Alterations in thrombin-induced protein tyrosine phosphorylation of platelets from patients with chronic myelogenous leukemia.

Thrombin is known to stimulate platelet protein tyrosine kinase (PTK). We studied thrombin-induced tyrosine-specific protein phosphorylation in normal platelets and those from patients with chronic myelogenous leukemia (CML) and other myeloproliferative disorders (MPD) using immunoblotting with antiphosphotyrosine (anti-P-Tyr) antibody. In resting platelets, two major phosphotyrosyl (P-Tyr) proteins with molecular masses of 120 kDa (p120) and 60 kDa (p60) were consistently detected both in normal subjects and in CML and other MPD patients. In addition to these P-Tyr proteins, a 36 kDa protein (p36) was predominantly phosphorylated only in CML platelets, using antilipocortin II antibody, we identified this p36 protein as lipocortin. Thrombin enhanced the tyrosine phosphorylation of p120 and p60, not only in normal platelets, but also in CML platelets, although the response was more delayed and the duration was shorter in CML platelets than those in normal platelets. Interestingly, decreased thrombin-induced aggregation was associated with a transient stimulation of p36 phosphorylation in CML platelets. These results suggest that the tyrosine phosphorylation of p36, which was probably identical to lipocortin, inhibits thrombin-induced platelet aggregation through anti-phospholipase A2 (anti-PLA2) activity.     

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

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.     

Glycoprotein IIIa is phosphorylated in intact human platelets

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that mediates platelet aggregation, but it is also involved in clot retraction, signal transduction, calcium transport, and other events. However, the mechanisms that regulate the functions of GP IIb-IIIa during platelet activation are largely unknown. One possible mechanism is phosphorylation, since several other receptors are regulated by this process. We found that GP IIIa, but not GP IIb, was phosphorylated in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP IIIa phosphorylation increased four-fold in platelets activated with thrombin or phorbol 12-myristate 13-acetate, but not at all in platelets treated with prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is an increase in the level or turnover of phosphate on GP IIIa during platelet activation, most likely involving protein kinase C. This phosphorylation may regulate some aspect(s) of GP IIb-IIIa function.     

Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and Shc in 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.     

BCR-ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 × 10⁹/l. P2l0^BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p2l0^BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.     

Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.     

Platelet activation via the collagen receptor GPVI is not altered in platelets from chronic myeloid leukaemia patients despite the presence of the constitutively phosphorylated adapter protein CrkL

In this study, we show that the adapter proteins CrkL and Cbl undergo increases in tyrosine phosphorylation and form an intracellular complex in platelets stimulated with the snake venom toxin convulxin, a selective agonist at the collagen receptor glycoprotein VI (GPVI). Constitutive tyrosine phosphorylation of CrkL has previously been reported in platelets from chronic myeloid leukaemia (CML) patients. This was confirmed in the present study, and shown to result in a weak constitutive association of CrkL with Cbl and a number of other unidentified tyrosine-phosphorylated proteins. There was no further increase in phosphorylation of CrkL in CML platelets in response to GPVI activation, whereas phosphorylation of Cbl and its association with CrkL were potentiated. In addition, this was accompanied by a small increase in p42/ 44 mapkinase (MAPK) activity in CML platelets. The functional consequence of the presence of constitutively phosphorylated proteins in CML platelets was investigated by measurement of aminophospholipid exposure and alpha-granule secretion. This revealed little alteration in the concentration-response curves for either in CML platelets stimulated via GPVI, although maximal levels of P-selectin were depressed. Despite the minimal effect on platelet activation in CML patients, we cannot exclude a role for CrkL or Cbl in signal transduction pathways stimulated via GPVI.     

Effect of neutrophil adhesion on the size of aggregates formed by agonist-activated platelets

In this study we have investigated the effect of human neutrophil on agonist-induced platelet aggregation by using the laser-light scattering method that can detect a two-phase process, formation of small aggregates followed by large aggregate formation. When nonstimulated neutrophils were added to agonist-stimulated platelet-rich plasma (PRP), the large platelet aggregates were decreased and the small ones were increased by using either collagen, thrombin or ADP as agonist. Scanning-electron microscopic observation showed marked adhesion of neutrophil to aggregated platelets. The supernatant from neutrophils cell lysate (neutrophil supernatant) showed inhibitory effect similar to that with intact neutrophils, suggesting that the inhibitory effect by neutrophils was due to soluble component(s) including proteases released from neutrophils adhered to activated platelets. We have examined the effect of inhibition of a major released protease, elastase. The addition of its potent inhibitor elafin to intact neutrophils or the neutrophil supernatant changed their antiaggregating activity. The treatment of platelets with genistein, an inhibitor of protein tyrosine kinase, decreased agonist-induced large aggregates and increased small ones, suggesting that certain protein tyrosine kinase would be involved in the transition from small to large platelet aggregates. It was also shown that the tyrosine phosphorylation induced by agonist stimulation of several high molecular-weight proteins of platelets was inhibited by coincubation with neutrophils, concurrent with increases in smaller phosphorylated proteins. In washed platelets, coincubation with neutrophils resulted in reduced formation of large aggregates when stimulated with collagen or thrombin and repressed agonist-induced activation of tyrosine protein kinases (Syk, Lyn, Src, and Pyk2), but not thrombin-induced ERK and p38 MAP kinase. These results suggest that the cleavage of platelet membrane glycoproteins at least in part by elastase which was released from neutrophils, is involved in the inhibition of the transition from small to large platelet aggregates.     

Effect of neutrophil adhesion on the size of aggregates formed by agonist-activated platelets

In this study we have investigated the effect of human neutrophil on agonist-induced platelet aggregation by using the laser-light scattering method that can detect a two-phase process, formation of small aggregates followed by large aggregate formation. When nonstimulated neutrophils were added to agonist-stimulated platelet-rich plasma (PRP), the large platelet aggregates were decreased and the small ones were increased by using either collagen, thrombin or ADP as agonist. Scanning-electron microscopic observation showed marked adhesion of neutrophil to aggregated platelets. The supernatant from neutrophils cell lysate (neutrophil supernatant) showed inhibitory effect similar to that with intact neutrophils, suggesting that the inhibitory effect by neutrophils was due to soluble component(s) including proteases released from neutrophils adhered to activated platelets. We have examined the effect of inhibition of a major released protease, elastase. The addition of its potent inhibitor elafin to intact neutrophils or the neutrophil supernatant changed their antiaggregating activity. The treatment of platelets with genistein, an inhibitor of protein tyrosine kinase, decreased agonist-induced large aggregates and increased small ones, suggesting that certain protein tyrosine kinase would be involved in the transition from small to large platelet aggregates. It was also shown that the tyrosine phosphorylation induced by agonist stimulation of several high molecular-weight proteins of platelets was inhibited by coincubation with neutrophils, concurrent with increases in smaller phosphorylated proteins. In washed platelets, coincubation with neutrophils resulted in reduced formation of large aggregates when stimulated with collagen or thrombin and repressed agonist-induced activation of tyrosine protein kinases (Syk, Lyn, Src, and Pyk2), but not thrombin-induced ERK and p38 MAP kinase. These results suggest that the cleavage of platelet membrane glycoproteins at least in part by elastase which was released from neutrophils, is involved in the inhibition of the transition from small to large platelet aggregates.     

A novel SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells

Because of the probable causal relationship between constitutive p210(bcr/abl) protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210(bcr/abl)-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain-containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210(bcr/abl)-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4, 5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210(bcr/abl)-mediated myeloid expansion.     

Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells

Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in clonally derived hematopoietic precursors and their progeny. The Ph chromosome arises from a translocation that deregulates the c-ABL protein tyrosine kinase, giving it transforming potential and increased kinase activity. We observed a unique 39-kD tyrosine phosphoprotein (pp39), previously reported in blastic CML cell lines, in neutrophils from 50 cases of chronic phase CML. This protein was prominently and constitutively tyrosine-phosphorylated in CML neutrophils and was not phosphorylated in normal neutrophils. Stimulation of normal neutrophils with cytokines and agonists did not induce tyrosine phosphorylation of proteins migrating in the region of pp39, and the phosphorylation state of pp39 in CML neutrophils was not affected by kinase inhibitors known to downregulate the ABL kinase. The pp39 was not phosphorylated in hematopoietic cells from healthy donors or from patients with Ph chromosome-negative myeloproliferative disorders. Using micro amino acid sequencing of purified preparations of pp39, we identified pp39 as CRKL protein, which is consistent with recent immunologic studies in the blastic K562 cell line. Immunoblotting with anti-CRKL antibodies showed the presence of CRKL protein in CML cells and cell lines as well as in antiphosphotyrosine immunoprecipitates from CML cells. Our results suggest that pp39 CRKL in CML neutrophils may be stably tyrosine-phosphorylated by the BCR/ABL kinase at an early stage of myeloid differentiation when the ABL kinase is active. CRK, CRKL, and other SH2 (SRC homology domain)/SH3-containing proteins function as adaptor molecules in nonreceptor tyrosine kinase signalling pathways. Although the CRKL protein is present in normal neutrophils, it is not tyrosine-phosphorylated, and the inability to induce such phosphorylation in normal neutrophils suggests a special role of this phosphoprotein in the pathogenesis of CML. Constitutive phosphorylation of CRKL is unique to CML, indicating that it may be a useful target for therapeutic intervention.     

Regulation and function of WASp in platelets by the collagen receptor, glycoprotein VI

Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin alpha2beta1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.     

Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C-gamma 1 in human platelets.

Thrombin activates phospholipase C in human platelets, but the specific isoenzymes activated and the signal pathway used are unknown. Using specific antibodies, we found that phospholipase C-gamma 1 and the p21ras GTPase-activating protein, rasGAP, are present in human platelets. Furthermore, phospholipase C-gamma 1 was detectable, based on enzyme activity and Western blot analysis, in immunoprecipitates of rasGAP, suggesting that these two proteins form tight complexes. The pool of phospholipase C-gamma 1 associated with rasGAP was phosphorylated but not through tyrosine phosphorylation. Although thrombin stimulation had no effect on the level of phosphorylation of phospholipase C-gamma 1 and only slightly increased the tyrosine phosphorylation of rasGAP, the agonist induced the association of rasGAP with rap1B, as indicated by the appearance of rap1B on a Western blot of rasGAP immunoprecipitates. Our results suggest the formation of a signaling complex involving rasGAP, phospholipase C-gamma 1, and rap1B that might be important in the cascade leading to platelet activation.     

Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion

In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.     

Thrombopoietin Induces Association of Crkl With STAT5 But Not STAT3 in Human Platelets

Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, isconstitutively tyrosine phosphorylated in hematopoietic cells fromchronic myelogenous leukemia (CML) patients. We recently reported thatthrombopoietin induces tyrosine phosphorylation of Crkl in normalplatelets. In this study, we demonstrate that thrombopoietin inducesassociation of Crkl with a tyrosine phosphorylated 95- to 100-kDprotein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, wedemonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. Thecoimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizingpeptide for Crkl antisera or phenyl phosphate (20 mmol/L). Afterdenaturing of Crkl immunoprecipitates, Crkl was stillimmunoprecipitated by Crkl antisera. However, coimmunoprecipitation ofSTAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity asdemonstrated by electrophoretic mobility shift assays (EMSA). Using a-casein promoter STAT5 binding site as a probe, we have alsodemonstrated that Crkl antisera supershift the STAT5-DNA complex,suggesting that Crkl is a component of the complex in the nucleus.Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in astimulation-dependent manner. In vitro, glutathione S-transferase(GST)-Crkl bound to STAT5 inducibly through its SH2 domain. Theseresults indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietincommonly induce association of STAT5 and Crkl and that the complextranslocates to the nucleus and binds to DNA. Interestingly, suchassociation between STAT5 and Crkl was not observed incytokine-stimulated murine cells, suggesting an intriguing possibilitythat components of the human STAT5-DNA complex may be different fromthose of the murine counterpart.     

The common pathway for alpha- and gamma-thrombin-induced platelet activation is independent of GPIb: a study of Bernard-Soulier platelets

The responses to alpha- and gamma-thrombin were studied in normal and Bernard-Soulier platelets labelled with [32P]phosphate, to investigate the relationship between thrombin binding to the platelet membrane glycoprotein Ib (GPIb) and thrombin-induced platelet activation. For this purpose we conducted parallel studies of the kinetics of platelet aggregation, granule secretion, hydrolysis of polyphosphoinositides, formation of phosphatidic acid, phosphorylation of the myosin light chain (p20) and of the 43 kDa protein (p43), and thromboxane B2 formation. Like alpha-thrombin, gamma-thrombin activated control platelets via all the above metabolic responses, but only after a prolonged lag. In Bernard-Soulier platelets, alpha-thrombin induced polyphosphoinositide hydrolysis and phosphatidic acid formation, p20 and p43 phosphorylation, thromboxane B2 formation, secretion and to a lesser extent aggregation, but only after a prolonged lag. The metabolic responses of Bernard-Soulier platelets to gamma-thrombin were very similar to those of control platelets. We have previously showed that GPIb which is not present in Bernard-Soulier platelets binds alpha- but not gamma-thrombin. The present results indicate that thrombin binding to GPIb is not directly coupled either with the activation of phospholipase C specific to polyphosphoinositides, or with the activation of protein kinase C and phospholipase A2. However, thrombin binding to GPIb appears to promote an early mechanism which accelerates all the platelet responses.     

Comparative studies on homocysteine and its metabolite-homocysteine thiolactone action in blood platelets in vitro

Homocysteine (Hcy), an intermediate formed during the catabolism of the essential dietary amino acid methionine, and its cyclic thioester, homocysteine thiolactone (TL) formed from Hcy in plasma, may be implicated in pathological haemostasis and atherosclerosis. The mechanism by which TL exerts the prothrombotic effect and influences blood platelets remains unclear. Activation of blood platelets plays an important role in prothrombotic events. The aim of our study was to establish and compare the influence of a reduced form of homocysteine (at final doses of 10-100 microM) and its cyclic thioester, homocysteine thiolactone (0.1-1 microM), on platelet activation induced by thrombin (platelet aggregation), on platelet protein modifications (determined by parameters such as level of protein carbonyl groups, 3-nitrotyrosine residues in proteins) and on superoxide anion radicals ( O2-*) generation using the model system in vitro. We have observed that TL, like its precursor, Hcy, stimulates the generation of O2* in platelets and causes an augmentation of platelet aggregation induced by thrombin. Our present results in vitro also demonstrate that Hcy (10-100 microM) and TL at lower doses than Hcy (0.1-1 microM) cause modification of platelet proteins: diminished formation of carbonyl groups and distinctly decreased tyrosine nitration in platelet proteins after thrombin stimulation, but increased platelet aggregation induced by thrombin. TL like Hcy (at concentrations corresponding to concentrations in blood during hyperhomocysteinemia) modifies platelet responses to an important physiological agonist--thrombin.     

Tyrosine phosphorylation of p62dok by p210bcr-abl inhibits RasGAP activity

The t(9;22) chromosomal translocation is found in almost all patients with chronic myelogenous leukemia. The resultant Bcr-Abl fusion gene expresses a chimeric fusion protein p210(bcr-abl) with increased tyrosine kinase activity. Hematopoietic progenitors isolated from chronic myelogenous leukemia patients in the chronic phase contain constitutively tyrosine-phosphorylated p62(dok) protein. p62(dok) associates with the Ras GTPase-activating protein (RasGAP), but only when p62(dok) is tyrosine phosphorylated. Here we have investigated the interaction between p62(dok) and RasGAP and the consequences of p62(dok) tyrosine phosphorylation on the activity of RasGAP. We have found that p62(dok) is directly tyrosine phosphorylated by p210(bcr-abl), and the sites of phosphorylation are located in the C-terminal half of the p62(dok) molecule. We have identified five tyrosine residues that are involved in in vitro RasGAP binding and have found that tyrosine-phosphorylated p62(dok) inhibits RasGAP activity. Our results suggest that p210(bcr-abl) might lead to the activation of the Ras signaling pathway by inhibiting a key down-regulator of Ras signaling.     

Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCdelta in platelets

Thrombin has been known to cause tyrosine phosphorylation of protein kinase C delta (PKCdelta) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCdelta and the role of this event in platelet function. PKCdelta was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCdelta did not occur in Galpha(q)-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5'-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ', N '-tetraacetic acid), suggesting a role for Galpha(q) pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCdelta. Inhibition of tyrosine phosphorylation or the kinase activity of PKCdelta dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCdelta in a calcium- and Src-family kinase-dependent manner in platelets, with functional implications in thromboxane A2 generation.