Distinguishing Sulfotyrosine Containing Peptides from their Phosphotyrosine Counterparts Using Mass Spectrometry.

Sulfotyrosine and phosphotyrosine are two post-translational modifications present in higher eukaryotes. A simple and direct mass spectrometry method to distinguish between these modifications is crucial to advance our understanding of the sulfoproteome. While sulfation and phosphorylation are nominally isobaric, the accurate mass of the sulfuryl moiety is 9.6 mDa less than the phosphoryl moiety. Based on this difference, we have used an Orbitrap Fusion Lumos mass spectrometer to characterize, resolve, and distinguish between sulfotyrosine and phosphotyrosine modifications using a set of model peptides. Multiple fragmentation techniques, namely HCD, CID, ETD, ETciD, and EThcD, have been used to compare the different fragmentation behaviors between peptides modified with these species. Sulfotyrosine undergoes neutral loss using HCD and CID, but the sulfuryl moiety is largely stable under ETD. In contrast, phosphotyrosine is stable during fragmentation using all these methods. This differential stability provides a mechanism to distinguish sulfopeptides from phosphopeptides. Based on the rigorous characterization presented herein, this work serves as a model for accurate identification of phosphotyrosine and, more challenging, sulfotyrosine, in complex proteomic samples. Graphical Abstract ᅟ.

Evaluation of erythrocyte band 3 phosphotyrosine level, glutathione content, CA-125, and human epididymal secretory protein E4 as combined parameters in endometriosis.

OBJECTIVE: To investigate the biochemical parameters of the erythrocyte response to diamide-induced oxidative stress, alone or as adjuncts to serum values of CA-125 and human epididymal secretory protein E4 (HE4), in the diagnosis and study of endometriosis. SETTING: University of Padova. DESIGN: Prospective study. PATIENT(S): Forty-five patients of reproductive age undergoing laparoscopy. INTERVENTION(S): All women were studied for endometriotic foci during laparoscopic surgery. Forty-one had laparoscopically and histologically confirmed endometriosis, and four did not. Twenty women with confirmed endometriosis were reassessed 1-4 months later. MAIN OUTCOME MEASURE(S): CA-125 and HE4 and two new parameters evaluated in erythrocytes after diamide-induced stress, that is, band 3 tyrosine phosphorylation (Tyr-P) level and decrease in total glutathione content (ΔGSH), were assessed in all patients. RESULT(S): In association with serum CA-125 levels but not with HE4, diamide-related erythrocyte band 3 Tyr-P and ΔGSH were significantly higher in patients with endometriosis and were able to discriminate with high sensitivity and specificity between patients before and after surgery. CONCLUSION(S): Endometriosis is associated with an increase in systemic oxidative stress, affecting the antioxidative defenses of circulating erythrocytes. All related implications, including evaluation of other oxidative stress-related changes, warrant further study.

Flow cytometric assay of phosphotyrosine levels in Bcr-Abl-positive chronic myelogenous leukemias: a potential prognostic marker.

Assay of phosphotyrosine levels using flow cytometry has been used to identify patients with chronic myelogenous leukemia positive for the Bcr-Abl fusion gene. We hypothesized that clinical monitoring could identify treatment response through reductions in intragranulocyte phosphotyrosine. Initially, we studied cell lines FDC-P1 and HL60 (Bcr-Abl-negative) and FDrv210 and K562 (Bcr-Abl-positive) with our technique. A fluorescein isothiocyanate-conjugated monoclonal antibody was used along with fluorescence-conjugated microspheres for reference (ratio of sample fluorescence: bead fluorescence = relative fluorescence unit [RFU]). Samples from 20 controls and 32 patients undergoing treatment were analyzed using the same method. Bcr-Abl status for each patient was confirmed using fluorescent in situ hybridization or polymerase chain reaction gene amplification (PCR). Testing of cell lines consistently produced expected results. Patient values were found to be significantly higher than control values (P < 0.001) and values for patients with advanced disease were significantly higher than for patients with chronic-phase disease (P < 0.05). Results of clinical monitoring were consistent with results from PCR. Two patients who received allogeneic stem cell transplantation had molecular remission confirmed by PCR and had a marked decrease in RFU value (from 62 to five and from 131 to 23). No such fluorescence change was observed in patients who achieved clinical remission. Flow cytometric analysis of phosphotyrosine levels is a reliable and convenient adjuvant technique for diagnosis of Bcr-Abl-positive leukemias and shows promise for serial evaluation of patients undergoing treatment.

Platelets possess functional TGF-beta receptors and Smad2 protein.

TGF-beta1 plays a main role in tissue repair by regulating extracellular matrix production and tissue granulation. Platelets are one of the main sources of this cytokine in the circulation. The aim of this study was to evaluate the presence of the TGF-beta receptors on platelets, the effect of TGF-beta1 on platelet aggregation and the underlying intracellular mechanisms. TGF-beta receptors on platelets were studied by flow cytometry and their mRNA by PCR. Platelet aggregation was assessed by turbidimetric methods and intracellular pathways by Western blot. TGF-beta receptor type II and mRNA codifying for TbetaRI and TbetaRII were found in platelets. We demonstrated that TGF-beta1 did not trigger platelet aggregation by itself but had a modulating effect on ADP-induced platelet aggregation. Either inhibition or increase in platelet aggregation, depending on the exposure time to TGF-beta1 and the ADP concentration used, were shown. We found that platelets possess Smad2 protein and that its phosphorylation state is increased after exposure to TGF-beta1. Besides, TGF-beta1 modified the pattern of ADP-induced tyrosine phosphorylation. Increased phosphorylation levels of 64-, 80- and 125-kDa proteins during short time incubation with TGF-beta1 and increased phosphorylation of 64- and 125-kDa proteins after longer incubation were observed. The modulating effect of TGF-beta1 on platelet aggregation could play a role during pathological states in which circulating TGF-beta1 levels are increased and intravascular platelet activation is present, such as myeloproliferative disorders. In vascular injury, in which platelet activation followed by granule release generates high local ADP concentrations, it could function as a physiological mechanism of platelet activation control.

Magnesium sulfate effect on erythrocyte membranes of asphyxiated newborns.

OBJECTIVES: Magnesium sulfate has been recognized as a neuroprotective agent against hypoxia-ischemia, mainly by the protection from the excitotoxicity associated with increased glutamate concentration. However, the mechanism of MgSO4 action is not fully understood and is considerably controversial. DESIGN AND METHODS: During the 2 first hours of life, the asphyxiated full-term newborns were treated intravenously with one dose of MgSO4 250 mg/kg body weight. At birth, after 6 and 48 h of life the activity of ATP-dependent enzymes in erythrocyte membranes: Mg2+-ATPase, Ca2+-ATPase, protein kinases A and C, were determined. Using monoclonal antibodies, the band 3 and its phosphotyrosine level were also assayed. RESULTS: The time-dependent decrease of Ca2+-ATPase activity was detected in untreated newborns, whereas MgSO4 prevented this reduction. After 48 h, protein kinases activities differed in MgSO4-treated and untreated groups. Magnesium therapy increased the amount of band 3 and diminished proteolytic degradation of this protein. CONCLUSION: Our results demonstrated, for the first time, that magnesium sulfate treatment significantly altered the activities of some important enzymes in erythrocyte membrane from asphyxiated newborns. It also reduced the post-asphyxial damages of membrane compounds. These data may partly explain the molecular mechanisms of MgSO4 action in asphyxiated newborns.

Inflammation and carnitine in hemodialysis patients.

The bioincompatibility of dialytic systems along with the loss of antioxidant substances via the dialysis may contribute to peripheral blood mononuclear cell (PBMC) activation and the production of inflammatory mediators, such as cytokines, oxygen radicals, and complement fragments, that may sustain a state of chronic microinflammation responsible for the pathogenesis of a variety of diseases, including atherosclerosis, anemia, and malnutrition. Moreover, during hemodialysis (HD), oxidative stress may influence several intracellular signaling enzymes, including some stress-activated kinases, such as jun-N-terminal kinase (JNK), potentially leading to PBMC activation and proinflammatory cytokine production. Recent reports suggest that L-carnitine may play an important role in balancing antioxidative systems. Therefore, we sought to evaluate the effect of L-carnitine supplementation on the PBMC responses to oxidative stress induced by different HD membranes. We observed in PBMC from cellulosic (C)-treated patients an increase in the amount of intracellular tyrosine-phosphorylated proteins and a striking activation of JNK, as compared with synthetic (S)-treated patients. On the contrary, 3 months of L-carnitine supplementation significantly lowered intracellular levels of phosphorylated proteins and JNK activity in PBMC from C-treated patients. In addition, after 180 minutes of HD, a significant decrease in global plasma antioxidant capacity was found, particularly in C-treated patients, whereas L-carnitine supplementation improved plasma antioxidant capacity levels in these patients. These observations were also confirmed by in vitro experiments, showing the ability of L-carnitine to reduce the JNK activation in normal PBMC exposed to different amounts of hydrogen peroxide. In conclusion, the uremic milieu is characterized by an enhanced inflammatory response and an increased oxidant load, affecting lipids, carbohydrates, and proteins. Regular L-carnitine supplementation in HD patients can improve cellular defense against chronic inflammation and oxidative stress, most likely by modulating the specific signal transduction cascade activated by an overproduction of proinflammatory cytokines and oxidative stress.

Plasma levels of HER-2/neu, tumor type M2 pyruvate kinase and its tyrosine-phosphorylated metabolite in advanced breast cancer.

INTRODUCTION: Tyrosine kinase signal transduction pathways are a focus of interest for therapeutic interventions. The oncoprotein HER-2/neu shows tyrosine kinase activity leading to phosphorylation and activation of numerous second-messenger systems. One target of phosphorylation processes is assumed to be the tumor type M2 isoenzyme of pyruvate kinase (TuM2-PK) which has been shown to be elevated in metastatic breast cancer. MATERIALS AND METHODS: We measured the plasma levels of HER-2/neu, TuM2-PK and tyrosine-phosphorylated TuM2-PK (p-TuM2-PK) in 69 patients (pts) with breast cancer and correlated these parameters to each other and to the classical tumor marker CA 27.29. The samples were measured with ELISA assays while CA 27.29 was determined with an automated chemiluminescence assay. For analysis, we formed 5 subgroups according to the plasma HER-2/neu levels (group 1: < 15 ng/ml, n = 28; group 2: 15 < or = x < 50 ng/ml, n = 21; group 3: 50 < or = x < 100 ng/ml, n = 9; group 4: 100 < or = x < 500 ng/ml, n = 7; group 5: > or = 500 ng/ml, n = 4). RESULTS: From the HER-2/neu group 1 to group 5, there was a statistically significant increase of CA 27.29 from 35.8 U/ml to 1095.8 U/ml (p < 0.001). There was also a trend for increasing TuM2-PK levels with increasing HER-2/neu levels (p = 0.126). From the lowest extinction (0.088) to the highest extinction result (2.167) of p-TuM2-PK we found a 25-fold increase, which was reproducible in spiking and dilution experiments proving that TuM2-PK is phosphorylated at tyrosine residues to a certain extent. However, there was no correlation between plasma HER-2/neu and p-TuM2-PK levels. CONCLUSION: TuM2-PK is phosphorylated at tyrosine residues in breast cancer patients. Using the shed antigen of HER-2/neu in plasma as a surrogate marker, we did not find any evidence that this phosphorylation is initiated by the oncoprotein HER-2/neu.

EDTA inhibits collagen-induced ATP+ADP secretion and tyrosine phosphorylation in platelets independently of Mg2+ chelation and decrease in pH.

The effects of Mg2+ and EDTA on collagen-induced platelet dense granule secretion and protein tyrosine phosphorylation were studied in the presence and absence of various inhibitors of autocrine agonists that are released from secretory granules. Addition of EDTA to gel-filtered platelets in Tyrode's solution caused a decrease in pH of 0.7-0.9 pH units, due to chelation of Mg2+, and a marked inhibition of cATP+ADP secretion induced by collagen (25 (micro)g/ml). Lowering pH of the platelet suspension to the same extent by HCl had no effect on secretion which also was the same in the absence of exogenous Mg2+ as in its presence. Similarly, secretion induced by collagen per se, i.e., isolated from autocrine agonists by inhibitors, which was about 50% less than with autocrine agonists, was not affected by the presence or absence of exogenous Mg2+. The level of tyrosine phosphorylation of several proteins in resting platelets was higher in the absence of Mg2+ than in its presence, but the onset of collagen-induced phosphorylation and dephosphorylation was more rapid in the absence of Mg2+. Tyrosine phosphorylation of p38 was specifically enhanced in the presence of inhibitors of autocrine phosphorylation and even more enhanced in the absence of Mg2+. EDTA inhibited the protein phosphorylation to the same extent in the presence as in the absence of exogenous Mg2+. These results show that EDTA inhibits collagen-induced dense granule secretion neither through chelation of Mg2+ nor lowering of pH, and thus probably through other effects on the platelet by the EDTA2- species that are reflected in protein tyrosine phosphorylation signalling.

Differential expression and regulation of GTPases (RhoA and Rac2) and GDIs (LyGDI and RhoGDI) in neutrophils from patients with severe congenital neutropenia.

Severe congenital neutropenia (SCN) or Kostmann syndrome is a disorder of myelopoiesis characterized by a maturation arrest at the stage of promyelocytes or myelocytes in bone marrow and absolute neutrophil counts less than 200/microL in peripheral blood. Treatment of these patients with granulocyte colony-stimulating factor (G-CSF) leads to a significant increase in circulating neutrophils and a reduction in infection-related events in more than 95% of the patients. To date, little is known regarding the underlying pathomechanism of SCN. G-CSF-induced neutrophils of patients with SCN are functionally defective (eg, chemotaxis, superoxide anion generation, Ca(++ )mobilization). Two guanosine triphosphatases (GTPases), Rac2 and RhoA, were described to be involved in many neutrophil functions. The expression of these GTPases and their regulation in patients' neutrophils were of interest. This study determined that the guanosine diphosphate (GDP)-dissociation inhibitor RhoGDI is overexpressed at the protein level in patients' neutrophils and that overexpression is a result of G-CSF treatment. RhoA and LyGDI are expressed at similar levels, whereas Rac2 shows a decreased expression. In addition, association of Rac2 and RhoGDI or LyGDI is abrogated or not detectable based on the low Rac2 expression in patients' neutrophils. (Blood. 2000;95:2947-2953)

Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton.

We have investigated the mechanism of Ca(2+) entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the presence, but not in the absence, of external Ca(2+), suggesting a relatively selective inhibition of Ca(2+) entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca(2+) entry evoked by the depletion of intracellular Ca(2+) stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca(2+) entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca(2+) entry in a time-dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca(2+) entry, indicating a cytoskeleton-independent component in the regulation of Ca(2+) entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store-regulated Ca(2+) entry through actin cytoskeleton-dependent and cytoskeleton-independent mechanisms in human platelets.

Apoptosis or plasma cell differentiation of CD38-positive B-chronic lymphocytic leukemia cells induced by cross-linking of surface IgM or IgD.

Previously, we demonstrated that B-chronic lymphocytic leukemia (B-CLL) cells could be divided into 2 groups depending on the expression of CD38 by the malignant cells. The 2 groups differed in their signal-transducing capacities initiated by cross-linking of surface IgM; only in CD38-positive cells was an efficient signal delivered, invariably resulting in cell apoptosis. In this study, we investigated the effect of surface IgD cross-linking in 10 patients with CD38-positive B-CLL. Exposure of the malignant cells to goat antihuman delta-chain antibodies (Gadelta-ab) caused [Ca(++)]i mobilization and tyrosine kinase phosphorylation in a manner not different from that observed after goat antihuman mu-chain antibody (Gamu-ab) treatment in vitro. However, Gadelta-ab-treated cells failed to undergo apoptosis and instead displayed prolonged survival in culture and differentiated into plasma cells when rIL2 was concomitantly present. Cross-linking of surface IgD failed to induce proliferation of the malignant cells in vitro. Moreover, treatment with Gadelta-ab did not prevent apoptosis of B-CLL cells induced by Gamu-ab. Collectively, these experiments demonstrated that IgM and IgD expressed by the same cell may deliver opposite signals under particular circumstances and provide some clues for the understanding of the pathophysiology of B-CLL. (Blood. 2000;95:1199-1206)

Thrombopoietin potentiates agonist-stimulated activation of p38 mitogen-activated protein kinase in human platelets.

Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.

SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 are dramatically increased at the protein level in neutrophils from patients with severe congenital neutropenia (Kostmann's syndrome).

Severe congenital neutropenia (SCN) or Kostmann's syndrome is characterized by a stop in differentiation of myeloid progenitor cells at the myelocytic or promyelocytic stage. The pathophysiology of SCN is still unclear. We previously showed that the tyrosine kinase JAK2 is phosphorylated and activated in neutrophils from patients with severe congential neutropenia. We investigated the role of tyrosine phosphatases in this disease. Expression of the SH2 domain-containing tyrosine phosphatases SHP-1 and SHP-2 was analyzed in myeloid cells from patients with SCN in comparison to healthy donors. We investigated tyrosine phosphatase expression in myeloid cells at the protein level by Western blot analysis using polyclonal antisera against SHP-1 and SHP-2. Whereas SHP-1 and SHP-2 were hardly detectable in neutrophils from healthy donors, neutrophils from patients with SCN revealed high amounts of these two proteins in Western blot analyses. Reverse transcriptase-polymerase chain reaction and Northern blot analyses demonstrated no dramatic differences of SHP-1 mRNA in neutrophils from congenital neutropenia patients as compared to healthy donors. SHP-2 mRNA was hardly detectable in the neutrophils from patients and in normal neutrophils. Increased expression of SHP protein correlated with elevated activity of both SHP-1 and SHP-2 in neutrophils of patients with SCN. Taken together, these data indicate differential regulation for SHP-1 and SHP-2 at the protein level in neutrophils from SCN patients in comparison to healthy donors. We suggest that overexpression of SHP-1 and SHP-2 protein in neutrophils and not in mononuclear cells from patients with SCN might be related to the disease, e.g., by defective dephosphorylation of proteins involved in intracellular signaling pathways.

Role of autocrine stimulation for the effects of cyclic AMP on protein and lipid phosphorylation in platelets activated by particles.

We have compared responses in platelets stimulated with the particulate materials, Intralipid (liposome-suspension) and a potential contrast medium IEEC (1'-(ethyloxycarbonyloxy)-ethyl-5-acetyl-amino-3-(N-methyl-acetyla mino)-2,4,6-triiodo-benzenecarboxylate coated with human serum albumin), with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP-removing system of creatine phosphate/creatine phosphokinase; RGDS to prevent fibrinogen/fibronectin binding to GPIIb/IIIa; SQ 29.548 as a TXA2 receptor antagonist; cyproheptadine as a serotonin receptor antagonist; BN 52021 as a platelet-activating factor receptor antagonist). The pattern of tyrosine-phosphorylated proteins, phosphorylation of initial lipids and phosphorylation of pleckstrin (P47) were used as markers for early signal transducing responses, while secretion of ADP+ATP and beta-N-acetyl-glycosaminidase were used as final responses. Intralipid showed no platelet activation except for some weak tyrosine protein phosphorylation that was inhibited by elevated cAMP. IEEC induced strong platelet activation that was partly inhibited by increased levels of cAMP and IAS. The inhibition of elevated cAMP seemed to be due to removal of the G protein-mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure effect from IEEC which is not affected by elevated cAMP.

Induction of 15-lipoxygenase expression by IL-13 requires tyrosine phosphorylation of Jak2 and Tyk2 in human monocytes.

The enzyme 15-lipoxygenase (15-LO) participates in the dioxygenation of polyenoic fatty acids. This activity leads to the degradation of mitochondrial membranes during reticulocyte differentiation, the production of pro- and anti-inflammatory mediators by a variety of cell types, and the oxidation of lipids in atherosclerotic lesions. The cytokines, IL-4 and IL-13, are reported to induce the expression of 15-LO in human peripheral blood monocytes. In this report we explore the signaling mechanisms involved in the IL-13-mediated induction of 15-LO expression. First we demonstrate that the delayed induction of 15-LO requires continuous stimulation of monocytes for a minimum period of 12 h. We also found that tyrosine kinase inhibitors blocked the induction of 15-LO in a dose-dependent manner. By immunoprecipitation and antiphosphotyrosine blotting experiments, IL-13 was shown to induce tyrosine phosphorylation of Jak2 and Tyk2, but not Jak1 or Jak3, within 5 min of treatment in human monocytes. To investigate whether the early induction of tyrosine phosphorylation of both Jak2 and Tyk2 was ultimately involved in 15-LO expression, we generated antisense oligodeoxyribonucleotides (ODNs) against Tyk2 and Jak2. We employed a cationic lipid-mediated delivery technique to transfect the monocytes and found that both antisense ODNs inhibited expression of their target proteins by 75-85%. The treatments were specific and did not affect the expression of each other. Furthermore, the antisense ODNs to Jak2 and Tyk2 both inhibited the induction of expression of 15-LO in monocytes treated with IL-13. Parallel experiments with sense ODNs to Jak2 and Tyk2 did not affect their protein levels or the induction of 15-LO by IL-13, and down-regulation of Jak1 also did not affect expression of 15-LO. Our results suggest the novel finding that IL-13 can induce tyrosine phosphorylation of both Jak2 and Tyk2 in primary human monocytes. This occurs as an early and essential signal transduction event for the IL-13-mediated induction of 15-LO expression. These data represent the first characterization of upstream kinases involved in the induced expression of 15-LO.

Identification of the Src family kinases, Lck and Fgr in platelets. Their tyrosine phosphorylation status and subcellular distribution compared with other Src family members.

We have identified the Src family members, Lck and Fgr in resting human and rodent platelets and compared their subcellular distributions and tyrosine phosphorylation status to those of the other Src family kinases to gain insights into the signal transduction pathways active in maintaining platelets in the circulation. Like Fyn, Lyn, and Yes, most of Fgr and Lck was detergent-insoluble in human and rat platelets. In comparison, Src showed higher detergent solubility than the Src-related kinases. Most all human platelet Src was detergent-soluble, while that of rodent platelets was present in all detergent fractions. We also compared the tyrosine-phosphorylation status of Lck and Fgr to other Src family members in resting platelets using immunoprecipitation and immunoblotting. All of these Src family members except Fgr exhibited substantial phosphotyrosine antibody labeling. The partitioning of these kinases, with the exception of Src, with the detergent-insoluble fraction, their tyrosine-phosphorylation status, and co-localization with endocytotic vesicles lead us to hypothesize that the Src family kinases are involved in signaling events that drive cytoskeletal reorganization and active endocytosis of plasma proteins by circulating platelets.

Rapid quantitative analysis of protein tyrosine residue phosphorylation in defined cell populations in whole blood and bone marrow aspirates.

We developed a rapid method for quantification of tyrosine phosphorylation in immunophenotypically defined cell populations in specimens of whole blood and unprocessed bone marrow. Samples were formaldehyde-fixed and cells were permeabilized. Phosphotyrosine residues and surface antigens were simultaneously stained by monoclonal antibodies and visualized by flow cytometry. The accuracy of the method was confirmed by demonstration of an increase of phosphotyrosine levels in pp60v-src transformed fibroblasts. In blood of healthy donors, monocytes and granulocytes showed higher levels of phosphotyrosine than lymphocytes. CD34+ peripheral blood stem cells showed slightly increased tyrosine phosphorylation compared to autologous lymphocytes. Significantly elevated levels of phosphotyrosine were demonstrated in leukaemic blasts compared to lymphocytes (P = 0.01).

Modulation of platelet activation in vitro by thrombopoietin.

Effect of human recombinant thrombopoietin (TPO) on platelet activation in vitro was studied. Although TPO 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 alpha-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 alpha-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.

Effect of cAMP and cGMP on endothelin-stimulated tyrosine phosphorylation in rabbit platelets.

Activation of platelets by different agents results in the increased tyrosine phosphorylation of several substrate proteins. Thus, the effect of endothelin-1 on the stimulation of tyrosine phosphorylation in rabbit platelets can be inhibited by preincubation with forskolin, which increase the cAMP level. However, incubations of platelets with 8-Bromo-cGMP showed lower inhibitory effect. Forskolin produced a dose-dependent inhibition on three different protein substrates, with an IC50 of approximately 12.8, 4.0 and 8.0 microM in the three molecular mass ranges of 50, 60 and 100-200 kDa, respectively. These results show that the endothelin-stimulated tyrosine phosphorylation in rabbit platelets can be regulated by a novel pathway of platelet signal transduction in which the cAMP level could be more relevantly involved than cGMP in some molecular mass ranges of tyrosine phosphorylated proteins.