© 1997 American Heart Association, Inc.
Articles |
Synthesis and Secretion of von Willebrand Factor and Fibronectin in Megakaryocytes at Different Phases of Maturation
From the Cardeza Foundation for Hematologic Research and the Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa.
Correspondence to Paul K. Schick, MD, Cardeza Foundation for Hematologic Research, Jefferson Medical College, Thomas Jefferson University, 1015 Walnut St, Philadelphia, PA 19107-5099.
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Abstract |
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Abstract Our goals have been to define the biochemical characteristics of megakaryocytes during maturation that are critical for platelet assembly and release into the circulation and to introduce biochemical markers for megakaryocytes. To achieve these goals, we have studied fibronectin (FN) and von Willebrand factor (vWF), which are large adhesive proteins that are synthesized by megakaryocytes, stored in alpha granules, and thought to have a fundamental role in hemostasis. The study demonstrated that vWF is primarily synthesized in mature megakaryocytes, which synthesized 7.5 times more vWF than immature megakaryocytes. Brefeldin A, which blocks the exit of proteins from the rough endoplasmic reticulum (RER), inhibited the formation of vWF multimers but did not affect the synthesis of monomers and dimers in mature megakaryocytes. These data are consistent with the formation of vWF dimers in the RER and the assembly of vWF multimers in the trans- and post-golgi. The synthesis of both the 260-kD and 275-kD pro-vWF was detected. However, the synthesis of 275-kD pro-vWF and 220-kD mature vWF was only evident after 2 hours, suggesting that the transit time of nascent vWF through the RER is about 2 hours. Constitutive secretion of vWF was demonstrated in megakaryocytes. About 14.5% and 4.6% of synthesized vWF was secreted by mature and immature megakaryocytes, respectively. In contrast, the synthesis of FN monomers and dimers was established in immature megakaryocytes, and their synthesis in mature megakaryocytes was very similar. Constitutive secretion of FN was not seen in megakaryocytes. Brefeldin A did not inhibit the synthesis of FN dimers; thus, formation of FN dimers occurs in the RER. The demonstration that vWF and FN are synthesized at different phases of megakaryocyte maturation and that only vWF is constitutively secreted by megakaryocytes provides new information relevant to alpha granule formation and possibly bone marrow matrix assembly.
Key Words: megakaryocytes • von Willebrand factor • fibronectin • synthesis • secretion
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Introduction |
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Megakaryocytes but not platelets are capable of protein synthesis and thus determine the content and structure of platelets. The bulk of protein synthesis most likely occurs in recognizable megakaryocytes that are approaching maturity. During the terminal phase of maturation, platelets are assembled and released into the circulation. Immature and mature recognizable megakaryocytes can be distinguished on the basis of cytoplasmic characteristics, but this morphological classification is a crude assessment of maturity.1
Delineating the biochemical changes that occur during megakaryocyte maturation would provide a more accurate assessment of maturity. There is evidence that immature recognizable megakaryocytes do not contain fully developed alpha granules and that demarcation membranes and granules are primarily evident in mature megakaryocytes. There are differences in the golgi, RER, and the demarcation membrane system in megakaryocytes at different phases of maturation.2 3
Our goals have been to define the biochemical characteristics of megakaryocytes during maturation and differentiation that are critical for platelet assembly and release into the circulation and to introduce biochemical markers for megakaryocyte maturity. We have developed techniques for studying protein synthesis and other biochemical characteristics of recognizable megakaryocytes at different phases of maturation.4 5 These approaches have been used to demonstrate differences in proteoglycan species,5 lipid synthesis,6 7 8 9 10 thromboxane synthesis,11 adenine nucleotides,12 and the expression of proteins in megakaryocytes at different phases of maturation.13
We have now extended these studies to FN and vWF. FN and vWF are large adhesive proteins that are synthesized by megakaryocytes, are stored in alpha granules, and have a fundamental role in hemostasis.14 15 16
FN is a large multifunctional interactive protein that is present in blood plasma and in a variety of cells types and extracellular matrices. It is thought to promote cell migration, adhesion, and differentiation and is involved in tissue remodeling and wound healing. FN is synthesized by fibroblasts and a number of other cells that can also secrete the protein. It is synthesized as a monomer (Mr 220 000) and processed to dimers composed of two similar monomers that are linked by disulfide bonds. FN is either constitutively secreted or stored in granules in most cells.17
vWF is a large multimeric protein that is present in plasma, endothelial cells, subendothelial cell matrix, megakaryocytes, and platelets. Immunoreactive vWF has been detected in mononuclear megakaryocytes, a progenitor stage that precedes the phase of recognizable immature megakaryocytes. Platelets adhere to vWF present in exposed subendothelium in injured vessels, and this represents an early phase of platelet activation. Endothelial cells synthesize vWF and are most likely the primary source of plasma vWF. vWF is synthesized as monomers that are processed into dimers in the RER. However, multimerization of vWF occurs in trans- and post-golgi. vWF is either constitutively secreted or stored in Weibel-Palade bodies in endothelial cells or in alpha granules in megakaryocytes and platelets.18
Previous studies have indicated that both FN and vWF are synthesized in megakaryocytes.14 15 16 It has been shown that there is marked similarity in the size of vWF multimers that are present in human platelets and guinea pig platelets and megakaryocytes19 and that the processing of vWF is similar in neoplastic megakaryocytes and endothelial cells.15 We now report that these proteins are synthesized at different phases of maturation in guinea pig megakaryocytes. Our study provides information about the processing of these proteins during megakaryocyte maturation. In addition, the study also demonstrates constitutive secretion of vWF but not secretion of FN in megakaryocytes.
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Methods |
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Preparation of Cells
Guinea pig megakaryocytes were isolated to about 85% purity by cell number and to >98% by protein content or cell volume, since megakaryocytes are considerably larger than other cells.20 Nonmegakaryocytic bone marrow hematopoietic cells were prepared and used as controls.5 Megakaryocytes at different phases of maturation were separated by the Celsep procedure, which separates cells by size and was used to prepare fractions that contained primarily mature megakaryocytes and fractions highly enriched with immature megakaryocytes.5 The viability of the isolated cells was about 89% based on trypan blue exclusion.
Antibodies
Affinity-purified rabbit anti-human FN that reacts with both plasma and cellular FN isoforms was used to assess general FN (F3648, Sigma Chemical Co). A rabbit anti-human vWF antibody was also used (Sigma). Both antibodies cross-reacted with guinea pig proteins.
Protein Synthesis
Isolated megakaryocytes were incubated with Expre35S 35S (New England Nuclear) at 100 µCi/mL in Dulbecco's modified Eagle's medium. The incubations were carried out for several periods up to 17 hours. Protein concentrations were measured using the Bio-Rad DC protein assay based on the Lowry method. Analysis of vWF and FN were performed on separate megakaryocyte preparations.
vWF: Immunoprecipitation and Electrophoresis
Megakaryocytes were pelleted by centrifugation and lysed at 4°C in 0.2% Tris-SDS, pH 8.3, in the presence of EDTA (4 mmol/L), PMSF (1 mmol/L), iodoacetic acid (2 mmol/L), and N-ethylmaleimide (NEM; 2 mmol/L). FN was removed by incubation of the lysate with gelatin–Protein A. Newly synthesized vWF was immunoprecipitated, and the immune complex was isolated by Protein A Sepharose CL-4B. Radiolabeled proteins were separated by 2% agarose gels prepared from Sigma Type V: high gelling temperature agarose21 and analyzed by autoradiography.
FN: Immunoprecipitation and Electrophoresis
Megakaryocytes were pelleted and lysed at room temperature in 0.2% Tris-SDS buffer, pH 8.3, in the presence of EDTA (5 mmol/L), PMSF (1 mmol/L), iodoacetic acid (2 µmol/L), NEM (2 mmol/L), leupeptin (0.1 mmol/L), and aprotonin (2.7 µmol/L). FN was immunoprecipitated, and the immune complex was isolated by Protein A Sepharose CL-4B. Newly synthesized FN was separated by reduced 7.5% SDS-PAGE, 2% agarose gels, or in some experiments by 1.5% Seakem Gold agarose gels (FMC).
Constitutive Secretion of vWF and FN
For the assessment of constitutive secretion of newly synthesized proteins, vWF or FN in the incubation medium was immunoprecipitated and separated by agarose gel electrophoresis and analyzed by autoradiography as described above.
Megakaryocytes were pretreated with brefeldin A3 (10 µg/mL) for 30 minutes before the addition of Expre35S 35S in several experiments.
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Results |
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The syntheses of vWF in immature and mature megakaryocyte subpopulations prepared by the Celsep procedure were compared. Fig 1
is representative of seven experiments demonstrating that considerably less vWF was synthesized by megakaryocytes in the immature than in the mature fraction. Brefeldin A, which blocks the exit of proteins from the RER,22 inhibited the formation of vWF multimers but did not affect the synthesis of monomers and dimers in mature megakaryocytes.
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The total radioactivity in multimers, dimers, and monomers was analyzed by scintillation spectrometry in megakaryocytes pooled from several animals in two experiments. The results of this analysis revealed that 7.5 times more vWF was synthesized in mature fractions than in fractions with immature megakaryocytes. However, both immature and mature cells synthesized multimers as well as dimers and monomers, although the ratio of dimer to monomer appeared to be greater in mature megakaryocytes.
The synthesis and secretion of vWF in megakaryocytes of differing maturities prepared by the Celsep procedure were investigated. Constitutive secretion was estimated by the analysis of newly synthesized vWF in the incubation medium. The data in Fig 2, representative of six experiments, demonstrate that the synthesis of vWF and constitutive secretion occurred primarily in mature megakaryocytes. vWF dimers and multimers but not monomers were secreted. The secretion of vWF was not due to cell damage because minimal amounts of LDH (5%) were detected in the incubation medium during these experiments. The analysis of radioactivity in the bands by scintillation spectrometry in megakaryocytes pooled from several animals in two experiments revealed that 14.5% of vWF was constitutively secreted by mature fractions and about 4.6% was secreted by immature fractions after correction for leakage (5% as estimated by LDH in the incubation medium).
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The synthesis of pro-vWF in megakaryocytes was studied. In the time course experiment shown in Fig 3, the 260-kD pro-vWF species and a trace of the 220-kD form were detected within 60 minutes after pulse labeling. However, at 120 minutes, the 220-kD mature vWF form was the primary band seen in the reduced 7.5% SDS-PAGE autoradiogram. The 260- and 275-kD species were also present. This suggests that nascent vWF has about a 2-hour transit time in the RER before exit into the golgi. As is the case in endothelial cells, the 220-kD mature vWF monomer and the 275-kD pro-vWF are formed in the trans- and post-golgi.
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To study the synthesis of pro-vWF at different phases of megakaryocyte maturation, immature and mature megakaryocytes were prepared by the Celsep procedure. Fig 4 demonstrates that after overnight metabolic labeling, both pro-vWF (260 and 275 kD) and mature vWF (220 kD) were synthesized and processed in mature and immature megakaryocytes. However, considerably more pro-vWF had been synthesized per total protein in mature than in immature megakaryocytes.
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The synthesis of FN in megakaryocytes was studied. After overnight metabolic labeling, FN was immunoprecipitated, and multimers, monomers, and dimers were separated on a 1.5% Seakem Gold agarose gel (FMC). Fig 5 is representative of three experiments. The dimers and multimers seen in the unreduced gel are not present in the reduced gel. Not shown are data indicating that FN dimers are synthesized within 30 minutes. Not shown are the results of eight experiments in which the incubation medium from megakaryocytes was immunoprecipitated, and no constitutive secretion of FN was seen.
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To compare FN synthesis during megakaryocyte maturation, immature and mature megakaryocyte subpopulations were prepared by the Celsep procedure. Fig 6 is representative of five experiments and shows that equivalent amounts of FN monomers, dimers, and multimers are synthesized in mature and immature megakaryocytes. Most of the synthesized FN in Figs 5
and 6 is monomeric or dimeric. However, some cross-linked FN multimers were also detected in unreduced gels.
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Not shown are data from experiments that demonstrated that brefeldin A did not affect the formation of FN dimers. This indicates that FN dimerization occurs in the RER. Brefeldin A would not be expected to inhibit FN multimerization that occurs in consequence of the assembly of FN on the cell surface and is not due to processing of FN in the golgi.17
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Discussion |
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Although the ratio of vWF monomers, dimers, multimers, and pro-vWF that are synthesized in immature megakaryocytes is similar to that in mature megakaryocytes, there is a marked quantitative difference in the synthesis of these vWF molecules during megakaryocyte maturation. vWF and pro-vWF are primarily synthesized by mature megakaryocytes. However, equivalent amounts of mRNA for vWF have been detected in both immature and mature recognizable megakaryocytes by Northern blotting and in situ hybridization.13 Thus, expression of mRNA appears to be established at an earlier phase of maturation than that in which maximal synthesis of vWF protein occurs. The study demonstrates that transcription does not necessarily correlate with the maximal synthesis of protein product.
One of the major actions of brefeldin A is to block the release of proteins from the RER.22 Treatment of megakaryocytes with brefeldin A permitted the formation of vWF monomers and dimers but blocked the formation of vWF multimers. Thus, as is the case in endothelial cells, multimerization of vWF in megakaryocytes occurs in the trans- and post-golgi. There are differences in the process of vWF dimerization and multimerization. vWF dimers are formed through disulfide bonds near the COOH terminus, whereas multimers are formed through disulfide bonds near the NH2 terminus.23 Also, vWF multimerization but not dimerization is dependent on an acid pH, which provides evidence that multimerization occurs in the trans- and post-golgi.24 Interestingly, vWF is the only protein known to form interchain disulfide bonds in trans- and post-golgi compartments.
The 260- and 275-kD pro-vWF have been detected in neoplastic megakaryocytes.15 Our study demonstrated that these pro-vWF proteins also occur in nonneoplastic megakaryocytes. In addition, the present study indicates that the transit time of nascent vWF through the RER is about 2 hours, since only the 260-kD pro-vWF can be detected in megakaryocytes at the 30- and 60-minute time points after metabolic labeling, whereas the formation of the 275-kD pro-vWF and the 220-kD mature vWF monomer occurs after 120 minutes. Thus, the processing of vWF is similar in megakaryocytes and endothelial cells.18
The detection of pro-vWF and vWF multimers in immature megakaryocytes is of interest because of its implications for the assembly of alpha granules. Pro-vWF is thought to be essential for vWF multimerization and the formation of vWF-containing storage granules.24 25 This is based on evidence that pro-vWF is involved in the formation of Weibel-Palade bodies in endothelial cells.18 Also, vWF storage granules can be detected in a mouse pituitary cell line (AtT-20 cells) in which pro-vWF has been expressed. Nontransfected AtT cells do not contain these storage granules.25 It is known that vWF is stored in platelet and megakaryocyte alpha granules.26 Therefore, the detection of pro-vWF and vWF multimers in immature megakaryocytes suggests that the mechanisms for the assembly of vWF-containing alpha granules exist in immature megakaryocytes but are only fully established in mature megakaryocytes, since the expression of pro-vWF and vWF multimers is considerably greater in mature than in immature megakaryocytes. This information is consistent with previous studies indicating that alpha granules are sparse and only partially developed in immature megakaryocytes and are fully developed in mature megakaryocytes.27
Our study compared the constitutive secretion of vWF in immature and mature megakaryocytes. The data indicate that constitutive secretion of vWF occurs in normal guinea pig megakaryocytes and that about 14.5% of synthesized vWF is secreted by mature cells. Also, constitutive secretion is established in immature megakaryocytes but occurs to a much greater degree in mature megakaryocytes.
FN is synthesized in a variety of cells, including fibroblasts, endothelial cells, and megakaryocytes.16 17 The present study showed that FN is processed to dimers in the RER based on the observation that brefeldin A did not inhibit the formation of FN dimers by megakaryocytes. The maximal amount of FN dimer is synthesized within 30 minutes in megakaryocytes, and thus time for FN processing is considerably shorter than for vWF.
The study showed that in contrast to vWF, FN is synthesized to the same extent in immature and mature recognizable megakaryocytes. New information about differences between mature and immature megakaryocytes is provided by the demonstration that vWF and FN are synthesized at different phases of megakaryocyte maturation and that only vWF is constitutively secreted by megakaryocytes. Although megakaryocytes do not appear to constitutively secrete FN, FN is constitutively secreted by other cells.17 To date, we have also shown that mRNA for P-selectin is expressed primarily in mature megakaryocytes, whereas mRNA for vWF and glycoprotein Ib-
are also expressed in immature megakaryocytes.13 Most aspects of lipid metabolism are maximally active in immature megakaryocytes.6 7 8 9 10 However, de novo lipid synthesis occurs primarily in mature megakaryocytes because of low levels of acetyl-CoA in immature megakaryocytes.8 9
FN synthesized by megakaryocytes is stored in alpha granules. Although FN is not constitutively secreted, we have previously reported that it can be released to the circulation in response to thrombin.16 This raises the possibility of a dual role for FN in platelet biology. FN may be released from platelets after injury and contribute to wound healing. Alternatively, FN may be released from megakaryocytes in response to specific stimuli and thereby modulate megakaryocyte interaction with matrix and megakaryocyte migration and maturation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The study was supported by grants from the National Institutes of Health (HL-25455 and HL-51481). We appreciate the expertise of Drew Likens in preparing the illustrations.
Received May 17, 1996; accepted December 17, 1996.
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References |
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3. Mazur E. Megakaryocytes and megakaryocytes. In: Lofcalzo J, Shafer AI, eds. Thrombosis and Hemorrhage. Cambridge, UK: Blackwell Scientific Publishers Inc; 1992.
4.
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5.
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8. Schick PK, He X. Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation. J Lipid Res. 1990;31:1645-1654.[Abstract]
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10.
Schick PK, Walker J. The acylation of megakaryocyte proteins: GpIX is primarily myristoylated while GpIb is palmitoylated. Blood. 1996;87:1377-1384.
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12. Wojenski C, Schick PK. Megakaryocyte storage granules: accumulation of adenine nucleotides during megakaryocyte maturation. J Lab Clin Med. 1993;121:479-485.[Medline] [Order article via Infotrieve]
13. Schick PK, Konkle BA, He X, Thornton RD. P-selectin mRNA expressed at a later phase of maturation than mRNAs for von Willebrand factor and glycoprotein Ib-alpha. J Lab Clin Med. 1993;121:714-721.[Medline] [Order article via Infotrieve]
14. Nachman RL, Levine RF, Jaffe EA. Synthesis of factor VIII antigen by cultured guinea pig megakaryocytes. J Clin Invest. 1977;60:914-921.
15. Sporn LA, Chavin SI, Marder VJ, Wagner DD. Biosynthesis of von Willebrand protein by human megakaryocytes. J Clin Invest. 1985;76:102-106.
16.
Schick PK, Wojenski C, Bennett VD, Ivanova T. The synthesis and localization of alternatively-spliced fibronectin EIIIB in resting and thrombin-treated megakaryocytes. Blood. 1996;87:1817-1823.
17. Hynes RO. Fibronectins. Springer Series in Molecular Biology. New York, NY: Springer-Verlag; 1990:335-348.
18. Wagner DD. Cell biology of von Willebrand factor. Annu Rev Cell Biol. 1990;6:217-246.
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20. Schick PK, Schick BP. Methods for the analysis of the biochemistry of recognizable megakaryocytes. In: Colman RW, Smith JB, eds. Methods in Pharmacology. New York, NY: Alan R Liss Inc; 1987:19-31.
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22. Pelham HHR. Multiple targets for brefeldin A. Cell. 1991;76:449-451.
23.
Dong Z, Thoma RS, Crimmins DL, McCourt DW, Tuley EA, Sadler JE. Disulfide bonds required to assemble functional von Willebrand factor multimers. J Biol Chem. 1994;269:6753-6758.
24.
Mayadas TN, Wagner DD. In vitro multimerization of von Willebrand factor is triggered by low pH. J Biol Chem. 1989;264:13497-13503.
25. Wagner DD, Saffaripour S, Bonfanti R, Sadler JE, Cramer EM, Chapman B, Mayadas TN. Induction of specific storage organelles by von Willebrand factor propolypeptide. Cell. 1991;64:403-413.[Medline] [Order article via Infotrieve]
26.
Cramer EM, Meyer D, le Menn R, Breton-Gorius J. Eccentric localization of von Willebrand factor in an internal structure of platelet alpha-granule resembling that of Weibel-Palade bodies. Blood. 1985;66:710-713.
27. Straneva JE, Goheen MP, Hui SL, Bruno E, Hoffman R. Terminal cytoplasmic maturation of human megakaryocytes. Exp Hematol. 1986;14:919-929.
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