scholarly journals Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Y Miyakawa ◽  
A Oda ◽  
BJ Druker ◽  
H Miyazaki ◽  
M Handa ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Human Platelet ◽  
Blood Platelets ◽  
Human Platelets ◽  
Genetically Engineered ◽  
Hematopoietic Growth Factors ◽  
Signal Transducers ◽  
Stat Proteins ◽  
Human Blood Platelets

Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently, we and others have shown that thrombopoietin induces rapid tyrosine phosphorylation of Jak2 and other proteins in human platelets and BaF3 cells, genetically engineered to express c- Mpl, a receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some of the effects of cytokines or hematopoietic growth factors by recruitment and tyrosine phosphorylation of a variety of Stat (signal transducers and activators of transcription) proteins. Hence, we have investigated whether Stat proteins are present in platelets and, if so, whether they become tyrosine phosphorylated in response to thrombopoietin. We immunologically identified Stat1, Stat2, Stat3, and Stat5 in human platelet lysates. Thrombopoietin induced tyrosine phosphorylation of Stat3 and Stat5 in these cells. Thrombopoietin also induced tyrosine phosphorylation of Stat3 and Stat5 in FDCP-2 cells genetically engineered to constitutively express human c-Mpl. Thus, our data indicate that Stat3 and Stat5 may be involved in signal transduction after ligand binding to c-Mpl and that this event may have a role in megakaryopoiesis/thrombopoiesis or possibly a mature platelet function such as aggregation.

scholarly journals p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1330-1338 ◽  
Author(s):  
A Oda ◽  
K Ozaki ◽  
BJ Druker ◽  
Y Miyakawa ◽  
H Miyazaki ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Blood Platelets ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Genetically Engineered ◽  
Jurkat Cells ◽  
Dependent Manner ◽  
Endogenous Substrate ◽  
Tyrosine Phosphorylated Protein ◽  
Triton X

To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-cbl in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-cbl was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120c-cbl was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-cbl may be involved in signal transduction following ligand binding to c- Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.

scholarly journals Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and Shc in human blood platelets

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Y Miyakawa ◽  
A Oda ◽  
BJ Druker ◽  
T Kato ◽  
H Miyazaki ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tyrosine Phosphorylation ◽  
Human Blood ◽  
Kinase Activity ◽  
Blood Platelets ◽  
Human Platelets ◽  
Janus Kinase ◽  
Janus Kinase 2 ◽  
Dose Dependent ◽  
Human Blood Platelets

A cDNA for the thrombopoietin has been cloned by several groups. The recombinant thrombopoietin has been reported to stimulate the megakaryocytopoiesis and thrombopoiesis. Little is known regarding the molecular basis of its effects. To elucidate the molecular mechanism involved in signal transduction, we have investigated the effects of thrombopoietin on platelet tyrosine phosphorylation. We report here that thrombopoietin induced time- and dose-dependent tyrosine phosphorylation of several proteins including Janus kinase 2 (Jak2) and a 52-kD protein, Shc, in human blood platelets. Both Jak2 and Shc were tyrosine phosphorylated within 15 seconds after stimulation. The tyrosine phosphorylation of Jak2 was accompanied by increased kinase activity, whereas Shc tyrosine phosphorylation induced its association with a 25-kD protein, Grb2. Thus, our data suggest that Jak2, Shc, and Grb2 may be involved in signal transduction after ligand binding to c- mpl in human platelets.

scholarly journals pp60c-src associates with the SH2-containing inositol-5-phosphatase SHIP1 and is involved in its tyrosine phosphorylation downstream of αIIbβ3 integrin in human platelets

2000 ◽  
Vol 348 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Sylvie GIURIATO ◽  
Stéphane BODIN ◽  
Christophe ERNEUX ◽  
Rüdiger WOSCHOLSKI ◽  
Monique PLANTAVID ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Tyrosine Phosphorylation ◽  
Blood Platelets ◽  
Human Platelets ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Cytoskeleton Reorganization ◽  
Haemopoietic Cells ◽  
Human Blood Platelets ◽  
Dependent Mechanism

SH2-containing inositol-5-phosphatase 1 (SHIP1) was originally identified as a 145 kDa protein that became tyrosine-phosphorylated in response to multiple cytokines. It is now well established that SHIP1 is specifically expressed in haemopoietic cells and is important as a negative regulator of signalling. We found recently that SHIP1 was present in human blood platelets as an Ins(1,3,4,5)P4-phosphatase and a PtdIns(3,4,5)P3-5-phosphatase that became tyrosine-phosphorylated and was relocated to the cytoskeleton in an integrin-dependent manner. Here we report biochemical and pharmacological evidence that the tyrosine kinase pp60c-src is constitutively associated with SHIP1 and is involved in its tyrosine phosphorylation downstream of integrin engagement in thrombin-activated human platelets. The use of cytochalasin D allowed us to demonstrate that the actin cytoskeleton reorganization induced on thrombin stimulation was not required for its integrin-mediated phosphorylation. Moreover, the integrin-dependent relocation of SHIP1 to the cytoskeleton did not require its tyrosine phosphorylation. These results suggest that SHIP1 is first recruited to the integrin-linked signalling complexes and then becomes tyrosine-phosphorylated through a Src-kinase-dependent mechanism but independently of the actin cytoskeleton reorganization.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF Mg++-DEPENDENT DINITROPHENOL-STIMULATED ADENOSINE TRIPHOSPHATASE IN HUMAN BLOOD PLATELETS

1967 ◽  
Vol 15 (5) ◽  
pp. 267-272 ◽  
Author(s):  
VICTOR G. VETHAMANY ◽  
SYDNEY S. LAZARUS
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Blood ◽  
Adenosine Triphosphatase ◽  
Blood Platelets ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Structural Localization ◽  
Adenosine Triphosphatase Activity ◽  
Human Blood Platelets

Fine structural localization of adenosine triphosphatase activity was studied in human platelets briefly fixed in cold formol calcium and then incubated in lead medium with added dinitrophenol. Under these conditions, the Mg++-dependent dinitrophenol-stimulated adenosine triphosphatase of platelet mitochondria was demonstrated, but neither granules nor plasma membrane showed enzyme activity.

Cinemicroscopic Visualization of Shape Change and Motility in Normal and Abnormal Living Human Platelets

1979 ◽  
Author(s):  
L. R. Zacharski ◽  
R. D. Allen ◽  
R. Rosenstein ◽  
S. Widirtsky
Keyword(s):  
Shape Change ◽  
Blood Platelets ◽  
Rapid Test ◽  
Human Platelets ◽  
Epifluorescence Microscopy ◽  
Optical Sectioning ◽  
Dense Bodies ◽  
Cine Film ◽  
20 Nm ◽  
Human Blood Platelets

Living human blood platelets (P) have been examined with a high extinction differential interference contrast (DIC) microscope capable of detecting structures as small as 20 nm in diameter. During the quarter hour required for complete transformation, the entire shape change sequence is clearly visible, including disk to sphere transformation, extension and retraction of pseudopodia, and spreading and ruffling of the hyalomere. The exocytosis of intact 5-hydroxy tryptamine (serotonin)-containing dense bodies has been observed both by DIC microscopy and by epifluorescence microscopy in P stained with mepacrine. The release of dense bodies is associated with the formation of one or more “craters” in the upper surface of the granulomere. With optical sectioning, it is evident that certain “craters” represent internal chambers of the open canalicular system. Using these techniques, abnormalities in P motility have been observed in hereditary P disorders. In summary, the ability to observe and record permanently on cine film the motility of living P provides a rapid test of P function which allows quantitation of normal vs. abnormal motile behavior.

Tachyphylaxis in Human Blood Platelets Induced by Various Agonists and the Effect on Response To other Agonists

1979 ◽  
Author(s):  
P.A. Ruggles ◽  
M.C. Scrutton
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Common Pathway ◽  
Stimulus Response ◽  
Reversible Aggregation ◽  
Agonist Concentration ◽  
Subsequent Addition ◽  
Human Blood Platelets

Tachyphylaxis of human platelets to ADP, adrenaline, thrombin, 5-HT and vasopressin (VP) was induced by preincubation of stirred citrated PRP with an agonist concentration which induced primary reversible aggregation. The effect was demonstrable within 2 mins, after addition of some of the agonists and persisted for at least 30 mins. The extent of tachyphylaxis showed a positive correlation with agonist concentration used to induce the initial reversible response. Partial aganists at the ADP (2’, 3’-dialcohol ADP) or α-adreno-(clonidine) receptors did not induce significant tachyphylaxis to subsequent addition of the full agonist. In most instances platelets iaade tachyphylactic to a given agonist showed an unchanged or enhanced response to all other agonists including arachidonate. However tachyphylaxis to ADP, 5HT or thrombin was associated with an attenuated response to collagen. Furthermore tachyphylaxis to thrombin also caused attenuation of the response to VP and arachidonate. Induction of tachyphylaxis to VP, or addition of oxytocin (an inhibitor of aggregation induced by VP) had no effect on the response to thrombin. Thus the region of the thrombin receptor responsible for induction of tachyphylaxis to this agonist is not identical with that at which VP interacts. If stimulus-response coupling involves a common pathway for most agonists these data suggest that development of tachyphylaxis depends on events which preceed the effect of the agonists en this common pathway.

scholarly journals Survival and recovery of human platelets stored for five days in a non- plasma medium

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 672-675 ◽  
Author(s):  
GA Adams ◽  
SD Swenson ◽  
G Rock
Keyword(s):  
Platelet Aggregation ◽  
Human Blood ◽  
Blood Platelets ◽  
Human Platelets ◽  
Plasma Medium ◽  
Release Reaction ◽  
In Vivo Recovery ◽  
Human Blood Platelets

Abstract Human blood platelets were stored for five days as concentrates in 60 mL of: (a) plasma; (b) non-plasma medium with anticoagulant; and (c) non-plasma medium without anticoagulant. All preparations were equally functional when tested for platelet aggregation and release reaction in response to single agonist or synergistic pairs of agonists in vitro. Platelets stored in non-plasma medium with anti-coagulant had lower kallikrein, fibrino(gen)peptide A, lactate, and beta-thromboglobulin than did plasma controls after five days. In vivo recovery and survival of platelets stored in non-plasma medium with anticoagulant were 51.2% +/- 4.3% and 8.7 +/- 0.3 days, respectively, which were not statistically different from plasma controls of 39.2% +/- 4.9% and 7.2 +/- 0.8 days, respectively. It is concluded that platelets can be stored for five days in a non-plasma medium and still have good in vivo recoveries and survivals.

scholarly journals Calcium Binding Sites on Human Blood Platelets

1977 ◽  
Author(s):  
S. Heptinstall
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Blood Platelets ◽  
Human Platelets ◽  
Extracellular Calcium ◽  
Scatchard Analysis ◽  
Magnesium Ions ◽  
Binding Data ◽  
Calcium Binding Sites ◽  
Human Blood Platelets

Extracellular calcium ions are required for platelets to aggregate in response to various aggregating agents. Although magnesium ions can sometimes stimulate aggregation they only do so when a small amount of calcium is present. The calcium bound to washed human platelets suspended in buffered saline containing 0-200μM+5CaCl2 depends upon the extracellular calcium concentration. Scatchard analysis of the binding data suggests that a few (0,8 × 106) relatively high affinity (K = 85,000) calcium binding sites are present on each platelet. When 2.5mM MgCl2 is included in the saline suspensions the calcium bound to the platelets is only reduced at the higher calcium concentrations. Magnesium ions do not displace the tightly bound calcium. It is suggested that these specific calcium binding sites are involved in platelet aggregation.

scholarly journals Accelerated uptake of 5-hydroxytryptamine by human blood platelets enriched in a sialic acid

1975 ◽  
Vol 148 (2) ◽  
pp. 335-336 ◽  
Author(s):  
L Szabados ◽  
L Mester ◽  
F Michal ◽  
G V R Born
Keyword(s):  
Sialic Acid ◽  
Human Blood ◽  
Blood Platelets ◽  
Human Platelets ◽  
Acetylneuraminic Acid ◽  
Transport Receptor ◽  
Human Blood Platelets

The enzymically catalysed incorporation of N-acetylneuraminic acid into human platelets, whether suspended in their own citrated plasma or in buffered saline containing 0.17 mM-sucrose, accelerated the uptake of 5-hydroxytryptamine. This acceleration decreased with time. The observations may be explained by assuming that N-acetylneuraminic acid is a component of a transport receptor for 5-hydroxytryptamine.

Influence of Blood Platelets on Fibrinolysis

1961 ◽  
Vol 6 (02) ◽  
pp. 196-214 ◽  
Author(s):  
Rudolf Holemans ◽  
Rudolf Gross
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Human Platelets ◽  
Bovine Blood ◽  
Platelet Surface ◽  
Inhibiting Effect ◽  
Platelet Concentration ◽  
Streptokinase Infusion ◽  
Human Plasminogen ◽  
Human Blood Platelets

Summary1. Human blood platelets contain a considerable amount of proactivator activity but no plasminogen activator activity or plasminogen. In test systems, containing streptokinase, the lysis of bovine fibrin is enhanced, depending upon the amount of platelets present.2. The platelet proactivator is completely destroyed by heating during 15 min at 70° C. Washing of platelets reduces their proactivator activity; however proactivator activity can still be found after ten washings.3. Bovine platelets do not contain proactivator activity. The similarity in the content in proactivator activity in plasma and platelets of both species, as well as the decrease of proactivator activity by washing, strongly suggests that the proactivator activity of human platelets is adsorbed on the platelet surface from the plasma.4. Both in human platelets and in bovine platelets, antifibrinolysin can be found. This factor is thermostable and is only slightly diminished by washing. The platelet antifibrinolysin seems for its larger part to be located in the platelets; only a small fraction may be adsorbed on their surface.5. The platelet antifibrinolytic activity can, depending upon the platelet concentration, be easily determined in systems containing urokinase or plasmin. Also in streptokinase-activated systems, bovine blood platelets have an inhibiting effect; human blood platelets inhibit streptokinase-induced fibrinolysis when their proactivator activity has been destroyed by heating. When streptokinase and unheated human blood platelets are tested on bovine fibrin the inhibitor effect is completely masked by the presence of proactivator.6. The clinical significance of these findings with regard to fibrinolysis occurring spontaneously or induced by streptokinase infusion, as well as their importance for the differentiation of proactivator and human plasminogen are discussed.

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