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Keywords:

  • cytoskeleton;
  • MYH9-related disease;
  • myosin;
  • platelets

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

Summary.  MYH9-related disease (MYH9-RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non-muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist-induced reorganization of the platelet cytoskeleton from seven MYH9-RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9-RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist-stimulated MYH9-RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9-RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

MYH9-related disease (MYH9-RD) is an autosomal dominant disorder caused by mutations of the MYH9 gene encoding for the non-muscle myosin heavy chain IIA (NMMHC-IIA), and characterized by thrombocytopenia with giant platelets, mild to severe bleeding diathesis, and clusters of NMMHC-IIA within granulocytes that in most cases are so large that they are identified by optical microscopy on May–Grünwald–Giemsa-stained blood films (Döhle-like bodies) [1,2]. In childhood or adult life, some of the affected patients develop the additional clinical findings of sensorineural hearing loss, cataracts, and/or glomeluronephritis that can lead to end-stage renal failure. Different phenotypic expressions have been described not only between families but also within families having the same mutation [1,3,4]. These thrombocytopenic patients were in the past classified as having different disorders, namely May–Hegglin anomaly (MHA, OMIM 155100) [4–8], Sebastian syndrome (SBS, OMIM 605249) [4,6,8,9], Fechtner syndrome (FTNS, 153640) [4,6,10,11], or Epstein syndrome (EPTS, OMIM 153650) [11,12]. However, careful re-evaluation of a large case series failed to identify correlations between MYH9 mutations and the clinical phenotypes of 77 affected families, suggesting that MHA, SBS, FTNS and EPTS are not allelic forms, but rather represent a unique disease with clinical symptoms variably expressed [1]. The reason for the phenotype variability is still unknown, although it has been suggested that it derives from the joint effect of specific mutations and environmental factors, as well as multiple gene products, such as polymorphic protein variants interacting with MYH9.

NMMHC-IIA is a 224-kDa subunit of the class II conventional myosin, which exists as a hexameric enzyme composed of two heavy chains and two pairs of light chains [13]. The N-terminal globular head domain of NMMHC-IIA contains both actin and ATP-binding sites, while the C-terminal tail represents a regulatory domain that forms a coiled-coil structure, and supports both dimerization of distinct myosin heavy chains and assembly of different myosin molecules into bipolar filaments [13]. Mutations of NMMHC-IIA so far identified in patients with MYH9-RD affect highly conserved positions. A few of them, such as the N93K, map in the N-terminal region, and destabilize the globular head domain by introducing positive charges and increasing electrostatic repulsion [6]. However, most mutations, such as E1841H, D1242H, and R1165C, are in the rod-like C-terminal region and may affect normal protein dimerization [6–8]. In this regard, it has been recently reported that rod mutations cause defects in NMMHC-IIA assembly resulting in the formation of aberrant aggregates [14]. The R1933X mutation introduces a stop codon resulting in a truncate myosin heavy chain unable to undergo proper self-assembly [6].

Non-muscle myosin IIA has been suggested to be involved in different events, including maintenance of cell shape, phagocytosis, cytokinesis, cell motility, and organelle/particle trafficking, but its precise biological function has not been completely elucidated [13,15–18]. It is interesting to note that, while most cells express multiple isoforms of myosin II (IIB and IIC, in addition to IIA), human platelets express exclusively myosin IIA [13,19,20], and thrombocytopenia and giant platelets, together with myosin clusters within leukocytes, are the only pathological features common to all MYH9-RD patients since birth [1,4]. These findings suggest that myosin IIA plays a key role in platelet formation.

In circulating platelets, myosin is an important component of the intracellular cytoskeleton. The platelet cytoskeleton is involved in the regulation of the morphology of the cells, but it also represents an intracellular network connecting several molecules involved in signal transduction [21,22]. In resting platelets, the cytoskeleton is mainly composed by polymerized actin and actin cross-linking proteins [21]. Upon stimulation with different agonists, actin undergoes further polymerization, and interacts with myosin and other contractile proteins to promote platelet shape change and granule secretion. When activated platelets bind fibrinogen and aggregate, outside-in signaling through integrin αIIbβ3 promotes the association of the actin-myosin filaments with membrane glycoproteins, such as integrin αIIbβ3 itself, and molecules involved in signal transduction such as tyrosine kinases, lipid metabolizing enzymes, and small GTP-binding proteins [23–25]. Although the role of the cytoskeleton in platelet function has been well recognized, the consequences of mutations of the NMMHC-IIA in the organization of this contractile structure has never been investigated, in that the rareness of this disorder, the low number of circulating platelets and difficulties in purification of these cells because of their very large volume have until now hampered biochemical investigations.

In this study, we have analyzed the composition and the agonist-induced reorganization of the cytoskeleton in platelets from seven individuals from four unrelated MYH9-RD families. The patients analyzed carried mutations in the C-terminal domain of the NMMHC-IIA, supposed to affect the coiled-coil structure resulting in an incorrect dimerization of the tail of the protein. We observed a number of anomalies, including the increased association of myosin with the cytoskeleton of resting platelets and the inability of the cytoskeleton to undergo proper reorganization upon platelet stimulation, that outline dramatic functional consequence of NMMHC-IIA mutations.

Materials

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

The thrombin receptor activating peptide specific for protease-activated receptor 1 (TRAP1) was custom synthesized by PRIMM (Milan, Italy). Anti-non-muscle myosin heavy chain monoclonal antibody F6 was a gift from S. Sartore (Department of Medical Sciences, University of Padua, Italy). Antibodies against glycoprotein Ibα antibody (SZ2) and integrin β3 subunit were from Immunotech (Marseille, France). Polyclonal anti-integrin α2 subunit (AB1936) was from Chemicon (Prodotti Gianni, Milano, Italy). Antibodies against Syk (N-19), FAK (A-17), Rap1B (121), Rap2B (V-19), and RhoA (119) were from Santa Cruz Biotechnology (Tebu-Bio, Magenta, Italy). Antiphosphotyrosine antibody was from Upstate Biotechnology (Lake Placid, NY, USA). Bicinchoninic acid assay kit for protein determination and chemiluminescence substrates were obtained from Pierce (Pero, Italy).

Patients

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

This study included seven patients from four different families with diagnosis of MYH9-RD. Clinical and laboratory findings of these patients, as well as genetic analysis of MYH9 gene mutations, have been previously reported [1,6] and are summarized in Table 1. Control subjects included nine different healthy donors, that were analyzed the same day a MYH9-RD patient was investigated.

Table 1.  Clinical and laboratory characteristics of seven MHA/SBS patients
Family/ patient (Ref.)Sex/ ageMYH9 mutationPlatelet count*, × 109 L−1Distribution of platelet diameters, %Bleeding time,minAggregation/ shape changeHearing impairment§Kidney impairmentCataracts
< 4 µm4–8 µm> 8 µm
  1. The seven patients analyzed in this study belong to four different families (indicated as A, B, C, and D). Mutations in the MYH9 gene have been previously described [1,6]. Essential clinical data of patients have been already published [1], with the only exception of patient C/4. M- and B-SNHLHT, mono- and bi-lateral sensorineural hearing loss for high tones; nd, not determined. *As determined by phase contrast microscopy. †Normal values: < 4 µm, 87–100%, 4–8 µm, 0–10%, > 8 µm, 0–1%, as determined by the analysis of 50 healthy subjects. In vitro platelet aggregation and shape change were evaluated in response to collagen, ADP and TRAP1. §As determined by audiogram.

A/1 (1)F/56E1945X6360.032.08.07Normal/ absentM-SNHLHTMild proteinuria, microscopic hematuriaNo
A/2 (1)F/29E1945X6869.721.78.69Normal/ absentM-SNHLHTMild proteinuria, microscopic hematuriaNo
B/3 (6)F/44R1165C8863.127.99.04Normal/ absentB-SNHLHTNoNo
C/4 (1)F/60D1424N5560.826.612.6ndNormal/ absentB-SNHLHTNoNo
C/5 (1)F/37D1424N3062.524.013.5ndNormal/ absentNoNoNo
D/6 (6)M/57E1841K8268.524.17.44Normal/ absentMild B-SNHLHTNoBilateral
D/7 (6)M/16E1841K12969.421.88.84Normal/ absentNoNoNo

Preparation of platelet-rich plasma and platelet stimulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

Blood from healthy volunteers and from MYH9-RD patients was taken into trisodium citrate (3.2% w/v) in a ratio of 9 : 1. Platelet-rich plasma (PRP) was isolated from whole blood by centrifugation at 200 × g for 8 min. The platelet count of the control healthy subjects was adjusted according to the platelet count of the MYH9-RD patient analyzed. PRP samples containing the same amount of platelets were incubated at 37 °C under constant stirring, and then stimulated by addition of 10 µm TRAP1 for 2 min. To prepare whole platelet lysates, platelets were recovered by centrifugation at 18 000 r.p.m. for 30 s, lyzed with 80 µL of 2% SDS and boiled for 5 min. Protein concentration was evaluated by bicinchoninic acid assay.

Cytoskeleton extraction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

Samples of PRP (typically 3–4 mL) were incubated at 37 °C under constant stirring, in the absence or presence of 10 µm TRAP1 for 2 min. Reaction was stopped by addition of an equal volume of extraction buffer 2 × (10 mm HEPES, 137 mm NaCl, 2.9 mm KCl, 12 mm NaHCO3, 2% Triton X-100, 10 mm EGTA, 4 mm PMSF, 10 µg mL−1 leupeptin, 10 µg mL−1 aprotinin, 2 mm Na3VO4, pH 7.4). The platelet cytoskeleton was isolated essentially as previously described [26]. Lysates were vigorously mixed and placed on ice for 15 min. Triton X-100-insoluble material was recovered by centrifugation at 18 000 r.p.m. for 5 min. The pellet was washed twice in 1 mL of extraction buffer, and finally resuspended in 80 µL of 4% SDS. The protein concentration of the cytoskeletal samples was determined by bicinchoninic acid assay. An equal volume of a mixture containing 1% dithiothreitol, 20% glycerol and 0.02% bromophenol blue was then added to the remaining of the samples and boiled for 5 min.

SDS-PAGE and immunoblotting

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

Cytoskeletal proteins (20 µg) from stimulated control and MYH9-RD platelets, and cytoskeletal proteins from a corresponding number of resting platelets (tipically 5–7 × 107) were separated on 5–15% gradient acrylamide gels. Proteins were either stained with Coomassie blue, or transferred to nitrocellulose membranes for further immunoblotting analysis. Nitrocellulose membranes were blocked overnight at 4 °C with 6% bovine serum albumin (BSA) in 20 mm Tris–HCl, pH 7.5, 0.5 m NaCl, and incubated with the appropriate antibody. The following antibodies and dilutions were used: antibody F6 against NMMHC-IIA (1 : 1000 dilution), anti-integrin β3 subunit (1 : 500 dilution), anti-integrin α2 subunit (1 : 200 dilution), anti-GPIbα (2 µg mL−1), anti-Syk, anti-Rap2B, and anti-Rho (0.4 µg mL−1), anti-FAK (0.5 µg mL−1), and anti-Rap1B (0.2 µg mL−1). Upon incubation for 2 h at room temperature, membranes were extensively washed with 50 mm Tris–HCl, pH 7.4, 0.2 m NaCl, 1 mg mL−1 polyethylene glycol 20000, 1% BSA, 0.05% Tween 20, and incubated with peroxidase-conjugated appropriate secondary antibody (1 : 4000 dilution) for 45 min. Upon extensive washing, reactive proteins were visualized with a chemiluminescence reaction. Nitrocellulose filters were then stripped by incubation with 62.5 mm Tris–HCl, pH 6.7, 2% SDS, 100 mmβ-mercaptoethanol for 30 min at 50 °C, and reprobed with a different antibody. Quantitative analysis of the intensity of the single bands was performed by densitometric scanning using a CAMAG TLC scanner. In some cases, immunoblotting analysis with different antibodies has been performed on different subsets of patients, depending exclusively on the different period every single patient has been analyzed and on the different platelet counts.

Cytoskeleton reorganization in MYH9-RD platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

The cytoskeleton was isolated as Triton X-100-insoluble material from resting and TRAP1-stimulated platelets from seven different patients affected by MYH9-RD, whose main clinical and laboratory features are summarized in Table 1. Because of the marked thrombocytopenia of the analyzed patients, PRP rather than washed platelets was used in this study, in order to obtain a sufficient number of platelets to isolate enough cytoskeletal proteins for biochemical analyses. Preliminary experiments performed with control platelets did not reveal any significant difference between the protein composition of the cytoskeleton extracted from washed platelets and platelets in PRP (data not shown). Thrombin is the most potent platelet activator and the strongest stimulator of cytoskeleton reorganization, and thus it was considered the best agonist for the current study. To avoid thrombin-mediated fibrin formation in the PRP samples, stimulation of a thrombin receptor was achieved using a well-characterized synthetic peptide, TRAP1, rather than the native enzyme. Although TRAP1 may be less potent than thrombin in the absence of secondary stimuli released by activated platelets, it is known that it can induce full platelet activation and aggregation in the absence of ADP scavengers or ADP receptor antagonists [27,28]. The amount of total proteins in the isolated cytoskeleton from resting or TRAP1-stimulated platelets was determined by a colorimetric assay. Typically, the cytoskeleton from resting MYH9-RD platelets was found to contain a greater amount of proteins than the cytoskeleton from normal platelets, when the same number of cells were considered (data not shown). However, this was mainly a consequence of the bigger size of MYH9-RD platelets, since the percentage of cytoskeletal proteins vs. total cell proteins was not significantly different between controls and patients (data not shown). Upon stimulation with 10 µm TRAP1 for 2 min, the total amount of cytoskeletal proteins increased, as expected, in both MYH9-RD and control platelets. However, such increase was variable in MYH9-RD platelets, and constantly reduced compared with that observed in control cells. Figure 1 reports the fold-increase of the protein content of the cytoskeleton from stimulated platelets. This increase was very small and almost hardly detectable in patients A/1–C/5, and more evident, although still reduced, in patients D/6 and D/7. When the increase of cytoskeletal proteins in stimulated platelets from nine different healthy subjects and from the seven MYH9-RD patients was analyzed with Student's t-test, the reduced increase in MYH9-RD platelets was found to be statistically significant (P < 0.001).

image

Figure 1. Agonist-induced cytoskeleton reorganization in MYH9-RD platelets. The cytoskeleton was extracted from normal platelets (control) or platelets from seven MYH9-RD patients (from A/1 to D/7) before of after stimulation with 10 µm TRAP1 for 2 min at 37 °C. The amount of total cytoskeletal proteins in resting or TRAP1-stimulated platelets was evaluated by the bicinchoninic acid assay. The figure shows the fold increase of total cytoskeletal proteins in TRAP1-stimulated vs. resting platelets. Patients C/4, D/6 and D/7 have been analyzed only once, while data concerning the other patients are representative of two (A/1, B/3, and C/5) or three (A/2) different observations performed on different days and giving similar results. Results concerning control platelets are the mean ± SD of nine different determinations with platelets from different donors.

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The composition of the isolated platelet cytoskeleton was investigated by SDS–PAGE and Coomassie blue staining to detect the main contractile proteins. Figure 2A shows a representative gel with the electrophoretic pattern of cytoskeletal proteins from healthy and MYH9-RD platelets before and after stimulation with TRAP1. The main bands have been identified as actin-binding protein (ABP), myosin, α-actinin, and actin according to their molecular mass, and on the basis of the previously well-described pattern of cytoskeletal proteins. Figure 2A shows the very small increase in the amount of the single main cytoskeletal proteins upon TRAP1 stimulation of MYH9-RD platelets compared with control platelets, in agreement with the data reported in Fig. 1. Moreover, an additional striking qualitative difference concerns a band with a Mr of about 200 kDa and corresponding to the myosin heavy chain, that was hardly detectable in the cytoskeleton from normal resting platelets, but was clearly evident in the cytoskeleton from resting MYH9-RD patients. This finding was constantly observed in all the patients analyzed, and quantified by densitometric analysis (Fig. 2B). Moreover, upon stimulation of normal platelets with TRAP1, the increase in the amount of this protein recovered in the cytoskeleton was much higher in control than in MYH9-RD platelets (Fig. 2A,B). The identity of this protein was confirmed by immunoblotting with specific antibody against NMMHC-IIA. Figure 3A confirms that in all the patients analyzed an abnormally high amount of myosin was present in the cytoskeleton from resting platelets, when compared with platelets from healthy donors. Moreover, while in control platelets the amount of actin-associated myosin dramatically increased upon cell stimulation, the amount of cytoskeletal-associated myosin in MYH9-RD platelets did not increase significantly upon treatment with TRAP1. Mutated NMMHC-IIA has been recognized to form aberrant aggregates that generate intracytoplasmic inclusions in leukocytes. Since the cytoskeleton has been isolated as Triton X-100-insoluble material, we verified whether the abnormal association of myosin to the cytoskeleton in resting platelets was actually due to the interaction with the actin filaments, or to a non-specific precipitation of the mutant protein. Control and MYH9-RD resting platelets were lyzed with Triton X-100 in the presence of cytochalasin D and DNase, a condition that caused the depolymerization of actin filaments. Figure 3B reports a representative result showing that depolymerization of actin abolished or greatly reduced the recovery of myosin in the Triton X-100-insoluble materials from MYH9-RD platelets. These results demonstrate that the increased association of myosin with the cytoskeleton of resting platelets is not due to an abnormal myosin aggregation. The observed increased amount of proteins in the cytoskeleton of resting MYH9-RD platelets, as well as the impaired agonist-induced cytoskeleton reorganization, may suggest that MYH9-RD platelets are preactivated and refractory to stimulation. Although this is unlikely, because of the minimal blood manipulation necessary for PRP preparation and because of the normal agonist-induced aggregation, we directly analyzed the level of protein tyrosine phosphorylation in the cytoskeleton from resting and stimulated platelets. Figure 4 shows a very weak and similar pattern of tyrosine-phosphorylated proteins in resting platelets from both healthy subjects and MYH9-RD patients. Moreover, upon stimulation with TRAP1 the number of tyrosine-phosphorylated proteins strongly increased in both control and MYH9-RD platelets. As evident from Fig. 4, reactivity to antiphosphotyrosine antibody was slightly weaker in MYH9-RD than in control platelets, and this is in agreement with reduced association of some tyrosine kinases with the cytoskeleton in these cells (see below). These results indicate that MYH9-RD platelets are neither preactivated nor refractory to stimulation.

image

Figure 2. Qualitative and quantitative analysis of the platelet cytoskeleton. Cytoskeletal proteins were separate by SDS–PAGE on 5–15% acrylamide gradient gel and stained with Coomassie blue. (A) A typical electrophoretic pattern of the cytoskeletal proteins from resting and TRAP1-stimulated platelets from a control subject and one of the analyzed MYH9-RD patients (B/3). The position of the molecular mass markers is reported on the left, while the identity of the main visualized bands is reported on the right. (B) Results of the quantitative analysis of cytoskeletal associated myosin from resting (□) or TRAP1-stimulaled (▪) platelets performed by densitometric scanning of Coomassie blue-stained gels. The results are expressed in arbitrary units, assuming 100 the area of the band corresponding to myosin in the cytoskeleton of TRAP1-stimulated control platelets.

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image

Figure 3. Immunological detection of myosin in the platelet cytoskeleton. (A) The cytoskeletal proteins from healthy subjects (control) and from the seven analyzed MYH9-RD patients before and after stimulation with TRAP1 for 2 min were separated by SDS–PAGE, transferred to nitrocellulose and analyzed by immunoblotting using a specific monoclonal antibody against non-muscle myosin heavy chain. Reactive myosin was detected by a chemiluminescence reaction. (B) The platelet cytoskeleton was extracted in the absence or presence of cytochalasin D (cytD) and DNase, which induced the rapid actin depolymerization. The presence of actin and myosin in the Triton-X-100-insoluble materials was investigated by immunoblotting using specific antibodies, as indicated on the right. Typical results obtained with a control subject and two different patients are reported.

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image

Figure 4. Analysis of agonist-induced protein tyrosine phosphorylation of cytoskeletal proteins. The cytoskeleton was isolated from normal (control) and MYH9-RD platelets (patients A/2 and B/3) before and after stimulation with 10 µm TRAP1 for 2 min. The tyrosine phosphorylated proteins were detected by immunoblotting with an antiphosphotyrosine antibody and revealed by a chemiluminescence reaction.

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Association of signaling proteins with the platelet cytoskeleton

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

It is known that, upon platelet stimulation, several membrane glycoproteins and intracellular molecules involved in signal transduction translocate to the reorganized cytoskeleton [23–26]. Since we revealed a defective cytoskeleton reorganization in MYH9-RD platelets, we investigated whether the interaction of some of these signaling proteins was also affected. The expression of all the analyzed signaling proteins in MYH9-RD total platelet lysates was comparable to that observed in control subjects (data not shown). Figure 5A shows that the fibrinogen receptor integrin αIIbβ3 failed to interact with the cytoskeleton of stimulated platelets in five out of seven MYH9-RD patients analyzed. Similarly, the collagen receptor integrin α2β1 did not associate with the cytoskeleton from MYH9-RD platelets in any the four patients we have been able to analyze with the anti-α2 antibody (Fig. 5B). By contrast, in all the patients analyzed, except for patient A/2, an evident, albeit reduced, translocation of the von Willebrand factor receptor GPIb-IX-V was detected (Fig. 5C).

image

Figure 5. Association of membrane glycoproteins with the cytoskeleton in TRAP1-stimulated platelets. Platelets from healthy subjects (control) and from different MYH9-RD patients were treated without or with 10 µm TRAP1 for 2 min, and the cytoskeleton was isolated upon lysis with Triton X-100. The association of membrane glycoproteins with the isolated cytoskeletons was analyzed by immunoblotting with specific antibodies. The figure shows representative results obtained using anti-integrin β3 (A) anti-integrin α2 (B), and anti-GPIbα (C) antibodies.

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Syk and FAK are non-receptor tyrosine kinases involved in several steps of platelet activation. In platelets from healthy subjects, both kinases associated to the cytoskeleton upon stimulation with TRAP1 (Fig. 6A). By contrast, in platelets from four different MYH9-RD patients, Syk was never recovered in the cytoskeleton (Fig. 6A). In patient B/3 a small amount of Syk was detected in the Triton X-100-insoluble material from resting platelets, but did not further increase upon stimulation with TRAP1. Figure 6B shows that the agonist-induced translocation of the tyrosine kinase FAK was totally absent in three MYH9-RD patients, but occurred normally in two patients (D/6 and D/7).

image

Figure 6. Association of tyrosine kinases with the platelet cytoskeleton. The presence of the tyrosine kinases Syk (A) and FAK (B) in the cytoskeleton from resting and TRAP1-stimulated control and MYH9-RD platelets was investigated by immunoblotting using specific antibodies.

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Small GTPases play crucial roles in cell activation, shape change, and integrin regulation. Figure 7 shows that none of the three most abundant small GTPases present in platelets, Rap1B, Rap2B and RhoA, able to interact with the cytoskeleton in normal agonist-stimulated platelets, was recovered in the Triton X-100-insoluble material isolated from all seven MYH9-RD patients.

image

Figure 7. Association of small GTPases with the platelet cytoskeleton. Antibodies against three small GTPases expressed in platelets, Rap1B (A), Rap2B (B), and RhoA (C) were used to reveal their association with the cytoskeleton in resting and TRAP1-stimulated control and MYH9-RD platelets.

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Altogether, these results indicate that mutations of the MYH9 gene dramatically affect the agonist-induced reorganization of the platelet cytoskeleton, and the interaction of different signaling proteins with the actin filaments.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References

Platelet formation starts when mature megakaryocytes convert the bulk of their cytoplasm into long protrusions, named proplatelets, which thin and branch repeatedly. Then, platelet granules and organelles are delivered from the megakaryocyte body to the proplatelet tips, which break off and give rise to platelets. It has been suggested that cytoskeletal proteins play a key role in platelet birth, in that proplatelets are extended in a microtubule-dependent fashion, and their surface is supported by an underlying membrane skeleton similar to that of mature platelets. Moreover, actomyosin assemblies could provide the forces required for the process [29].

MYH9-RD is an inherited disorder constantly characterized by thrombocytopenia and marked platelet macrocytosis deriving from mutations of MYH9, the gene encoding for NMMHC-IIA [1,2]. In this illness, megakaryocyte number and maturation as well as platelet survival are normal [30–32], thus suggesting that platelet abnormalities mainly derive from a defect of platelet formation. Although it is reasonable to hypothesize that mutant myosin alters the organization of cytoskeletal contractile structures and interferes with the process of platelet formation, the actin-myosin-based intracellular structures in cells from MYH9-RD patients have never been investigated.

In this work we used circulating platelets as a useful and easy model to analyze the intracellular cytoskeleton and its ability to undergo modifications in response to extracellular agonists in MYH9-RD patients. This study benefits from the fact that the platelet cytoskeleton is a very well-characterized structure, which has been investigated for years in many different laboratories [21–26]. Because of this, possible anomalies in MYH9-RD platelets can be easily recognized. Moreover, while other cells express multiple isoforms of NMMHC-II (IIA, IIB, or IIC), only NMMHC-IIA is present in platelets [13,19,20], and therefore, functional consequences of protein mutations may result in a more evident altered phenotype. We made a number of interesting observations, revealing evident differences between the platelet cytoskeleton in normal and MYH9-RD platelets. The most intriguing one is that, in all the patients analyzed, an increased association of myosin with the actin filaments was observed in resting platelets. This was paralleled by an impaired increase of the cytosketal associated myosin upon platelet stimulation, which is one of the most evident events occurring in normal platelets. We cannot, at the moment, provide chemical explanations for the increased association of myosin with the platelet cytoskeleton. It is noteworthy, however, that all the seven patients analyzed carried mutations in the C-terminal tail of NMMHC-IIA, which includes the regulatory domain responsible for the proper organization of myosin into bipolar filaments. It is important to note that the increased recovery of myosin in the cytoskeleton from MYH9-RD platelets was not due to a non-specific precipitation of aberrant aggregates of this protein, but reflected the interaction of myosin (either as a single molecule or organized bipolar filaments) with the intracellular actin filaments, as it was found to be prevented by treatment of the cells with actin-depolymerizing agents. Increased interaction with actin filaments was not due to an increased expression of myosin in the analyzed patients, as revealed by immunoblotting analysis on whole platelet lysates (data not shown). By contrast, a reduced expression of myosin has been recently reported in a patient with MYH9-RD [33]. We have been able to confirm this observation (data not shown), and a more complete study involving a greater number of patients with different mutations is currently being performed in our laboratory. The occurrence of haploinsufficiency implies that the increased association of myosin with actin filaments observed in resting platelets involves a large fraction of the available myosin population.

In control platelets, agonist stimulation induced a strong increase of the total cytoskeletal proteins, which was supported by an increase of the amount of polymerized actin and by the association of a number of proteins, including myosin, with the newly formed actin filaments. In MYH9-RD platelets, agonist-induced increase of polymerized actin (revealed by its intrinsic insolubility in Triton X-100) was comparable to that observed in control cells. However, myosin failed to associate further with the reorganized cytoskeleton. Based on the electrophoretic pattern of the main contractile proteins, it appears that in resting MYH9-RD platelets the cytoskeleton is more similar to that observed in normal agonist-stimulated, rather than in resting platelets. This premature and constitutive partial reorganization may prevent further modifications induced by extracellular agonists. This is supported by the inability of a number of signaling molecules to be incorporated into the cytoskeleton of MYH9-RD platelets, as revealed by immunoblotting with specific antibodies. The incorporation of tyrosine kinases, small GTP-binding proteins, as well as membrane glycoproteins into the cytoskeleton of stimulated platelets is known to be relevant for the correct development of late events during platelet activation, associated with the irreversibility of platelet aggregation and thrombus consolidation [22–26]. Therefore, the impaired cytoskeletal reorganization could have functional consequences not only on the morphological changes of stimulated cells, but also on their ability to integrate intracellular signaling processes for the correct response to extracellular agonists. Surprisingly, all the patients analyzed in this study, like the vast majority of those reported in the literature, presented normal platelet aggregation ‘in vitro’. Because of this, it is reasonable to conclude that the observed cytoskeletal anomalies, and the lack of translocation of several signaling molecules, do not interfere with this process. Our results therefore question the real importance of cytoskeleton association of membrane glycoproteins and signaling molecules for platelet response to extracellular agonists, and may suggest that a re-evaluation of these events is needed. Nevertheless, it has been shown that in MYH9-RD patients, platelets no longer have their disc shape and are unable to undergo shape change, pseudopodal formation and spreading on surfaces [31,34,35]. Because all these events are cytoskeleton-dependent, our study offers a possible pathogenic explanation for the reported functional abnormalities of platelets. The normal platelet aggregation without shape change in MYH9-RD platelets is not surprising and is consistent with the notion that the intracellular actin-myosin network is differently involved in these processes. In this regard, it should be noted that platelet shape change requires the phosphorylation of myosin light chain to activate actin-myosin-driven contractility, while depolymerization of actin filaments does not alter agonist-induced platelet aggregation [36,37]. In addition, normal aggregation without shape change has been reported in different experimental conditions [38,39]. Moreover, platelets originate from megakaryocytes, and it is reasonable to suppose that the cytoskeletal defects affect also their progenitors and represent the biochemical basis for the altered platelet formation resulting in thrombocytopenia and giant platelets. Finally, cytoskeleton interacts with platelet membrane, and it is therefore possible that its deranged organization is responsible for the abnormal surface expression of membrane glycoproteins that has been described in platelets of patients with MYH9 mutations [40].

Patients with MYH9 mutations often present glomerulonephritis and/or hearing loss. Immunomorphological and ultrastructural analyses of patients from a family with D1424H substitution in NMMHC-IIA revealed that kidney podocytes expressing mutant NMMHC-IIA had focal and segmental fusion of the foot processes and no interpodocyte slit diaphragm [41]. Podocyte foot processes represent key components of the glomerular filtration barrier, and through their contractile structure composed of actin, myosin and other cytoskeletal proteins modulate ultrafiltration in response to different factors and stresses. Moreover, NMMHC-IIA is expressed in several structures of the inner ear [42,43], and it has been suggested that actomyosin cytoskeletal abnormalities of one or more cells of these structures could be responsible for hearing loss of patients with MYH9 mutations [42]. In this context, it seems reasonable to hypothesize that the cytoskeletal defects we observed in platelets could occur also in these cells, and could have a role in the pathogenesis of renal failure and deafness.

Finally, although severe cytoskeleton abnormalities have been observed in all investigated patients, some quantitative and/or qualitative differences have been detected not only between families, but also within the same family. These discrepancies might derive from polymorphisms of yet unknown NMMHC-IIA interactors and might be related to the aforementioned variability of the clinical phenotype. Further investigation in a larger number of patients is required to test this hypothesis. In conclusion, our results report for the first time that mutations of the NMMHC-IIA result in an altered composition of the platelet cytoskeleton, and impaired cytoskeletal reorganization induced by extracellular agonists. Further investigations are required to examine the role of these anomalies in the pathogenesis of MYH9-RD.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Patients
  7. Preparation of platelet-rich plasma and platelet stimulation
  8. Cytoskeleton extraction
  9. SDS-PAGE and immunoblotting
  10. Results
  11. Cytoskeleton reorganization in MYH9-RD platelets
  12. Association of signaling proteins with the platelet cytoskeleton
  13. Discussion
  14. Acknowledgements
  15. References
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