Effect of human recombinant thrombopoietin (TPO) on platelet activation in vitro was studied. Although TPO by itself did not cause platelet aggregation, it upregulated ADP-induced aggregation, especially the second wave of aggregation. This effect was dose-dependent for up to 5 ng/ml of TPO. When platelets were activated by epinephrine, collagen, or α-thrombin, similar effect was observed. However, TPO did not affect A23187- or PMA-induced aggregation, suggesting that TPO may have modulated the signal transduction pathway upstream of inositol 1,4,5-trisphosphate and diacylglycerol production. TPO also upregulated thrombin-induced α-granule secretion. To clarify the involvement of protein tyrosine phosphorylation, platelets were activated by TPO and/or suboptimal concentration of ADP, then tyrosine phosphorylation was detected by immunoblot analysis, using anti-phosphotyrosine monoclonal antibody. TPO by itself caused significant tyrosine phosphorylation of 146,130,122,108, 97,94, and 88 kDa proteins. Further, by using antibodies against signal transduction molecules for immunoprecipitation, we observed the significant tyrosine phosphorylation in Jak2 and Tyk2 molecules after TPO-stimulation. The results of the present experiment clearly indicate that TPO directly activated platelets and modulated intracellular signal transduction pathway.
References
1
McDonald TP.
Thrombopoietin: its biology, purification, and characterization. Exp Hematol 1988; 16: 201-205
2
Hill RJ,
Levin J,
Levin FC.
Correlation of in vitro and in vivo biological activities during the partial purification of thrombopoietin. Exp Hematol 1992; 20: 354-360
3
Mazur EM,
South K.
Human megakaryocyte colony-stimulating factor in sera from aplastic dogs: partial purification, characterization, and determination of hematopoietic cell lineage specifity. Exp Hematol 1985; 13: 1164-1172
5
Methia N,
Louache F,
Vainchenker W,
Wendling F.
Oligodeoxynucleotide antisense to c-mpl protooncogene inhibits human megakaryopoiesis in vitro. Blood 1993; 82: 1395-1401
6
Vigon I,
Mornon JP,
Cocault L,
Mitjavila MT,
Tambourin P,
Gisselbrecht S,
Souyri M.
Molecular cloning and characterization of Mpl, the human homolog of the v-mpl oncogene. Proc Natl Acad Sci USA 1992; 89: 5640-5644
10
Wendling FW,
Maraskovsky E,
Debili N,
Florindo C,
Teepe M,
Titeux M,
Methia N,
Breton-Gorius J,
Cosman D,
Vainchenker W.
c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 1994; 369: 571-574
11
Batley TD,
Bogenberger PH,
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,
Jacobsen 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,
Castillo JD,
Biron J,
Cole S,
Hu C-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: 1117-1124
12
Sohma Y,
Akahori H,
Seki N,
Hori T,
Ogami K,
Kato T,
Shimada Y,
Kawamura K,
Miyazaki H.
Molecular cloning and chromosomal localization of the human thrombopoietin gene. FEBS Letters 1994; 353: 57-61
13
Kuramochi S,
Chiba T,
Amanuma H,
Tojo A,
Todokoro K.
As growth signal erythropoietin activates the same tyrosine kinases as interleukin 3, but activates only one tyrosine kinase as differentiation signal. Biochem Biophys Res Commun 1991; 181: 1103-1109
14
Miyakawa Y,
Oda A,
Druker BJ,
Kato T,
Miyazaki H,
Handa M,
Ikeda Y.
Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and She in human blood platelets. Blood 1995; 86: 23-27
16
Golden A,
Nemeth SP,
Brugge JS.
Blood platelets express high levels of the pp60c-scr-specific tyrosine kinase activity. Proc Natl Sci USA 1986; 85: 852-856
17
Nakamura S,
Takeuchi F,
Tomizawa T,
Takasaki N,
Kondo H,
Yamamura H.
Two separate tyrosine kinases in human platelets. PUBS Letters 1985; 184: 56-59
18
Nakamura S,
Yamamura H.
Thrombin and collagen induce rapid phosphorylation of a common set of cellular proteins on tyrosine in human platelets. J Biol Chem 1989; 264: 7089-7091
19
Golden A,
Brugge JS,
Shattil SJ.
Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins. J Cell Biol 1990; 111: 3117-3127
20
Witthuhn BA,
Quelle FW,
Silvennoinen O,
Yi T,
Tang B,
Miura O,
Ihle JN.
Jak2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 1993; 30: 227-236
21
Silvennoinen O,
Witthuhn BA,
Quelle FW,
Cleveland JL,
Yi T,
Ihle JN.
Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction. Proc Natl Acad Sci USA 1993; 90: 8429-8433
22
Quelle FW,
Sato N,
Witthuhn BA,
Inborn RC,
Eder M,
Miyajima A.
Jak2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol Cell Biol 1994; 14: 4335-4341
23
Shimoda K,
Okumura S,
Harada N,
Kondo S,
Okamura T,
Niho Y.
Identification of a functional receptor for granulocyte colony-stimulating factor on platelets. J Clin Invest 1993; 91: 1310-1313
26
Daniel JL,
Dangelmaier C,
Smith JB.
Evidence for a role for tyrosine phosphorylation of phospholipase Cγ2 in collagen-induced platelet cytosolic calcium mobilization. Biochem J 1994; 302: 617-622
27
Kavanaugh WM,
Klippel A,
Escobendo JA,
Williams LT.
Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells. Mol Cell Biol 1992; 12: 3415-3424
