scholarly journals Vinculin is a major platelet protein that undergoes Ca2+-dependent tyrosine phosphorylation

1993 ◽  
Vol 294 (3) ◽  
pp. 675-680 ◽  
Author(s):  
J G Vostal ◽  
N R Shulman
Keyword(s):  
Monoclonal Antibody ◽  
Molecular Mass ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Soluble Fraction ◽  
Phosphorylated Protein ◽  
Platelet Protein ◽  
Activated Platelets ◽  
Sds Page ◽  
Tyrosine Phosphorylated Protein

When intracellular Ca2+ pools are released during platelet stimulation by thrombin, elevation of platelet cytosolic Ca2+ concentration induces tyrosine phosphorylation of a 130 kDa protein, and refilling the pools mediates dephosphorylation of this protein [Vostal, Jackson and Shulman (1991) J. Biol. Chem. 266, 16911-16916]. In the present work the 130 kDa protein was identified as vinculin by the following criteria. (1) It is detected on protein immunoblots of thrombin-activated platelets by both monoclonal anti-phosphotyrosine and anti-vinculin antibodies. (2) Removal of N-linked sugars with peptide-N-glycosidase or reduction did not change the molecular mass of vinculin or of the 130 kDa protein on SDS/PAGE. (3) The 130 kDa tyrosine-phosphorylated protein associates with Triton-soluble fraction of platelets as does vinculin. (4) The 130 kDa protein immunoprecipitated by anti-vinculin monoclonal antibody reacts with anti-phosphotyrosine antibody; when immunoprecipitated by anti-phosphotyrosine antibody it reacts with anti-vinculin antibody. (5) The 130 kDa tyrosine-phosphorylated protein and vinculin focus isoelectrically at pI 5.4-5.8. Our finding that vinculin is a major platelet protein that undergoes Ca(2+)-dependent tyrosine phosphorylation during platelet activation may provide clues to the function of this protein.

scholarly journals CD40 signaling pathway: anti-CD40 monoclonal antibody induces rapid dephosphorylation and phosphorylation of tyrosine-phosphorylated proteins including protein tyrosine kinase Lyn, Fyn, and Syk and the appearance of a 28-kD tyrosine phosphorylated protein.

1994 ◽  
Vol 179 (6) ◽  
pp. 1923-1931 ◽  
Author(s):  
M Faris ◽  
F Gaskin ◽  
J T Parsons ◽  
S M Fu
Keyword(s):  
Monoclonal Antibody ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Protein Tyrosine Kinase ◽  
Immunoglobulin M ◽  
Protein Tyrosine ◽  
Phosphorylated Protein ◽  
Tyrosine Phosphorylated Protein

CD40 plays an important role in B cell activation, proliferation, and Ig class switching. The signal transduction pathway mediated by CD40 was studied using monoclonal antibody (mAb) 626.1 to CD40. Burkitt's lymphoma and Epstein-Barr virus-transformed B cell lines and tonsilar B lymphocytes were treated with the anti-CD40 mAb for various lengths of time. The early events triggered by CD40 were examined by monitoring the changes in tyrosine phosphorylation of cellular proteins with anti-phosphotyrosine mAb. Dephosphorylation of specific proteins ranging between 50-110 kD and the appearance of a 28-kD tyrosine phosphorylated protein were seen within 30 s in human B cell lines. The dephosphorylation was reversed and the 28-kD protein was dephosphorylated in cells stimulated for 1 min. In resting B cells, the appearance of the 28-kD phosphoprotein was observed in 30 s after the addition of the anti-CD40 mAb. The tyrosine phosphorylation of this protein persisted. The patterns of protein tyrosine phosphorylation differed from those induced by an anti-immunoglobulin M mAb. The changes in the state of tyrosine phosphorylation induced by the anti-CD40 mAb were obviated by mAb to CD45, a protein tyrosine phosphatase (PTP) or by the addition of sodium orthovanadate, a broad PTP inhibitor. They were also blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, and PKC and protein serine/threonine kinase inhibitors, H7 and HA1004. In addition, the alteration in the tyrosine phosphorylation of PTKs Lyn, Fyn, and Syk was directly demonstrated. Engagement of CD40 for 30 s induced a transient decrease in tyrosine phosphorylation of these PTKs. These results indicate that the early events in CD40 signaling involve the complex interaction between PTP and protein kinases.

A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma

1994 ◽  
Vol 72 (05) ◽  
pp. 745-749 ◽  
Author(s):  
Elza Chignier ◽  
Maud Parise ◽  
Lilian McGregor ◽  
Caroline Delabre ◽  
Sylvie Faucompret ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Peripheral Blood ◽  
Vascular Trauma ◽  
Activated Platelets ◽  
Group I ◽  
Group Ii ◽  
Rat Platelets

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.

Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4304-4313 ◽  
Author(s):  
A Oda ◽  
Y Miyakawa ◽  
BJ Druker ◽  
A Ishida ◽  
K Ozaki ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Genetically Engineered ◽  
Myelogenous Leukemia ◽  
Insoluble Residue ◽  
Consistent Difference ◽  
Clot Retraction ◽  
Phosphorylated Protein ◽  
Tyrosine Phosphorylated Protein

Abstract Platelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crid, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crid was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crk1 in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crk1 can be phosphorylated by a kinase other than p210bcr-abl and that crk1 may have a role in signaling by thrombopoietin.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Abstract Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

A new monoclonal antibody, mAb 204-11, that influences the binding of platelet GPVI to fibrous collagen

2003 ◽  
Vol 89 (06) ◽  
pp. 996-1003 ◽  
Author(s):  
Jun Mizuguchi ◽  
Sachiko Kawashima ◽  
Michiko Nagamatsu ◽  
Yoshiki Miura ◽  
Tomohiro Nakagaki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Site ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Collagen Binding ◽  
Glycoprotein Vi ◽  
Phosphorylation Of Proteins ◽  
Dimeric Form ◽  
Collagen Receptor

SummaryThe newly identified platelet collagen receptor glycoprotein VI binds to fibrous collagen, inducing platelet activation. Several antibodies against GPVI have been reported, including a patient’s auto-antibodies, that activates platelets through their ability to crosslink this glycoprotein. We have developed a monoclonal antibody (mAb) against GPVI using the recombinant extracellular domain of GPVI as an antigen. This antibody, mAb 204-11, induced platelet aggregation and tyrosine phosphorylation of proteins similar to those induced by GPVI-reactive proteins, collagen and convulxin. Its interaction with GPVI was analyzed by measuring the effect of the antibody on GPVI binding to collagen using a dimeric form of recombinant GPVI, GPVI-Fc2. MAb 204-11 inhibited the binding of GPVI-Fc2 to fibrous collagen particles, but enhanced the GPVI binding to immobilized collagen, suggesting that the antibody binds to a region near the collagen binding site of GPVI. MAb 204-11 also inhibited the GPVI binding to convulxin at a low concentration, but not completely. Since mAb 204-11 reacts specifically with GPVI and is applicable for immunoblotting and immunoprecipitation, this antibody would be useful for studies on GPVI.

scholarly journals Erythropoietin stimulates the tyrosine phosphorylation of Shc and its association with Grb2 and a 145-Kd tyrosine phosphorylated protein

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2296-2303 ◽  
Author(s):  
JE Damen ◽  
L Liu ◽  
RL Cutler ◽  
G Krystal
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Consensus Sequence ◽  
Erythropoietin Receptor ◽  
Phosphorylated Protein ◽  
Molecular Masses ◽  
Epidermal Growth ◽  
Tyrosine Phosphorylated Protein

Although the erythropoietin receptor (EpR) lacks a tyrosine kinase consensus sequence within its proline-rich intracellular domain, addition of its ligand to Ep-responsive cells stimulates the rapid and transient tyrosine phosphorylation of a number of cellular proteins. The characterization of these phosphorylatable substrates, which include 5 major phosphoproteins with molecular masses of approximately 145, 130, 97, 72, and 56 Kd is an essential step in understanding the signal transduction pathways used by Ep. Recently, we and others have shown that the major 72-Kd tyrosine phosphorylated protein is the EpR itself. We now report, using both murine DA-3 and human MO7E cell lines engineered to express high levels of biologically responsive EpRs (and designated DA-ER and MO7-ER, respectively), that the major 56-Kd tyrosine phosphorylated protein is the recently identified SH2- containing protein, p52shc. Interestingly, in Ep-stimulated cells, anti- Shc antibodies coprecipitate the major 145-Kd tyrosine phosphorylated protein in both DA-ER and MO7-ER cells. Tyrosine phosphorylation of both proteins is detectable within 30 seconds of incubation with Ep at 37 degrees C, reaches a maximum between 2 and 5 minutes, and declines by 30 minutes. In addition, tyrosine phosphorylated Shc appears capable of associating with the activated EpR, but this could only be shown in MO7-ER cells. Lastly, as has been shown previously with the tyrosine kinase containing receptors for epidermal growth factor, platelet derived growth factor, and insulin, activation of the EpR leads to the association of p52shc with the 25-Kd polypeptide, Grb2. Taken together, our data suggest that the previously reported increases in rasGTP observed with Ep result, in part, from the tyrosine phosphorylation of Shc and its association with Grb2 and/or a tyrosine phosphorylated 145- Kd protein.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

Heparin-Associated Thrombocytopenia: The Effects of Various Intravenous lgG Preparations on Antibody Mediated Platelet Activation - A Possible New Indication for High Dose i.v. IgG

1994 ◽  
Vol 71 (05) ◽  
pp. 641-645 ◽  
Author(s):  
A Greinacher ◽  
U Liebenhoff ◽  
V Kiefel ◽  
P Presek ◽  
C Mueller-Eckhardt
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Activation ◽  
Manufacturing Process ◽  
Inhibitory Effect ◽  
Serotonin Release ◽  
Low Ph ◽  
High Dose ◽  
Human Igg ◽  
Platelet Protein

SummaryThe immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-re- ceptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP Ilb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT. We used normal platelets and platelets from a patient with Glanzmann’s thrombasthenia (GT) lacking GP Ilb/IIIa. Heparin and sera from patients with HAT stimulated GT platelets in the same manner as determined by 14C-serotonin release and the changes in phosphorylation of p20 and p47. Platelet activation could be inhibited by an anti FcRII monoclonal antibody (IV. 3, Fab-fragments), and by Fc-fragments, but not by F(ab’)2-fragments of human IgG. The effect of four different, commercially available preparations of intact i.v. IgG on the platelet activation by six HAT sera was investigated by 14C-seroto- nin release. The inhibitory effect was strongly dependent upon the manufacturing process. At a concentration of 20 mg/ml only IgG that had been subjected to low pH and traces of pepsin sufficiently inhibited platelet activation. IgG treated with polyethylenglycol or sulfitolysis was less effective, whereas beta-propiolactone-treated IgG almost completely lost the ability to inhibit platelet activation by antibodies related to HAT. We conclude that inhibition of GP Ilb/IIIa-fibrinogen interaction is insufficient for preventing platelet activation in HAT. This is, however, possible by high dose i.v. IgG, whereby inhibition of FcRII on platelets strongly depends upon the process by which the i.v. IgG preparation was manufactured.

USE OF A MONOCLONAL ANTIBODY TO MEASURE THE SURFACE EXPRESSION OF THROMBOSPONDIN FOLLOWING PLATELET ACTIVATION

1987 ◽  
Author(s):  
C Legrand ◽  
V Dubernard ◽  
N Kieffer ◽  
A T Nurden
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Activation ◽  
Surface Expression ◽  
Ionophore A23187 ◽  
Normal Range ◽  
Platelet Surface ◽  
Platelet Protein ◽  
Platelet Binding ◽  
Chloramine T ◽  
Gray Platelet Syndrome

A radiolabelled monoclonal antibody (mAb) against native thrombospondin (TSP) has been used to quantitatively assess the surface exposure of intracellular TSP following platelet stimulation. This mAb, designated 5G11, was purified from ascitic fluid by ammonium sulfate precipitation followed by chromatogloghy on DEAE Trisacryl. The isolated IgG were labelled with I by the chloramine T method (sp.act. 200-500 cpm/ ng). The specificity of the mAb was established by immunoblot-ting and crossed immunoelectrophoresis using platelet protein extracts. When the labelled IgG (20 μg/ml) were incubated with resting platelets in Tyrode's buffer binding was of the order of 2,000 molecules per platelet. Binding was increased 2 fold and 5-7 fold respectively upon ADP- and thrombin-(or ionophore A23187) stimulation. Unactivated platelets from 2 patients with the Gray Platelet Syndrome bound baseline levels of 5G11, but binding did not increase after platelet activation. In the presence of saturating concentrations of mAb 5G11, an average of 30,000 molecules of IgG were bound by normal platelets stimulated by thrombin. This binding was strongly reduced in the presence of EDTA. It was not significantly affected by AP-2, an anti-GP IIb-IIIa monoclonal antibody which inhibited by more than 85% the binding of plasma fibrinogen but which did not inhibit the surface expression of platelet fibrinogen. It was decreased but not prevented by the presence of an excess of rabbit anti-fibrinogen Fab fragments during the stimulation, while binding at the lower end of the normal range was observed on two different occasions using platelets isolated from an afibrinogenemic patient lacking platelet fibrinogen. These results suggest that while platelet fibrinogen may contribute to the surface organization of TSP other component(s) are required for the full expression of TSP on the platelet surface.

scholarly journals Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4304-4313 ◽  
Author(s):  
A Oda ◽  
Y Miyakawa ◽  
BJ Druker ◽  
A Ishida ◽  
K Ozaki ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Genetically Engineered ◽  
Myelogenous Leukemia ◽  
Insoluble Residue ◽  
Consistent Difference ◽  
Clot Retraction ◽  
Phosphorylated Protein ◽  
Tyrosine Phosphorylated Protein

Platelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crid, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crid was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crk1 in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crk1 can be phosphorylated by a kinase other than p210bcr-abl and that crk1 may have a role in signaling by thrombopoietin.

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