Collagen or Collagen-related Peptide Cause [Ca²⁺]_i Elevation and Increased Tyrosine Phosphorylation in Human Megakaryocytes

Summary

Since megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in mega-karyocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.

^* Current address: Dr. J. C. Mountford, Department of Medicine, University of Birmingham, Q. E. Medical Centre, Birmingham, B15 2TH, UK.

• References

• 1 Choi ES, Nichol JL, Hokom MM, Hornkohl AC, Hunt P. Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional. Blood 1995; 85: 402-13.
• 2 Guerriero R, Testa U, Gabbianelli M, Mattia G, Montesoro E, Macioce G, Pace A, Ziegler B, Hassan HJ, Peschle C. Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture. Blood 1995; 86: 3725-36.
• 3 Birkmann J, Oez S, Smetak M, Kaiser G, Kappauf H, Gallimeier WM. Effects of recombinant human thrombopoietin alone and in combination with erythropoietin and early-acting cytokines on human mobilized purified CD34+ progenitor cells cultured in serum-depleted medium. Stem Cells 1997; 15: 18-32.
• 4 Broudy VC, Lin NL, Kaushansky K. Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Blood 1995; 85: 1719-26.
• 5 Burstein SA. Cytokines, platelet production and hemostasis. Platelets 1997; 8: 93-107.
• 6 Bartley TD, Bogenberger J, Hunt P, Li Y-S, Lu HS, Martin F, Chang M-S, Samal B, Nichol JL, Swift S, Johnson MJ, Hsu R-Y, Parker VP, Suggs S, Skrine JD, Merewether LA, Clogston C, Hsu E, Hokom MM, Hornkohl A, Choi E, Pangelinan M, Sun Y, Mar V, McNinch J, Simonet L, Jacobson F, Xie C, Shutter J, Chute H, Basu R, Selander L, Trollinger D, Sieu L, Padilla D, Trail G, Elliott G, Izumi R, Covey T, Crouse J, Garcia A, Xu W, Del Castillo J, Biron J, Cole S, Hu MC-T, Pacifici R, Ponting I, Saris C, Wen D, Yung YP, Lin H, Bosselman RA. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 1994; 77: 1117-24.
• 7 de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang W-J, Oles KJ, Hultgren B, Solberg Jr LA, Goeddel DV, Eaton DL. Stimulation of megakaryo-cytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994; 369: 533-8.
• 8 Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort PJ, Grant FJ, Heipel MD, Burkhead SK, Kramer JM, Bell LA, Sprecher CA, Blumberg H, Johnson R, Prunkard D, Ching AFT, Mathewes SL, Bailey MC, Forstrom JW, Buddle MM, Osborn SG, Evans SJ, Sheppard PO, Presnell SR, O’Hara PJ, Hagen FS, Roth GJ, Foster DC. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994; 369: 565-8.
• 9 Kaushansky K. Thrombopoietin: basic biology and clinical promise. Leukemia 1997; 11 (S3) 426-7.
• 10 Drachman JG, Sabath DF, Fox NE, Kaushansky K. Thrombopoietin signal transduction in purified murine megakaryocytes. Blood 1997; 89: 483-92.
• 11 Uneyama C, Uneyama H, Narisawa K, Takahashi M, Akaike N. Kinetic characterisation of thrombin receptor-mediated responses in rat megakaryocytes. Eur J Pharmacol 1997; 319: 299-305.
• 12 Tertyshnikova S, Fein A. [Ca²⁺]_i oscillations and [Ca²⁺]_i waves in rat megakaryocytes. Cell Calcium 1997; 21: 331-44.
• 13 Melford SK, Turner M, Briddon SJ, Tybulewicz VLJ, Watson SP. Syk and Fyn are required by mouse megakaryocytes for the rise in intracellular calcium induced by a collagen-related peptide. J Biol Chem 1997; 272: 27539-42.
• 14 Poole A, Gibbins JM, Turner M, van Vugt MJ, van de Winkel JGJ, Saito T, Tybulewicz VLJ, Watson SP. The Fc receptor γ-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J 1997; 16: 2333-41.
• 15 Chiang TM, Kang AH. Isolation and purification of collagen alpha 1(I) receptor from human platelet membrane. J Biol Chem 1982; 2257: 7581-6.
• 16 Santoro SA, Rajpara SM, Staatz WD, Woods Jr VL. Isolation and characterization of a platelet surface collagen-binding complex related to VLA-2. Biochem & Biophys Res Comm 1988; 153: 217-23.
• 17 Tandon NN, Kralisz U, Jamieson GA. Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion. J Biol Chem 1989; 264: 7576-83.
• 18 Deckmyn H, van Houtte E, Vermylen J. Disturbed platelet aggregation to collagen associated with an antibody against an 85-90 Kd platelet glycoprotein in a patient with prolonged bleeding time. Blood 1992; 79: 1466-71.
• 19 Ryo R, Yoshida A, Sugano W, Yasunaga M, Nakayama K, Saigo K, Adachi M, Yamaguchi N, Okuma M. Deficiency of P62, a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation. Am J Hematol 1992; 39: 25-31.
• 20 Kehrel B, Wierwille S, Clemetson KJ, Anders O, Steiner M, Knight CG, Farndale RW, Okuma M, Barnes MJ. Glycoprotein VI is a major collagen receptor for platelet activation: it recognises the platelet-activating quaternary structure of collagen, whereas CD36, glycoprotein IIb/IIIa, and von Willebrand factor do not. Blood 1998; 91: 491-9.
• 21 Ichinohe T, Takayama H, Ezumi Y, Arai M, Yamamoto N, Takahashi H, Okuma M. Collagen-stimulated activation of syk but not c-src is severely compromised in human platelets lacking membrane protein glycoprotein VI. J Biol Chem 1997; 272: 63-8.
• 22 Tsuji M, Ezumi Y, Arai M, Takayama H. A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets. J Biol Chem 1997; 272: 23528-31.
• 23 Gibbins JM, Okuma M, Farndale R, Barnes M, Watson SP. Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor gamma-chain. FEBS Lett 1997; 413: 255-9.
• 24 Gibbins J, Asselin J, Farndale R, Barnes M, Law C-L, Watson SP. Tyrosine phosphorylation of the Fc receptor 7-chain in collagen-stimulated platelets. J Biol Chem 1996; 271: 18095-9.
• 25 Arai M, Yamamoto N, Moroi M, Akamatsu N, Fukutake K, Tanoue K. Platelets with 10% of the normal amount of glycoprotein VI have an impaired response to collagen that results in a mild bleeding tendancy. B J Haematol 1995; 89: 124-30.
• 26 Blake RA, Schieven GI, Watson SP. Collagen stimulates tyrosine phosphorylation of phospholipase c-gamma 2 but not phospholipase C-gamma 1 in human platelets. FEBS Lett 1994; 353: 212-6.
• 27 Daniel JL, Dangelmaier C, Smith JB. Evidence for a role for tyrosine phosphorylation of phospholipase C gamma 2 in collagen-induced platelet cyto-solic calcium mobilization. Biochem J 1994; 302: 617-22.
• 28 Fugman DA, Witte DP, Jones CLA, Aronow BJ, Lieberman MA. In vitro establishment of a human megakaryoblastic cell line. Blood 1990; 75: 1252-61.
• 29 Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Nagura H, Saito H. Establishment of a novel human megakaryoblastic leukaemia cell line, MEG-01, with positive Philadelphia chromosome. Blood 1985; 66: 1384-92.
• 30 Alitalo R. Induced differentiation of K562 leukaemia cells: A model for studies of gene expression in early megakaryoblasts. Leuk Res 1990; 14: 501-14.
• 31 Martin P, Papayannopoulou T. HEL cells: a new human erythroleukaemia cell line with spontaneous and induced globin expression. Science 1982; 216: 1233-5.
• 32 Mouthon MA, Freund M, Titeux M, Katz A, Guichard J, Breton-Gorius J, Vainchenker W. Growth and differentiation of the human megakaryoblastic cell line (ELF-153): a model for early stages of megakaryocytopoiesis. Blood 1994; 84: 1085-97.
• 33 Saito H. Megakaryocytic cell lines. Baill Clin Haematol 1997; 10: 47-63.
• 34 Morton LF, Hargreaves PG, Farndale RW, Young RD, Barnes MJ. Integrin alpha₂beta₁-independant activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (ploymeric) structures are sufficient alone for alpha₂ beta₁-independant platelet reactivity. Biochem J 1995; 306: 337-44.
• 35 Nunn DL, Watson SP. A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets. Biochem J 1987; 243: 809-13.
• 36 Wu CD, Medeiros LJ, Miranda RN, Mark HF, Rintels P. Chronic myeloid leukaemia manifested during megakaryoblastic crisis. Southern Med J 1996; 89: 422-7.
• 37 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-50.
• 38 Poenie M. Alteration of intracellular Fura-2 fluorescence by viscosity: a simple correction. Cell Calcium 1990; 11: 85-91.
• 39 Poole A, Watson SP. Regulation of calcium by collagen in single human platelets. Br J Pharmacol 1995; 115: 101-6.
• 40 Asselin J, Gibbins JM, Achison M, Lee J-H, Morton LF, Farndale RW, Barnes MJ, Watson SP. A collagen-like peptide stimulates tyrosine phosphorylation of syk and phospholipase Cγ2 in platelets independent of integrili α₂β₁ . Blood 1997; 89: 1235-42.
• 41 Watson SP, Hambleton S. Phosphorylation-dependent and -independent pathways of platelet aggregation. Biochem J 1989; 258: 479-85.
• 42 Puri RN, Colman RW. ADP-induced platelet activation. Crit Rev in Biochem & Mol Biol 1997; 32: 437-502.
• 43 Mossuz P, Schweitzer A, Molla A, Berthier R. Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro. B. Haematol 1997; 98: 819-27.
• 44 Cramer EM, Norol F, Guichard J, Breton-Gorius J, Vainchenker W, Masse JM, Debili N. Ultrastructure of platelet formation by human megakaryocytes cultured with Mpl ligand. Blood 1997; 89: 2336-46.