the cell body to the proplatelets where they move bidirectionally until they are captured at proplatelet ends. Movement occurs at approximately 0.2 microm/min, but pauses and changes in direction are frequent. At any given time, approximately 30% of organelles/granules are in motion. Actin poisons do not diminish organelle motion, and vesicular structures are intimately associated with the microtubules. Therefore, movement appears to involve microtubule-based forces. Bidirectional organelle movement is conveyed by the bipolar organization of microtubules within the proplatelet, as kinesin-coated beads move bidirectionally on the microtubule arrays of permeabilized proplatelets. Movement of organelles along proplatelets involves 2 mechanisms: organelles travel along microtubules, and the linked microtubules move relative to each other. These studies demonstrate that the components that form platelets are delivered to and assembled de novo along proplatelets.     

Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis

To produce blood platelets, megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P(2), a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P(2) in the DMS occurs in proximity to PI-5-P-4-kinase alpha (PIP4Kalpha), and short hairpin (sh) RNA-mediated loss of PIP4Kalpha impairs both DMS development and expansion of megakaryocyte size. Thus, PI-4,5-P(2) is a marker and possibly essential component of internal membranes. PI-4,5-P(2) is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P(2) in the megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P(2) might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis.     

Immune thrombocytopenia: antiplatelet autoantibodies inhibit proplatelet formation by megakaryocytes and impair platelet production in vitro

Primary immune thrombocytopenia is an autoimmune disease mediated by antiplatelet autoantibodies that cause platelet destruction and suppression of platelet production. In vitro effects of autoantibodies on megakaryocyte production and maturation have been reported recently. However, the impact of these autoantibodies on crucial megakaryocyte functions, proplatelet formation and subsequent platelet release, has not been evaluated. We examined the effects of serum and IgG from 19 patients with immune thrombocytopenia using day 8 or 9 megakaryocytes (66.3 ± 10.6% CD41⁺), derived from cord blood hematopoietic stem cells (CD34⁺). The number of proplatelet-bearing megakaryocytes, the number of platelets released in the culture, total megakaryocyte numbers, ploidy pattern and caspase activation were measured at various times after treatment. After 5 days of treatment the number of proplatelet-bearing megakaryocytes was significantly decreased by 13 immune thrombocytopenia autoantibodies relative to the control group (P<0.0001) and this decrease was accompanied by a corresponding reduction of platelet release. Other features, including total megakaryocyte numbers, maturation and apoptosis, were not affected by immune thrombocytopenia antibodies. Treating the megakaryocytes with the thrombopoietin receptor agonists romiplostim and eltrombopag reversed the effect of the autoantibodies on megakaryocytes by restoring their capacity to form proplatelets. We conclude that antiplatelet antibodies in immune thrombocytopenia inhibit proplatelet formation by megakaryocytes and hence the ability of the megakaryocytes to release platelets. Treatment with either romiplostim or eltrombopag regenerates proplatelet formation from the megakaryocytes.     

Absence of GPIbalpha is responsible for aberrant membrane development during megakaryocyte maturation: ultrastructural study using a transgenic model

OBJECTIVE: The glycoprotein Ib/IX/V complex (GPIb-IX-V) mediates platelet attachment to von Willebrand factor in exposed subendothelium. Molecular defects in the genes for GPIbalpha, GPIbbeta, and GPIX give rise to the Bernard-Soulier syndrome, in which thrombocytopenia and giant platelets suggest that this receptor also is involved in platelet production. To study how giant platelets are produced in vivo, we used a model of GPIbalpha-deficient mice (GPIbalpha(null)) and mice rescued with the human GPIbalpha transgene (GPIbalpha(null;hTg)). MATERIALS AND METHODS: Using electron microscopy and immunogold labeling, we examined megakaryocytopoiesis in the bone marrow of these mice and developed a method to quantify the membranes of megakaryocytes (MK) and proplatelets by computer analysis. RESULTS: Abnormal membrane development in the perinuclear zone was found in immature MK of GPIbalpha(null) mice. This led to a poorly developed demarcation membrane system and other ultrastructural changes. As a result, well-organized platelet territories were rarely seen within the cytoplasm of mature MK. Membrane quantification confirmed that MK of GPIbalpha(null) mice had a reduced internal membrane pool. Whereas these MK normally crossed the endothelial barrier, their migration was accompanied by the production of unusually large MK fragments or proplatelets in the vascular sinus with an approximately 50% decrease in internal membrane content compared to wild-type. In the rescued GPIbalpha(null;hTg) model, GPIbalpha was normally localized in MK, and there was a total correction of the ultrastructural defects. CONCLUSIONS: GPIbalpha is essential for membrane development and distribution in maturing MK. Its absence leads to abnormal partitioning of the membrane systems and abnormal proplatelet production.     

Rho GTPases and their downstream effectors in megakaryocyte biology

Megakaryocytes differentiate from hematopoietic stem cells in the bone marrow. The transition of megakaryocytes to platelets is a complex process. Thereby, megakaryocytes extend proplatelets into sinusoidal blood vessels, where the proplatelets undergo fission to release platelets. Defects in platelet production can lead to a low platelet count (thrombocytopenia) with increased bleeding risk. Rho GTPases comprise a family of small signaling G proteins that have been shown to be master regulators of the cytoskeleton controlling many aspects of intracellular processes. The generation of Pf4-Cre transgenic mice was a major breakthrough that enabled studies in megakaryocyte-/platelet-specific knockout mouse lines and provided new insights into the central regulatory role of Rho GTPases in megakaryocyte maturation and platelet production. In this review, we will summarize major findings on the role of Rho GTPases in megakaryocyte biology with a focus on mouse lines in which knockout strategies have been applied to study the function of the best-characterized members Rac1, Cdc42 and RhoA and their downstream effector proteins.     

Megakaryocytes from CYCS mutation‐associated thrombocytopenia release platelets by both proplatelet‐dependent and ‐independent processes

Thrombocytopenia Cargeeg is a rare autosomal dominant disorder and one of three thrombocytopenias caused by mutation of cytochrome c (Online Mendelian Inheritance in Man entry THC4). Our previous observations of platelet‐like structures in the marrow space and early platelet production in vitro suggested that the low platelet phenotype in Thrombocytopenia Cargeeg subjects is caused by premature release of platelets into non‐vascular regions of the bone marrow. We now show that two processes of platelet release occur in Thrombocytopenia Cargeeg subjects. Circulating platelets have a normal marginal microtubule coil, and cultured megakaryocytes derived from peripheral blood cells of Thrombocytopenia Cargeeg subjects form proplatelets normally and release platelets containing a marginal microtubule coil, consistent with effective platelet release via the proplatelet mechanism. In contrast, platelet‐like structures within the extravascular bone marrow space have the dimensions of platelets but lack the marginal microtubule coil, suggesting abnormal proplatelet‐independent platelet release. The mechanism of extravascular platelet release remains unclear. The failure to recapitulate this mechanism in vitro implies that the phenotype is not simply an intrinsic property of CYCS mutation‐carrying megakaryocytes, but is dependent on the interaction between these cells and their environment.     

Recombinant human c-Mpl ligand is not a direct stimulator of proplatelet formation in mature human megakaryocytes

To evaluate the effect of the c-Mpl ligand on platelet production by megakaryocytes, we investigated proplatelet formation in isolated human megakaryocytes cultured in serum-free medium, with or without the c-Mpl ligand, interleukin-6 and erythropoietin. When interleukin-6 was added to the culture medium, the percentage of megakaryocytes displaying proplatelets was approximately 1.5-fold the control value; whereas, in the presence of the c-Mpl ligand, the percentage of megakaryocytes displaying proplatelets decreased in a dose-dependent manner and did not increase compared to control values at any dose tested. However, the viability of megakaryocytes after a 4 d culture in the presence of the c-Mpl ligand was significantly higher than that of the cells cultured without it. The c-Mpl ligand did not stimulate the proplatelet formation in megakaryocytes in vitro     

Regulation of proplatelet formation and platelet release by integrin alpha IIb beta3

Mature megakaryocytes form structures called proplatelets that serve as conduits for platelet packaging and release at vascular sinusoids. Since the megakaryocyte expresses abundant levels of integrin alpha IIb beta3, we have examined a role for fibrinogen in proplatelet development and platelet release alongside that of other matrices. Primary mature murine megakaryocytes from bone marrow aspirates readily formed proplatelets when plated on fibrinogen at a degree that was significantly higher than that seen on other matrices. In addition, alpha IIb beta3 was essential for proplatelet formation on fibrinogen, as megakaryocytes failed to develop proplatelets in the presence of alpha IIb beta3 antagonists. Interestingly, inhibition of Src kinases or Ca2+ release did not inhibit proplatelet formation, indicating that alpha IIb beta3-mediated outside-in signals are not required for this response. Immunohistochemical studies demonstrated that fibrinogen is localized to the bone marrow sinusoids, a location that would allow it to readily influence platelet release. Further, thrombopoietin-stimulated alpha IIb-/- mice had a reduced increase in platelet number relative to controls. A similar observation was not observed for platelet recovery in alpha IIb-/- mice in response to antibody-induced thrombocytopenia, indicating the existence of additional pathways of regulation of proplatelet formation. These results demonstrate that fibrinogen is able to regulate proplatelet formation via integrin alpha IIb beta3.     

Recombinant human c-Mpl ligand is not a direct stimulator of proplatelet formation in mature human megakaryocytes

To evaluate the effect of the c-Mpl ligand on platelet production by megakaryocytes, we investigated proplatelet formation in isolated human megakaryocytes cultured in serum-free medium, with or without the c-Mpl ligand, interleukin-6 and erythropoietin. When interleukin-6 was added to the culture medium, the percentage of megakaryocytes displaying proplatelets was approximately 1.5-fold the control value; whereas, in the presence of the c-Mpl ligand, the percentage of megakaryocytes displaying proplatelets decreased in a dose-dependent manner and did not increase compared to control values at any dose tested. However, the viability of megakaryocytes after a 4 d culture in the presence of the c-Mpl ligand was significantly higher than that of the cells cultured without it. The c-Mpl ligand did not stimulate the proplatelet formation in megakaryocytes in vitro     

Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes

Megakaryocytes are terminally differentiated cells that, in their final hours, convert their cytoplasm into long, branched proplatelets, which remodel into blood platelets. Proplatelets elongate at an average rate of 0.85 microm/min in a microtubule-dependent process. Addition of rhodamine-tubulin to permeabilized proplatelets, immunofluorescence microscopy of the microtubule plus-end marker end-binding protein 3 (EB3), and fluorescence time-lapse microscopy of EB3-green fluorescent protein (GFP)-expressing megakaryocytes reveal that microtubules, organized as bipolar arrays, continuously polymerize throughout the proplatelet. In immature megakaryocytes lacking proplatelets, microtubule plus-ends initiate and grow by centrosomal nucleation at rates of 8.9 to 12.3 microm/min. In contrast, plus-end growth rates of microtubules within proplatelets are highly variable (1.5-23.5 microm/min) and are both slower and faster than those seen in immature cells. Despite the continuous assembly of microtubules, proplatelets continue to elongate when net microtubule assembly is arrested. One alternative mechanism for force generation is microtubule sliding. Triton X-100-permeabilized proplatelets containing dynein and its regulatory complex, dynactin, but not kinesin, elongate with the addition of adenosine triphosphate (ATP) at a rate of 0.65 microm/min. Retroviral expression in megakaryocytes of dynamitin (p50), which disrupts dynactin-dynein function, inhibits proplatelet elongation. We conclude that while continuous polymerization of microtubules is necessary to support the enlarging proplatelet mass, the sliding of overlapping microtubules is a vital component of proplatelet elongation.     

In vitro culture of murine megakaryocytes from fetal liver-derived hematopoietic stem cells

Megakaryocytes (MKs) are specialized precursor cells committed to producing and proliferating platelets. In a cytoskeletal-driven process, mature MKs generate platelets by releasing thin cytoplasmic extensions, named proplatelets, into the sinusoids. Due to knowledge gaps in this process and mounting clinical demand for non-donor-based platelet sources, investigators are successfully developing artificial culture systems to recreate the environment of platelet biogenesis. Nevertheless, drawbacks in current methods entail elaborate procedures for stem cell enrichment, extensive growth periods, low MK yield, and poor proplatelet production. We propose a simple, robust method of primary MK culture that utilizes fetal livers from pregnant mice. Our technique reduces expansion time to 4 days, and generates ~15,000–20,000 MKs per liver. Approximately, 20–50% of these MKs produce structurally dense, high-quality proplatelets. In this review, we outline our method of MK culture and isolation.     

TUBB1 mutation disrupting microtubule assembly impairs proplatelet formation and results in congenital macrothrombocytopenia

This report describes a family with TUBB1‐associated macrothrombocytopenia diagnosed based on abnormal platelet β1‐tubulin distribution. A circumferential marginal microtubule band was undetectable, whereas microtubules were frayed and disorganized in every platelet from the affected individuals. Patients were heterozygous for novel TUBB1 p.F260S that locates at the α‐ and β‐tubulin intradimer interface. Mutant β1‐tubulin was not incorporated into microtubules with endogenous α‐tubulin, and α‐tubulin expression was decreased in transfected Chinese hamster ovary cells. Transduction of mutant β1‐tubulin into mouse fetal liver‐derived megakaryocytes demonstrated no incorporation of mutant β1‐tubulin into microtubules with endogenous α‐tubulin and diminished proplatelet formation, leading to the production of fewer, but larger, proplatelet tips. Furthermore, mutant β1‐tubulin was not associated with endogenous α‐tubulin in the proplatelets. Deficient functional microtubules might lead to defective proplatelet formation and abnormal protrusion‐like platelet release, resulting in congenital macrothrombocytopenia.     

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

Background

Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia.

Design and Methods

To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbβ. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells.

Results

The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbβ^−/− bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbβ^−/− megakaryocytes could be differentiated in culture from Lin⁻ fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbβ^−/− cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbβ^−/− released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbβ^−/− proplatelet buds in cultured and circulating platelets.

Conclusions

Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.     

Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration

Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1(-/-) mouse model. Cib1(-/-) mice have more platelets and BM megakaryocytes than wild-type (WT) controls (P < .05). Furthermore, subsequent analysis of megakaryocyte-CFU production revealed an increase with Cib1 deletion compared with WT (P < .05). In addition, BM from Cib1(-/-) mice, cultured with thrombopoietin (TPO) for 24 hours, produced more highly polyploid megakaryocytes than WT BM (P < .05). Subsequent analysis of TPO signaling revealed enhanced Akt and ERK1/2 phosphorylation, whereas FAK(Y925) phosphorylation was reduced in Cib1(-/-) megakaryocytes treated with TPO. Conversely, platelet recovery in Cib1(-/-) mice after platelet depletion was attenuated compared with WT (P < .05). This could be the result of impaired adhesion and migration, as adhesion to fibrinogen and fibronectin and migration toward an SDF-1α gradient were reduced in Cib1(-/-) megakaryocytes compared with WT (P < .05). In addition, Cib1(-/-) megakaryocytes formed fewer proplatelets compared with WT (P < .05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in megakaryopoiesis, initially by negatively regulating TPO signaling and later by augmenting proplatelet production.     

Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis, and pyropoikilocytosis

The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.     

The demarcation membrane system of the megakaryocyte: a misnomer?

The concept that the demarcation membrane system delineates platelets within the cytoplasm of megakaryocytes has been examined. In short-term culture of mouse bone marrow, mature megakaryocytes extended long, attenuated processes that were found by electron microscopy to have a limited amount of invaginated membrane. When such megakaryocytes were exposed to microtubule depolymerizing agents, the attenuated processes retracted, became thicker, and an extensive demarcation membrane reappeared. It is suggested from the results that the demarcation membrane system functions to provide a membrane reserve that undergoes evagination during the formation of attenuated processes and thereby envelops putative platelets, rather than to demarcate platelets in the maturing megakaryocyte. The term "invaginated membrane system" is considered more appropriate than "demarcation membrane system."     

A novel method for automated assessment of megakaryocyte differentiation and proplatelet formation

Transfusion of platelet concentrates represents an important treatment for various bleeding complications. However, the short half-life and frequent contaminations with bacteria restrict the availability of platelet concentrates and raise a clear demand for platelets generated ex vivo. Therefore, in vitro platelet generation from megakaryocytes represents an important research topic. A vital step for this process represents accurate analysis of thrombopoiesis and proplatelet formation, which is usually conducted manually. We aimed to develop a novel method for automated classification and analysis of proplatelet-forming megakaryocytes in vitro. After fluorescent labelling of surface and nucleus, MKs were automatically categorized and analysed with a novel pipeline of the open source software CellProfiler. Our new workflow is able to detect and quantify four subtypes of megakaryocytes undergoing thrombopoiesis: proplatelet-forming, spreading, pseudopodia-forming and terminally differentiated, anucleated megakaryocytes. Furthermore, we were able to characterize the inhibitory effect of dasatinib on thrombopoiesis in more detail. Our new workflow enabled rapid, unbiased, quantitative and qualitative in-depth analysis of proplatelet formation based on morphological characteristics. Clinicians and basic researchers alike will benefit from this novel technique that allows reliable and unbiased quantification of proplatelet formation. It thereby provides a valuable tool for the development of methods to generate platelets ex vivo and to detect effects of drugs on megakaryocyte differentiation.