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Offprint requests to: Alan T. Nurden, Ph.D., Centre de Référence des Pathologies Plaquettaires, Plateforme Technologique et d'Innovation Biomédicale, Hôpital Xavier Arnozan, 33604 Pessac, France
Expression of tissue inhibitors of matrix metalloproteinases (TIMPs) is one way that activated platelets intervene in tissue remodeling and angiogenesis. Our study was designed to investigate their synthesis in megakaryocytes (MKs) and their storage in platelets.
Materials and Methods
TIMP expression in MKs derived from blood CD34+ progenitor cells of normal donors and a megakaryocytic cell line (CHRF-288-11) grown in serum-free conditions and platelets from normal donors or two patients with gray platelet syndrome was studied by immunofluorescence labeling, reverse transcription-polymerase chain reaction, and western blotting.
Results
Biosynthesis of TIMPs 1−4 in MKs was indicated by presence of their messenger RNAs as shown by polymerase chain reaction and of their proteins. Immunofluorescence labeling suggested a primarily granular localization of TIMPs in MKs and platelets. But when colocalization with von Willebrand factor, fibrinogen, P-selectin, and other α-granule proteins was assessed in platelets by confocal microscopy, TIMP-1, -2, and -4 were localized as distinct fluorescent patches apart from the established α-granule markers and largely independent of platelet metalloproteinases. TIMP-3 differed for it also had an α-granule location. Western blotting confirmed the presence of TIMPs 1−4 in platelets and thrombin activation resulted in their extensive release to the medium. Platelets from two patients with gray platelet syndrome, congenitally deficient in α-granules, showed sparse labeling of von Willebrand factor and fibrinogen confined to vestigial α-granules; however, localization of the TIMPs was unchanged.
Conclusions
TIMPs are synthesized and organized in MKs and platelets independently of other secreted proteins present in α-granule pools.
Blood platelets accumulate at sites of vessel injury; form platelet-rich plugs, and release biologically active compounds essential for wound repair [
]. Platelets contain storage pools of proteins in α-granules, although the true extent and diversity of the secretome is only now being recognized. As well as adhesive proteins such as von Willebrand factor (VWF) and fibrinogen (Fg), platelets store clotting and fibrinolytic factors, proteases and anti-proteases, growth factors, chemokines and cytokines, and other proteins of diverse function [
]. Biogenesis of α-granules occurs in megakaryocytes (MKs), although the mechanisms for protein storage are still being elucidated. Granule proteins are either synthesized in MKs (e.g., VWF) or captured by endocytosis from the external medium (e.g., Fg). Although it has been assumed that α-granules represent a homogeneous pool of storage organelles, this view has been challenged by the discovery that discrete granule populations may be enriched in specific proteins [
Evidence that differential packaging of the major platelet granule proteins von Willebrand factor and fibrinogen can support their differential release.
Angiogenesis is regulated by a novel mechanism: pro- and anti-angiogenic proteins are organized into separate platelet α-granules and differentially released.
]. ADAM-10 (α-secretase) and ADAM-17 (tumor necrosis factor−α converting enzyme) mediate Ca2+-dependent shedding of platelet adhesion receptors, including glycoprotein (GP) VI and components of the GPIb-IX complex [
]. TIMPs have been studied little in platelets, although TIMP-4 release has been reported after platelet activation by strong agonists, while a trimolecular complex between membrane type I−MMP, TIMP-2, and MMP-2 mediates the transformation of MMP-2 from its latent to proteolytically active form [
]. Despite this information, there is no clear picture of the storage of MMPs and TIMPs in platelets whose localization has been variously described as cytoplasmic and/or granular.
Taking the TIMPs as a model, we now describe their synthesis and storage in MK-derived from CD34+ cells and a MK-derived cell line. We have used confocal microscopy to localize TIMPs 1−4 in platelets and to compare their distribution with that of other well-characterized stored proteins, including MMPs. Furthermore, we have examined their presence in the platelets of two patients with gray platelet syndrome (GPS), a rare inherited platelet disease characterized by a severely decreased platelet content of α-granules and their contents and marrow fibrosis [
]. GPS is a very rare disorder, these patients represent the only known cases of GPS in Southwest France. In brief, patient 1 is an elderly woman whose current platelet count is around 30,000/μL; her platelets show typical GPS ultrastructure in electron microscopy (EM) and are severely deficient in α-granule proteins. Patient 2 is a middle-aged woman from a French Gypsy tribune whose current platelet count is about 50,000/μL. Her platelets also had the typical GPS appearance when studied by EM. Control donors were adult healthy individuals taking no medication. Ethical approval was obtained in the context of the activities of the French national network GIS-Maladies Rares.
Antibodies
Rabbit anti-human VWF antibody and a phycoerythrin-labeled monoclonal antibody (mAb) to GPIbα were from DAKO (Glostrup, Denmark); mouse mAbs against human TIMPs 1−4, MMPs 1−3, and ADAM-17 were from R&D Systems (Minneapolis, MN, USA), as was a rabbit antibody to platelet-derived growth factor. A second mouse mAb against TIMP-3 and rabbit antibodies to TIMP-1 and ADAM-10 were from Calbiochem (Darmstadt, Germany). Rabbit antibody to MMP-2 (hinge region) was from Affinity BioReagents (Golden, CO, USA) and against the C-terminal domain of MMP-9 from Thermo Fischer Scientific (Fremont, CA, USA). VH10, a murine mAb against P-selectin was prepared by us [
]. Wm23, a murine mAb against GP1bα was a gift from Dr. M. Berndt (Melbourne, Australia). A fluorescein isothiocyanate (FITC)−labeled mAb to αIIbβ3 was from Beckman Coulter (Villepinte, France). Rabbit antibodies to fibrinogen were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-human β-actin antibody was from Sigma-Aldrich (Saint Quentin Fallaviers, France). FITC-labeled F(ab′)2 fragments of a goat antibody to mouse immunoglobulin G (IgG) and FITC-labeled F(ab′)2 fragments of a swine antibody to rabbit IgG were from DAKO. Alexa Fluor 488 (green) goat anti-rabbit IgG (H+L), Alexa Fluor 568 (red) goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-mouse IgG (H+L), all highly cross-adsorbed antibodies, were obtained from Interchim (Montluçon, France). Secondary IRDye 680 goat anti-mouse and anti-rabbit IgG were obtained from LICOR Biosciences (Lincoln, NE, USA).
Megakaryocyte liquid cultures and theCHRF-288-11 cell line
MKs were obtained by in vitro differentiation of CD34+ progenitors derived from peripheral blood or bone marrow from each of three informed adult control donors. Mononuclear cells were isolated by Ficoll gradient cell separation and CD34+ cells selected by magnetic-activated cell sorting (Miltenyi, Paris, France). A cell purity superior to 95% was assessed by flow cytometry using an anti-CD34+ mAb (clone 8G12-PerCP) on a FACSCalibur flow cytometer (Becton Dickinson, San José, CA, USA). Purified CD34+ cells were plated in a 48-well microtiter plate (5×104/500 μL/well) and cultured in SYN.H serum-free medium (AbCys Synergie, Paris, France) containing the following human recombinant cytokines: stem cell factor (5 ng/mL), interleukin (IL)-3 (2 ng/mL), IL-6 (1 ng/mL), IL-11 (40 ng/mL), and thrombopoietin (50 ng/mL) (Tebu, Le Perray en Yvelines, France or AbCys Synergie) for the first week of culture. For the second week of culture, cells were transferred to a 24-well plate and the culture volume was adjusted to 1 mL in Stem Alpha A serum-free medium (Stem Alpha; St Genis l'Argentière, France) containing the same concentrations of IL-3, IL-6, IL-11, and thrombopoietin. Cultures were stopped after 14 days at 37°C in a humid atmosphere containing 5 % CO2. The CHRF-288-11 megakaryocytic cell line has been described elsewhere [
Reverse transcription-polymerase chain reaction and sequencing analysis
Total RNA was extracted from MKs using the commercial RNA extraction kit NucleoSpin RNAII (Macherey-Nagel EurL, Hoerdt, France) according to manufacturer's instructions. Complementary DNA synthesis was performed following the instructions of the First Strand cDNA Synthesis Kit for reverse transcription-polymerase chain reaction (RT-PCR) using avian myeloblastosis virus (Roche Diagnostics, Meylan, France) and oligo-dT primers. PCR reactions were performed using selected primers for TIMP-1 (NCBI access number NM_003254), TIMP-2 (NM_003255), TIMP-3 (NM_000362), and TIMP-4 (NM_003256). The structure of the primers is available on request. RT-PCR products were sequenced after electroelution from agarose gel and purification with the Qiaquick PCR purification Kit (Quiagen, Courtaboeuf, France). Sequencing reactions were performed with the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) using an ABI-prism sequencer and sequencing analysis 3.4.1 software (Applied Biosystems).
Localization of proteins in platelets and MKs using immunofluorescence and confocal microscopy
Platelet-rich plasma was prepared by centrifuging citrated blood at 150g for 10 minutes at room temperature. Apyrase (0.05 U/mL; Sigma-Aldrich, St Louis, MO, USA) was added and the platelet-rich plasma incubated for 30 minutes at 37°C. Platelets were fixed by adding an equal volume of 2% paraformaldehyde (PFA). After 30 minutes at room temperature, Triton X-100 (0.1%) was added and the incubation continued for 5 minutes. The permeabilized platelets were washed three times in 1% bovine serum albumin (BSA; Eurobio, Les Ulis, France) in phosphate-buffered saline (pH 7.2; BSA-PBS) with centrifugation at 800g for 10 minutes. Platelets were then smeared on glass slides and left to air-dry. After blocking for 45 minutes in BSA-PBS, slides were incubated for 1 hour with selected primary antibodies in a humidity chamber. For each primary antibody, serial dilutions were effected to find the optimal conditions (see Figure legends). Slides were washed three times in 0.25% BSA in PBS and incubated for 1 hour with the appropriate FITC- or Alexa Fluor−labeled secondary antibodies diluted in BSA-PBS in a dark humidity chamber at room temperature. In two-color microscopy, paired primary antibodies were from different species. Washed slides were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA, USA). FITC-labeled anti-mouse or rabbit antibodies were used at 1/50 or 1/100, while the Alexa Fluor−labeled antibodies were all used at 1/200. Negative controls included the use of a primary isotype-matched mouse IgG or nonimmune rabbit IgG followed by secondary antibody or by direct labeling with the secondary antibody alone. MKs or the CHRF-288-11 cell line were treated similarly after plating on glass slides although an additional step here was the use of 4′, 6′ diamidino-2-phenyl indole (Merck KGaA, Darmstadt, Germany). FITC- or phycoerythrin-labeled mAbs to αIIbβ3 and GPIbα were used as an aid to MK characterization. Samples were visualized using a Zeiss Axioplan 2 microscope equipped for epifluorescence using a 63× objective and Isis software (Carl Zeiss, Le Pecq, France) or a Leica SP5 confocal microscope using a 63× objective and Leica Microsystem LAS AF software (Leica Microsystèmes SAS, Rueil Malmaison, France).
TIMP release from platelets
Washed platelets were prepared as described previously [
]; after resuspension at 5×108/mL, they were incubated for different times with 0.5 U/mL thrombin (Hyphen, BioMed, Neuville sur Oise, France) or 25 μM thrombin receptor activating peptide (TRAP) peptide (Neosystem, Strasbourg, France) at room temperature. Platelets lysates were prepared by the addition of radio immunoprecipitation assay buffer (Sigma-Aldrich) added with protease inhibitors (Sigma-Aldrich) to platelet pellets. Proteins in platelet lysates or releasates (equivalent of 5×106 platelets) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 12% minigels and detected by western blotting. Proteins were transferred to nitrocellulose membranes and the membranes blocked using 2.5% BSA in Tris-buffered saline and incubated overnight with murine mAbs to TIMPs 1−4 following antibody specification sheets. A rabbit polyclonal to β-actin was used as a control of loading intensity. Bound IgG was detected using the appropriate IRDye 680−conjugated secondary antibodies at a 1/5,000 dilution. Fluorescent bands were visualized using the Odyssey Infrared Imaging System (LICOR Biosciences).
Results
Localization and expression of TIMPs in megakaryocytes
We localized TIMPs by three-color confocal microscopy in MKs derived from CD34+ progenitor cultures of normal donors in serum-free medium (Fig. 1A ). Results were compared with those obtained for the CHRF-288-11 cell line (Fig. 1B). Nuclei were stained with 4′, 6′ diamidino-2-phenyl indole in these experiments and are blue; the organization of TIMPs was compared to VWF, a known α-granule protein. Confocal microscopy demonstrated the presence of all four TIMPs in both mature MKs and the CHRF-288-11 cell line. The TIMPs were by and large localized independently from VWF, while distribution of TIMP-2 in particular appeared to be more peripheral. Presence of RNA for all four TIMPs in both the cultured MKs and the CHRF-288-11 cell line was confirmed by RT-PCR (Fig. 1C), although the band for TIMP-1 was faint in in vitro cultured MKs. These results show that TIMPs are synthesized in the MK, from which they are ready to be packaged into platelets.
Figure 1Tissue inhibitors of matrix metalloproteinases (TIMPs) are synthesized and stored in megakaryocytes (MKs). In (A) shows the presence of TIMPs 1−4 in mature MKs examined at day 12 after in vitro culture of CD34+ peripheral blood cells in serum-free medium. In (B), the same proteins have been localized in CHRF-288-11 cells also grown in culture in serum-free media. The localization of the TIMPs was by three-color confocal microscopy using monoclonal antibodies (mAbs) (green; dil 1/10−1/25) and compared to that of von Willebrand factor (VWF) detected with a rabbit antibody (red; dil 1/500). Nuclei are visualized using 4′, 6′ diamidino-2-phenyl indole (DAPI, blue). Merged images are shown on the right of each series of illustrations. In (C), RNA was isolated from both sets of cultured cells (day 12) and analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis. RT-PCR was performed with (+) or without (−) RNA template. Sequencing of the amplified products confirmed the identity of the RNA of each TIMP. The sizes for each amplified product are 491 bp (TIMP-1), 562 bp (TIMP-2), 502 bp (TIMP-3), and 544 bp (TIMP-4).
Location ofTIMPs in normal platelets and those from two patients with GPS
We next proceeded to ascertain to what extent TIMPs were found in α-granules of circulating platelets (Fig. 2). Comparison of GPIbα and VWF showed how a membrane glycoprotein and an α-granule constituent could clearly be distinguished by two-color confocal microscopy. TIMPs 1−4 all continued to show a patchwork-like pattern of fluorescence in platelets. While TIMPs-1, -2, and -4 were organized largely independently from the VWF-containing α-granules, there was a colocalization of TIMP-3 with VWF (confirmed with mAbs to TIMP-3 from two different sources). Studies were extended to platelets from the two GPS patients. The deficit in α-granules containing VWF was confirmed for platelets of both patients. The deficit is not total, some residual granules are occasionally seen, although many of these appeared smaller and vestigial. To our surprise, TIMPs 1−4 were retained in the platelets of both patients and labeling was of comparable intensity to the controls. TIMP-2 and -4, especially, tended to have a peripheral organization in the GPS platelets; occasionally, what appeared to be high-density patches were observed on the GPS platelets. Identical results were obtained when Fg was used as a marker of α-granules (illustrated for TIMP-2 in Fig. 2).
Figure 2Bicolor confocal microscopy showing the localization of tissue inhibitors of matrix metalloproteinases (TIMPs) 1 to 4 and the comparison of their distribution with α-granules in the platelets of control donors and two patients with gray platelet syndrome (GPS). In this series of experiments, monoclonal antibodies (mAbs) specific for TIMPs 1 to 4 (dil 1/10−1/25) or GPIbα (1 μg/mL) were detected using Alexa Fluor 488 (green) goat anti-mouse immunoglobulin G (IgG) and rabbit antibodies to von Willebrand factor (VWF) (dil 1/500) or fibrinogen (Fg) (dil 1/100) with Alexa Fluor 568 (red) goat anti-rabbit IgG. An occasional surface-localized large patch of fluorescence is highlighted for GPS patient 2 and TIMP-3 by the white arrow. Quite clearly the TIMPs are not stored in α-granules.
TIMPs 1−4 can be secreted from normal platelets on platelet activation
We next confirmed that TIMPs could be secreted from normal platelets after platelet activation. Western blotting revealed that the bulk of each of TIMPs 1−4 was released into the supernatant following activation of washed platelets with thrombin for 5 minutes (Fig. 3). In contrast, no β-actin was present in the supernatant as a control of lysis. A similar secretion of TIMPs was seen when platelets were stimulated by TRAP through PAR-1 (data not shown).
Figure 3Rapid release of tissue inhibitors of matrix metalloproteinases (TIMPs) from platelets. Washed control platelets resuspended in Tyrode−ethylenediamine tetraacetic acid were incubated with (+) or without (−) 0.5 U/mL thrombin for 5 minutes; this was followed by rapid centrifugation and preparation of the platelet pellet and releasate for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer to nitrocellulose membrane, TIMPs were revealed using murine monoclonal antibodies in Western blotting and are mostly found in the supernatant after platelet activation with this strong agonist.
Quantitative assessment of the organization of a series of stored proteins in normal platelets
We next examined the localization of different combinations of stored proteins in platelets by immunofluorescence microscopy and assessed the degree of colocalization of paired proteins. Figure 4A illustrates further selected images and Figure 4B a quantitative analysis of a larger number of combinations including MMPs. Interestingly, MMPs 1−3 as well as ADAM-17 (tumor necrosis factor−α converting enzyme) showed a substantial presence in α-granules identified through their labeling with VWF or P-selectin. However, MMP-9 and ADAM-10 only weakly colocalized with α-granule markers. We further show that the bulk of TIMP-2 and TIMP-1 failed to colocalize respectively with platelet-derived growth factor (another α-granule protein) and P-selectin, and that TIMPs 1−4 were distributed independently of each other, showing the absence of a universal TIMP storage organelle in platelets. Except for TIMP-3, the other TIMPs in platelets are localized in submembranous structures separated from α-granules. However, none of them were accessible to antibodies in intact platelets by flow cytometry unless the platelets were fixed and permeabilized (data not shown).
Figure 4Further immunofluorescence detection of stored proteins in unstimulated platelets (A) and a quantitative assessment of the colocalization of paired proteins (B). Fixed and permeabilized platelets were dried onto glass slides and incubated with combinations of mouse monoclonal antibodies to P-selectin (1 μg/mL), a disintegrin and metalloprotease (ADAM)-17 (dil 1/25), tissue inhibitors of matrix metalloproteinases (TIMPs) 1−4 (dil 1/10−1/25), matrix metalloproteases (MMPs) 1-3 (dil 1/10-1/50), or rabbit antibodies to von Willebrand factor (VWF) (dil 1/500), ADAM-10 (dil 1/25), MMP-9 (dil 1/50), TIMP-1 (dil 1/40), and platelet-derived growth factor (PDGF) (dil 1/25). Bound primary antibody (see Materials and Methods) was then located with fluorescein isothiocyanate (FITC)-labeled F(ab′)2 fragments of goat antibody to mouse immunoglobulin G (IgG) (green), FITC-labeled F(ab′)2 fragments of swine antibody to rabbit IgG (green), or appropriate combinations of species-specific Alexa Fluor 488 (green) or Alexa Fluor 568 (red) goat anti-mouse or anti-rabbit IgG. In (B), selected images from a minimum of 10 platelets for each antibody combination were superimposed on the computer screen over a grid and the color in each square evaluated as green, red, or merged. Results were then expressed as percent colocalization. Transposing the fluorochromes on the secondary antibodies as shown in (B) for TIMP-3 and VWF did not modify the results.
It is important to study the distribution and secretion of TIMPs/MMPs/ADAMs in platelets because of their role in tissue remodeling and repair processes [
]. Our studies have shown that these proteins are mostly organized independently of each other. MMPs are largely known for their role in the degradation of the extracellular matrix especially collagen. They are implicated in a wide range of biological processes, including development and wound healing [
]. Mostly, MMPs are soluble proteins grouped into specific categories stored as latent proenzymes with unblocking of a catalytic domain required for Ca2+-dependent enzymatic activity [
]. Our results show that MMP-1, MMP-2, MMP-3, and MMP-9 (to a lesser degree), colocalize with the α-granule markers VWF and P-selectin, although our approaches do not distinguish between latent and active protein forms. Our results for MMP-9 are compatible with those of Sheu et al. [
] showed an essentially cytosolic localization of MMP-2, others using confocal microscopy localized MMP-2 in a patchwork-like immunofluorescence similar to that now shown by us [
]. Furthermore, these authors showed that the proenzyme was transiently translocated to the platelet surface after TRAP-induced platelet activation, where it bound in an αIIbβ3-dependent manner prior to release of the active enzyme. Galt et al. [
] published similar results for MMP-1 and confirmed its presence in platelets by Western blotting. Similar to our work, these authors showed that in resting platelets, MMP-1 gave a patchwork-like pattern in confocal microscopy.
ADAM-10 and ADAM-17 are also present in cells as latent forms that require activation [
]). We show that ADAM-17 has an α-granule location, but that ADAM-10 is organized independently of α-granule markers. As both are membrane glycoproteins (reviewed in [
]), their appearance on the platelet surface requires trafficking after platelet activation prior to their exercising sheddase activity. While all TIMPs have a generalized inhibitory activity against MMPs, TIMP-3 is distinctive in inhibiting members of the ADAMs family [
]. Colocalization of TIMP-3 and ADAM-17 to α-granules leaves open the possibility that TIMP-3 is a natural regulator of ADAM-17. Significantly, our evidence strongly suggests that TIMP-1, TIMP-2, and TIMP-4 are stored elsewhere in the platelet and independently of each other. Yet, upon stimulation of platelets by either TRAP or thrombin, all TIMPs are rapidly released from platelets. This is not the first time that proteins that are secreted from platelets are reported not to have a designated granular location; thrombopoietin taken up by and stored in platelets was shown to be mostly cytoplasmic in distribution [
While platelets contain a spliceosome and are capable of some protein synthesis, the bulk of the platelet storage pool of proteins are either synthesized during megakaryocytopoiesis or taken up by endocytosis [
The production of tissue inhibitors of metalloproteinases [TIMPs] in megakaryopoiesis: possible role of platelet- and megakaryocyte- derived TIMPs in bone marrow fibrosis.
Matrix metalloproteases and tissue inhibitors of metalloproteinase secretion by haematopoietic and stromal precursors and their production in normal and leukemic long-term marrow cultures.
]. Our current studies confirm that MKs may be a contributory source of these TIMPs. The detection of TIMP-2 messenger RNA in resting platelets has also been reported [
]. At present, we have no explanation for these different findings.
GPS remains a somewhat enigmatic disorder of α-granule biogenesis with considerable phenotypic heterogeneity (see introduction). Here we confirm for two GPS patients that the α-granule deficiency in this rare inherited disease extends to both Fg- and VWF-containing organelles. The presence of small residual VWF-labeled structures in platelets of both patients is compatible with the hypothesis that α-granule biogenesis is a multistep process and that in GPS there is a block in their maturation [
]. In the case of Fg, endocytic vesicles would form but would not fuse with the missing α-granules, so again the presence in the platelets of small Fg-containing vesicles has a logical explanation. In the clear absence of α-granules, the TIMPs continued to be found in distinct fluorescent patches in the GPS platelets. Despite the fact that platelets of patient 1 (but not patient 2) have a sheddase-induced loss GPVI and TREM-like transcript (TLT)-1 [
], we obtained no evidence for a TIMP deficiency in the platelets of this patient that could account for the phenotypic heterogeneity. One possibility is that GPVI degradation in platelets of patient 1 relates to secondary changes within the marrow. An imbalance between TIMPs and MMPs has been implicated in diseases such as arthritis, cancer, atherosclerosis, aneurysms, nephritis, tissue ulcers, and fibrosis [
Until now, the importance of platelet secretion to platelet function has been thought to be the release of the contents of dense granules and/or α-granules [
]. Examination of EM images of platelets from many GPS patients, including the two studied here, has consistently shown a severe absence of α-granules [
Defective α-granule production in megakaryocytes from gray platelet syndrome: ultrastructural studies of bone marrow cells and megakaryocytes growing in culture from blood precursors.
]. So, how are the TIMPs and perhaps also ADAM-10 stored? Dense granules or lysosomal granules remain candidates, but the somewhat peripheral organization of at least some TIMPs as well as the fact that the TIMPs segregate independently of each other, points to other vesicular structures. One possibility is that these proteins remain in transport vesicles released from the Golgi apparatus but unable to fuse with α-granules. An independent organization may be necessary to prevent continued MMP activation and glycoprotein shedding from platelets. Studies are continuing to investigate this hypothesis. We also plan to complete our work by looking at the organization of ADAMTS-13 in GPS (ADAMTS-13 is an α-granule protease responsible for cleavage of VWF multimers) [
]. In the meantime, our conclusion is that the organization of the platelet secretome is even more complex than previously thought.
Acknowledgments
We acknowledge financial support from the French GIS-Maladies Rares (Paris, France) and INSERM (Paris, France). This work was also performed in the context of the French National Reference Center for platelet disorders sponsored by the French Health Ministry. We thank Robert Combrié for excellent technical assistance and Jean-Max Pasquet (Bordeaux University, Bordeaux, France) for giving us the CHRF-288-11 megakaryocytic cell line. No financial interest/relationships with financial interest relating to the topic of this article have been declared.
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Evidence that differential packaging of the major platelet granule proteins von Willebrand factor and fibrinogen can support their differential release.
Angiogenesis is regulated by a novel mechanism: pro- and anti-angiogenic proteins are organized into separate platelet α-granules and differentially released.
The production of tissue inhibitors of metalloproteinases [TIMPs] in megakaryopoiesis: possible role of platelet- and megakaryocyte- derived TIMPs in bone marrow fibrosis.
Matrix metalloproteases and tissue inhibitors of metalloproteinase secretion by haematopoietic and stromal precursors and their production in normal and leukemic long-term marrow cultures.
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