Mauro Torti, Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy. Tel.: +39 0382 987238; fax: +39 0382 987240. e-mail: email@example.com
Summary. Background: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized. Objective: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors. Methods: Human megakaryocytes were obtained by differentiation of cord blood-derived CD34+ cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: We found that human megakaryocytes extended proplatelets in a time-dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin αIIbβ3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb-IX-V, or upon cleavage of GPIbα by the metalloproteinase mocarhagin. Membrane-associated VWF was detected exclusively on proplatelet-forming megakaryocytes, but not on round mature cells that do not extend proplatelets. Conclusions: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio-temporal regulation orchestrated by adhesive proteins, GPIb-IX-V and myosin IIA.
Platelets are formed and released into the bloodstream by the megakaryocytes, within the bone marrow, which extend long, branching filaments called proplatelets that assemble nascent platelets at their tips [1,2]. The signals that initiate and regulate proplatelet formation (PPF) are still poorly understood, but the characteristics of the microenvironment surrounding megakaryocytes may play an important role [3–5]. Recently, it has been demonstrated that interaction of primary murine megakaryocytes with fibrinogen stimulates PPF, while other adhesive proteins, including von Willebrand factor (VWF) are less efficient . Nevertheless, antibodies against the main VWF receptor GPIb-IX-V strongly inhibited proplatelet production by human cultured megakaryocytes , and abnormalities of GPIb-IX-V expression or interaction with VWF cause impaired megakaryocytopoiesis and thrombocytopenia [8,9]. In contrast, type I collagen is unable to support PPF, and may prevent premature release of platelets within the bone marrow [6,10,11]. The negative regulation of PPF by collagen is mediated by the interaction with integrin α2β1, and involves the Rho/ROCK pathway [10,12]. As ROCK is implicated in the phosphorylation of the myosin light chain , these findings point to a negative role of the actin-myosin cytoskeleton in the regulation of PPF. This is consistent with the evidence that PPF is increased in megakaryocytes differentiated from mouse embryonic stem cells deficient in the myosin heavy chain IIA . Interestingly, mutations in the MYH9 gene encoding for myosin heavy chain IIA results in different autosomal dominant disorders characterized by thrombocytopenia with giant platelets . In these patients, a reduced expression and an abnormal reorganization of myosin IIA may be responsible for a defective platelet production [16,17].
In this study, we analyze the role of different adhesive proteins and membrane receptors in the regulation of PPF by human megakaryocytes. We propose that the interaction with different adhesive proteins does not stimulate but rather represses the otherwise spontaneous process of PPF, thus resulting in a spatio-temporal regulation of the entire process of platelet release. We also demonstrate that myosin IIA and GPIb-IX-V play essential roles in the adhesive protein-regulated PPF.
Materials and methods
Differentiation of megakaryocytes from human umbilical cord blood-derived CD34+ cells and evaluation of PPF
Upon informed consent of the parents, human umbilical cord blood was collected after normal pregnancies and deliveries, and processed within 48 h. CD34+ cells were separated as previously described , and cultured for 12 days in Stem Span medium (Stem-Cell Technologies, Vancouver, BC, Canada) supplemented with 10 ng mL−1 TPO, interleukin-6 (IL-6), IL-11 (PeproTech EC Ltd, London, UK) at 37 °C in a 5% CO2 fully-humidified atmosphere .
To evaluate PPF using megakaryocytes grown in suspension, cells at day 12 of culture were harvested and plated in fresh Stem Span medium supplemented with TPO in 24-well plates (1 × 105 cells per well). After increasing times, proplatelet-bearing megakaryocytes were counted by phase-contrast microscopy. Moreover, cells were harvested from parallel samples, cytospun on glass coverslips and double-stained with antibodies against CD41 and α-tubulin. Proplatelets-forming megakaryocytes were identified as large CD41+ cells extending α-tubulin-positive long filamentous structures. The extent of PPF was calculated as the percentage of proplatelet-bearing CD41+ cells with respect to total CD41+ cells.
To analyze PPF onto different adhesive substrates, 12-mm glass coverslips were coated with 100 μg mL−1 fibrinogen or 25 μg mL−1 fibronectin (Sigma, Milan, Italy), 10 μg mL Sigma VWF (Haemate P; Aventis-Behring, Milan, Italy) for 2 h at room temperature (RT), or with 25 μg mL−1 type I, type III or type IV collagen (provided by Prof. Tira and Dr Gruppi, University of Pavia or by Sigma) overnight at 4 °C, and subsequently blocked with 1% bovine serum albumin (BSA) for 1 h at RT. Cells at day 12 of culture were harvested, plated onto substrate-coated coverslips in 24-wells plates (1 × 105 cells per well) and allowed to adhere for 4 h at 37 °C and 5% CO2. PPF was then evaluated by phase-contrast microscopy, as well as by fluorescence microscopy, as described above. Cell spreading was determined by double staining of cells, in parallel coverslips, with anti-CD41 antibody and TRITC-conjugated phalloidin. CD41+ cells exhibiting actin stress fibres were counted as spreaded megakaryocytes.
In some experiments, megakaryocytes were pre-incubated with 1 mm RGDS, anti-GPIbα monoclonal antibodies AK2 (5 μg mL−1; Serotec, Oxford, UK), AN51 (0.3 μg mL−1; Dako, Glostrup, Denmark), the anti-integrin β3 subunit monoclonal antibody SZ21 (5 μg mL−1; Immunotech, Marseille, France) or with the metalloproteinase mocarhagin (10 μg mL−1), purified from the crude venom of the snake Mocambique mocambique (Sigma) as described , prior to being plated or allowed to adhere to different matrices. Negative controls were prepared with cells incubated with identical concentrations of unrelated isotype-matched IgG.
Immunofluorescence and confocal microscopy analysis
Megakaryocytes were fixed in 3% paraformaldehyde for 20 min at RT, permeabilized by 0.5% Triton X-100 for 5 min, and subsequently blocked with 3% BSA and 10% fetal bovine serum (FBS) in phosphate-buffered saline (PBS) for 1 h at RT. Cells were then incubated with the following primary antibodies diluted in PBS for 1 h at RT: anti-CD41, clone P2, 1:100 (Immunotech); anti-α-tubulin, clone DM1A, 1:700 (Sigma); anti-GPIbα, clone AK2, 1:100; anti-GPV, clone CLB-SW16, 1:50 (Sanquin, Amsterdam, the Netherlands). To evaluate stress fibres formation, cells were stained with TRITC-conjugated phalloidin, 1:500 (Sigma). After washing with PBS, cells were incubated with 10 μg mL−1 of the appropriate secondary antibody conjugated with either Alexa Fluor 594 or Alexa Fluor 488 (Invitrogen, Milan, Italy) in PBS for 1 h at RT. Nuclear counterstaining was performed with Hoechst 33258 (100 ng mL−1 in PBS). Specimens were mounted in Mowiol 4–88. Negative controls were routinely performed by omitting the primary antibody.
Conventional fluorescence microscopy was performed through an Olympus BX51 (Hamburg, Germany) microscope, using a 63×/1.25 or a 100×/1.30 UplanF1 oil-immersion objective. For each specimen at least 100 megakaryocytes were observed.
Confocal analysis was performed using the TCS SPII confocal laser scanning microscopy system (Leica, Heidelberg, Germany), equipped with a Leica DM IRBE inverted microscope, as described . For each specimen at least 20 megakaryocytes were examined.
anova was used to analyze data, with a significant difference set at P < 0.05. Data are presented as mean ± SD.
Kinetics of PPF by human megakaryocytes
When plated in fresh medium, mature cord blood-derived megakaryocytes extended proplatelets in a time-dependent manner (Fig. 1A). PPF was detectable only after 8 h, and increased progressively up to 24 h, when about 25% of megakaryocytes extended proplatelets. Prolonged incubation did not result in any further increase of PPF. However, as the entire process of PPF was typically completed in only a few hours, leading to the complete fragmentation of the parental megakaryocyte and to the concomitant release of single platelets-like fragments (Fig. 1B,C), evidence that the percentage of proplatelet-forming megakaryocytes remained constant, indicated that the process was not arrested but continued, at an approximately constant rate, up to 72 h. Prolonged analysis revealed that PPF rapidly declined afterwards, and terminated within 120 h (Fig.1A).
Regulation of PPF by megakaryocyte adhesion
Megakaryocytes were then plated on fibrinogen, VWF, fibronectin or collagens, and PPF was monitored over 72 h. Adhesion to fibrinogen, fibronectin or VWF efficiently supported PPF (Fig. 2A–C), but the kinetics and the magnitude of this process was definitively different from that of non-adherent megakaryocytes. Upon adhesion to fibrinogen, PPF was already evident after 4 h, but involved a lower percentage of cells (about 10%) and did not increase further over time. As a consequence, at 24 h the percentage of PPF was about 2.5-fold higher in megakaryocytes in suspension than among fibrinogen-adherent cells (Figs 1A and 2A). Finally, PPF on fibrinogen progressively decreased after 24 h, revealing that the process was temporally restricted. The kinetics of PPF by megakaryocytes adhering to VWF shared many similarities with that observed on the fibrinogen matrix, but revealed important peculiarities (Fig. 2B). After 4 h, PPF was evident, but involved only 2% of the adherent megakaryocytes. This process increased progressively with time, and reached the maximum after 16 h when about 15% of the VWF-adherent megakaryocytes were found to extend proplatelets. As for fibrinogen, the formation of proplatelets on VWF appeared to decline afterwards and was almost completely terminated after 72 h. Different preparations of VWF were used in our study and comparable results were obtained (data not shown). Similar to fibrinogen and VWF, fibronectin was also found to support PPF, although the rate and the magnitude of this process were reduced (Fig. 2C). In particular, we found that PPF by megakaryocytes adhering to fibronectin was evident only after 8 h, and the maximal percentage of proplatelet-forming cells was observed after 24 h but never exceeded 5% (Fig. 2C). We found that megakaryocytes adhering to type I collagen were unable to extend proplatelets (Fig. 2D). However, PPF was observed upon adhesion to type III and type IV collagens (Fig. 2E,F). The kinetics of this process were slower than that seen on fibrinogen or on VWF, but were comparable to that seen on fibroncetin.
The role of GPIb-IX-V and integrin αIIbβ3 on PPF
We next evaluated the contribution of the main platelet adhesive receptors, integrin αIIbβ3 and GPIb-IX-V, on PPF by adherent cells. Megakaryocytes were plated on VWF or fibrinogen in the presence of unrelated IgG or monoclonal antibodies AN51 or AK2, raised against different epitopes of GPIbα . The monoclonal antibody SZ21 and the RGDS peptide were used to antagonize αIIbβ3. Table 1 shows that all of these antibodies and the RGDS peptide inhibited the adhesion of megakaryocytes to VWF by about 30–40%. Interestingly, spreading of adherent cells was not affected by the two anti-GPIbα antibodies, but was strongly reduced by the anti-αIIbβ3 antibody SZ21, and almost totally suppressed by the RGDS peptide. Antagonists of αIIbβ3 did not significantly alter PPF by megakaryocytes adherent to VWF (Fig. 3B). In contrast, virtually no proplatelets were produced in the presence of the anti-GPIbα antibodies AN51 and AK2 (Fig. 3B). When megakaryocytes were plated on fibrinogen, the anti-GPIbα antibodies did not alter adhesion or spreading, which were reduced or almost completely suppressed by the anti-αIIbβ3 antibody SZ21 and by the RGDS peptide (Table 1). Nevertheless, αIIbβ3 antagonists did not cause any significant reduction of PPF, which was totally suppressed by the two anti-GPIbα antibodies (Fig. 3A). These results indicate that, independent of the nature of the extracellular matrix, PPF is regulated through GPIbα, rather than αIIbβ3. This conclusion was also supported by the finding that PPF using non-adhering megakaryocytes was completely abolished by anti-GPIbα monoclonal antibodies, but not by αIIbβ3 antagonists (Fig. 3C).
Table 1. Role of GPIb-IX-V and αIIbβ3 on megakaryocyte adhesion and spreading on fibrinogen and von Willebrand factor (VWF)
Results are reported as the percentage of adherent or spread out megakaryocytes treated with the anti-GPIbα antibodies AN51 or AK2, the anti-β3 subunit antibody SZ21 or the peptide RGDS compared with the corresponding samples treated with isotype-matched IgG (control). Measurements have been performed after 16 h of adhesion, and results are the mean ± SD of three different experiments.
% of adhesion relative to control
90 ± 5
95 ± 5
70 ± 3
65 ± 20
57 ± 2
60 ± 1
68 ± 7
60 ± 17
% of spreading relative to control
76 ± 10
88 ± 2
20 ± 3
10 ± 5
113 ± 38
104 ± 50
40 ± 25
5 ± 2
To confirm the essential role of GPIα in PPF, we treated megakaryocytes with the metalloproteinase mocarhagin, which cleaves the extracellular portion of GPIbα. Incubation with mocarhagin removed GPIbα from the megakaryocyte surface, without altering the expression of αIIbβ3 or GPV (Fig. 4A,B). Mocarhagin reduced adhesion to VWF and fibrinogen by about 90% (±10%, n = 3) and 50% (±5%, n = 3), respectively, without affecting cell viability, which remained as high as 95% (±5%n = 3). Figure 4C shows that mocarhagin-treated megakaryocytes were almost completely unable to extend proplatelets, not only when plated on VWF or fibrinogen, but also when maintained in suspension. These results clearly indicate an essential role for the extracellular domain of GPIbα in PPF. As GPIbα contains binding sites for VWF, which is stored into intracellular α-granules and may be released in the extracellular environment, we investigated whether VWF could be detected on the surface of cultured megakaryocycets. By immunostaining of intact mature megakaryocytes with a specific antibody, we have been able to detect membrane-associated VWF exclusively on proplatelet-forming megakaryocytes, but not on mature megakaryocytes not extending proplatelets (Fig. 5). Similarly, we also obtained evidence indicating P-selectin exposure on proplatelet-forming megakaryocytes (data not shown). These results indicate that GPIb-IX-V may be ligand occupied during the process of proplatelet extension.
Myosin IIA is required for collagen-induced inhibition of PPF
Recent works have reported that intracellular myosin IIA may negatively regulate PPF both in mice and humans [12,14]. We investigated whether myosin IIA could also modulate proplatelet extension by adherent megakaryocytes, using the selective antagonist of myosin IIA ATPase activity blebbistatin. Figure 6A shows that PPF by megakaryocytes in suspension, or adherent to fibrinogen, VWF or type III collagen was not significantly altered by blebbistatin. However, we observed that blebbistatin-treated megakaryocytes were able to extend proplatelets even upon adhesion to type I collagen (Fig. 6B). In particular, after 16 h, the percentage of PPF on type I collagen was comparable to that observed on fibrinogen. Therefore, blebbistatin overcome the inhibitory effect of type I collagen of PPF.
In this study, we have compared the kinetics of PPF using human umbilical cord blood-derived megakaryocytes in suspension or adherent to immobilized fibrinogen, fibronectin, VWF or collagens. We demonstrate that the interaction with adhesive proteins does not stimulate, but actually represses and temporally restricts the spontaneous extension of proplatelets. In addition, we have demonstrated that PPF requires the expression of a functional GPIb-IX-V, and is negatively regulated through intracellular myosin IIA.
We found that megakaryocyte adhesion to fibrinogen, fibronectin or VWF initially appeared to accelerate PPF, as this process was observed earlier compared with cells cultured in suspension. However, while the frequency of PPF in suspension substantially increased with time, continued at a constant rate up to 72 h and declined afterwards, this process became exhausted within 16 h with adherent cells, and it slowly decreased afterwards. Moreover, the maximal extent of PPF was definitely lower in adherent cells.
In contrast to our results, it has been recently proposed that fibrinogen and, to a lesser extent, VWF stimulate PPF by primary murine megakaryocytes . Although these discrepancies may reflect differences in the species analysed, it should be noted that the previous study limited the analysis of PPF at 4 h, and that the analysis of our own results at that single time point could lead to similar conclusions. However, our kinetic study over a longer time period produced a more comprehensive characterization of the temporal development of this process and disclosed a more complex picture in which proplatelet extension on fibrinogen or VWF was restricted and transient, while a higher percentage of cells in suspension continued to generate proplatelets at least up to 72 h. Therefore, it can be concluded that megakaryocytes adhesion to fibrinogen or VWF similarly regulates, by restricting, rather than stimulating, spontaneous PPF.
In this work, we have confirmed that adhesion to type I collagen completely prevented PPF, but we have also documented that PPF actually occurs upon megakaryocytes adhesion to type III and type IV collagens, demonstrating, for the first time, that different collagen types may differently regulate this process. Importantly, while type I collagen, which completely suppresses PPF, is an abundant extracellular protein in the osteoblastic niche, type IV, and possibly also type III, collagens, which support PPF, are located around the marrow vessels [21,22]. Our results, thus, are consistent with a model in which interaction with type I collagen in the osteoblastic niche refrains megakaryocytes from extending proplatelets and prevents the premature release of platelets, a process which is subsequently allowed to occur when megakaryocytes interact with permissive adhesive proteins in the vascular niche. The mechanism for the differential effects of collagens on PPF is still unknown. Our preliminary evidence indicates that these differences may be ascribed to peculiar structural properties of the collagens, as well as to differences in receptor engagement. The inhibitory effect of type I collagen on PPF is mediated by the interaction with integrin α2β1 . By using a specific antibody, we found that blockade of α2β1 reduced megakaryocyte adhesion to type I collagen by about 60%, while inhibition of adhesion to type IV or type III collagen was only 20% and 30%, respectively (data not shown). In addition, preliminary electron microscopy analysis of collagen-coated coverslips indicated that while immobilized type I collagen formed ordered fibrils, type IV and, surprisingly, also type III collagens organized reticular structures (A. Balduini, A. Malara, E. Tira, C. Gruppi, M. Torti, unpublished data). It is therefore possible that all these factors distinguish the effect of type I from that of type III and type IV collagens. More detailed investigations are certainly required to verify this possibility.
We found that the inhibitory action of type I collagen on PPF is dependent on myosin IIA, the only isoform expressed in megakaryocytes and platelets , encoded by the MYH9 gene, whose mutations cause thrombocytopenia [15,24]. Two recent works have implicated myosin IIA in the Rho/ROCK-mediated inhibition of PPF [12,14]. Our results support these previous findings, and extend this model as we demonstrated that inhibition of myosin IIA efficiently counteracted the ability of type I collagen to repress PPF. Therefore, type I collagen can negatively control PPF as long as the functionality of myosin is preserved. This may suggest a mechanism for the thrombocytopenia induced by MYH9 mutations in humans, as the inability of type I collagen to repress PPF in the presence of a functionally altered myosin IIA may cause the premature platelet release before megakaryocytes reach the vascular niche.
A previous work has indicated a role for integrin αIIbβ3 in the regulation of PPF in mice . Here we demonstrate that antagonists of integrin αIIbβ3 block spreading of human megakaryocytes to both fibrinogen and VWF but do not alter PPF, which, by contrast, strictly requires GPIb-IX-V. This discrepancy may be indicative of a different contribution of integrin αIIbβ3 in PPF by human and murine megakaryocytes. We propose here that, in humans, GPIb-IX-V is a major regulator of PPF. This is in line with a previous work showing inhibition of PPF by the anti-GPIbα antibody HIP1 . Our findings extend and consolidate these early observations, and definitely demonstrate the essential role of GPIb-IX-V on PPF not only in suspension, but also upon adhesion to different matrices. GPIb-IX-V clearly plays multiple roles on platelet production. A defective development of internal demarcation membranes associated with megakaryocyte maturation has been described in GPIbα-deficient mice . Here we showed, in agreement with previous findings [7,25], that in normally differentiated, mature human megakaryocytes, GPIb-IX-V is also important for the terminal process of PPF. All these observations point to a central role of GPIb-IX-V in megakaryocyte biology, and are consistent with the notion that the absence or the reduced expression of this receptor, as seen in the Bernard–Soulier syndrome, results in a defective platelet production [8,24]. The exact mechanism of proplatelet regulation through GPIb-IX-V is not completely understood. We have reported in this work that membrane-bound VWF can be detected exclusively in proplatelet-forming megakaryocytes, indicating that release of α-granules had occurred in these cells, in association with PPF. Therefore, it is possible that GPIb-IX-V mediates a sort of autocrine stimulation through secreted VWF, essential for PPF. This hypothesis, however, clearly deserves further investigation.
In conclusion, our results have provided important new elements in the understanding of the regulatory pathways for PPF and for platelet production and release.
This work was supported by grants from the Italian Telethon Foundation (grant GGP06177), the Cariplo Foundation and from the Banca del Monte di Lombardia Foundation.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.