Impact of electronic-cigarette refill liquid on rat testis.

Electronic cigarettes (e-cigarettes) are becoming the fashionable alternative to decrease tobacco smoking, although their impact on health has not been fully assessed yet. The present study was designed to compare the impact of e-cigarette refill liquid (e-liquid) without nicotine to e-liquid with nicotine on rat testis. For this purpose, e-liquid with nicotine and e-liquid without nicotine (0.5 mg/kg of body weight) were administered to adult male Wistar rats via the intraperitoneally route during four weeks. Results showed that e-liquid with or without nicotine leads to diminished sperm density and viability, such as a decrease in testicular lactate dehydrogenase activity and testosterone level. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) analysis identified a reduction in cytochrome P450 side-chain cleavage (P450 scc) and 17 beta-hydroxysteroid dehydrogenase (17ßHSD) mRNA level, two key enzymes of steroidogenesis. Following e-liquid exposure, histopathological examination showed alterations in testis tissue marked by germ cells desquamation, disorganization of the tubular contents of testis and cell deposits in seminiferous tubules. Finally, analysis of oxidative stress status pointed an outbreak of antioxidant enzyme activities such as superoxide dismutase, catalase and gluthatione-S-transferase, as well as an important increase in sulfhydril group content. Taken together, these results indicate that e-liquid per se induces toxicity in Wistar rat testis, similar to e-liquid with nicotine, by disrupting oxidative balance and steroidogenesis.

Evora® chromium-cobalt dual mobility socket: results at a minimum 10 years' follow-up.

INTRODUCTION: The Evora chromium-cobalt alloy dual mobility socket claims to display a large articulation tribology different from that of stainless steel models, limiting the risk of intraprosthetic dislocation and wear. The present study reports a minimum of 10years' follow-up in a multicenter prospective series of 200 sockets previously reported on at 5years. HYPOTHESIS: The use of chromium-cobalt in dual mobility sockets provides a low rate of failure at 10years, especially as regards to osteolysis and intraprosthetic dislocation. MATERIALS AND METHODS: Two hundred hydroxyapatite-coated molded chromium-cobalt sockets without titanium interface were implanted without cement in 194 patients with a mean age of 70 years (range, 32-91 years). Clinical results were assessed on Postel Merle d'Aubigné and Harris scores, plain radiographs and survival analysis. RESULTS: At a mean 11 years' follow-up (10-13 years), 56 patients had died and 31 were lost to follow-up. Four underwent surgical revision (3 femoral components, and 1 socket for migration at 9 years with complete disappearance of the hydroxyapatite). A total of 109 implants were analyzable in 107 patients with a mean age of 81 years (55-93 years). At follow-up, the mean Harris score was 90 (75-96) and the PMA score 16.3 (14-18). There were no cases of loosening (except for the case reoperated on at 9 years) and no acetabular radiolucency or cysts. There were 2 cases of non-evolutive femoral radiolucency and 10 of femoral granuloma, involving head size > 22 mm (P<0.0001) and a cemented titanium stem (P=0.004) as risk factors. There were no dislocations in the large or small articulation. Ten-year survival was 99% (95% CI: 97.3%-100%) with socket revision as censorship criterion. DISCUSSION: The absence of dislocation in both small and large articulations confirmed the efficacy of the dual mobility concept and suggested an advantage for chromium-cobalt sockets in reducing the rate of intraprosthetic dislocation and preventing blockage of the large articulation by a better performance in the friction couple. Granulomas were associated with wear in cemented titanium stems and with heads greater than 22 mm in diameter. Ten-year survival was 99% (censorship criterion: revision for socket failure); there was, however, one case of socket loosening with disappearance of the hydroxyapatite, indicating that surveillance should be continued in this cohort.

Michelangelo’s art on the Sistine Chapel ceiling: sacred representation or anatomy lessons?

The Renaissance was a period of extensive scientific and cultural production, which occurred between the fourteenth and sixteenth centuries. One of the exponents of this artistic period was the poet, architect, sculptor and painter Michelangelo Buonarroti, who was born and lived in Italy between 1475 and 1564. Among his best known artworks are the frescoes painted on the Sistine Chapel ceiling. Currently, there is discussion if the paintings are only representations made from the sacred guidance of the church at the time, or if there are other meanings hidden in the images. From this context, we analyzed studies that associated the frescoes painted on the Sistine Chapel ceiling with anatomical structures hidden in the images, taking into account their significance, importance, and if these structures are not simply an imaginative interpretation of the researchers. This study was performed aiming to complement the work published by Ellwanger, Mohr and Campos (2012) in this journal.

Palatoglossus muscle neuroanatomy: a review

The palatoglossus muscle is classically described as an extrinsic muscle of the tongue. However, this descriptionis not consensus among the researchers, is one that sometimes it is not considered a muscle of the tongue.Thus, the objective of this study is to discuss some neuroanatomical aspects of palatoglossus muscle that mayhelp explain this aspect. Furthermore, this study shall be useful for clinicians, surgeons and academics thatmanipulate and keep particular interest for this anatomical site.

Chronic exercise promotes alterations in the neuroendocrine profile of elderly people.

Aging and physical inactivity are 2 factors that favour the development of cardiovascular disease, metabolic syndrome, obesity, and diabetes. In contrast, adopting a habitual moderate exercise routine may be a nonpharmacological treatment alternative for neuroendocrine aging disorders. We aimed to assess the effects of moderate exercise training on the metabolic profiles of elderly people with sedentary lifestyles. Fourteen sedentary, healthy, elderly male volunteers participated in a moderate training regimen for 60 min/day, 3 days/week for 24 weeks at a work rate equivalent to their ventilatory aerobic threshold. The environment was maintained at a temperature of 23±2°C, with a humidity of 60±5%. Blood samples for analysis were collected at 3 intervals: at baseline (1 week before training began), and 3 and 6 months after training. The training promoted increased aerobic capacity (relative VO(2), and time and velocity to VO(2)max; (p<0.05)) and reduced serum α-MSH (p<0.05) after 3 months of training when compared with the baseline data. In addition, serum thyroid hormone (T3 and T4) was reduced after 6 months of training compared with baseline levels. Our results demonstrate that a moderate exercise training protocol improves the metabolic profile of older people, and metabolic adaptation is dependent on time.

Experimental photophysical characterization of fluorophores in the vicinity of gold nanoparticles.

We propose an experimental-based tool for dealing with fluorescence modulation close to nanoparticles for application in studies of fluorophores in the vicinity of gold nanoparticles (AuNPs), typically addressed via theoretical models. We performed a photophysical characterization of fluorophores in the vicinity of AuNPs, showing that correct Φ(F) determination suffers from a local pH effect, and address the observed radiative enhancement. Our approach is based on the experimental assurance that the reference fluorophores are in the same optical conditions as those of the AuNP-fluorophore conjugates. We demonstrate the relevance for introducing corrections for the inner filter effect and the reabsorption of the emitted light caused by AuNPs. The proposed approach could circumvent the need for theoretical based corrections and allow for more accurate determination of fluorescence emission in the vicinity of gold nanoparticles.

Results of the Evora dual-mobility socket after a minimum follow-up of five years.

PURPOSE OF THE STUDY: Dislocation is a well-known complication of total hip arthroplasty. The risk can be reduced to one or two cases per thousand using a dual-mobility cup. The survival rate achieved with the Bousquet implant is 95% at 10 years. The complications with this implant are early mobilization and inguinal pain. An overly-large cup and insufficient primary and secondary fixation can be implicated. The design of the original implant was later modified to limit these early complications. The purpose of this study was to check the validity of these design changes. PATIENTS AND METHODS: The chromium-cobalt moulded cementless cup was used. The outer surface of this cup presents large geometric striations and is coated with hydroxyapatite. The cup has the shape of a 180 degrees half sphere and a posterior wall prolongation measuring 6.5mm. Three mechanisms were used for the primary fixation: an asymmetrical growth ring, four anchorage stems and a superior screw. Two hundred cementless cups were implanted in 194 patients. The femoral piece was a Charnley stainless-steel implant (n=139), a titanium SEM implant (n=59) or another implant (n=12). Cement was used for femoral fixation in 193 implantations. The series included 97 women and 103 men with osteoarthritis (n=180), necrosis (n=16) and surgery for fracture and primary arthroplasty (n=9). The Harris and Postel-Merle-d'Aubigné scores were noted. Eight radiographic criteria were analyzed to assess the position of the cup and the radiological course of the interface. RESULTS: The mean follow-up was six years and the minimum was five years. The mean age at surgery was 70 years (range, 32 to 91). At last follow-up, 17 patients had died, eight were lost to follow-up and five were bedridden. Three patients underwent revision surgery. Thus, this analysis included 170 prostheses followed for more than five years (mean, six years; range, five to seven years). The Harris score improved from 48 to 92 and the Postel-Merle-d'Aubigné score from 2/5/4 to 5.8/5.9/5.5 (range, 4 to 6/5 to 6/1 to 6). None of the patients complained of anterior pain during active hip flexion in supine position (related to ilio-psoas irritation). Cup inclination was 46 degrees on average (range, 62 to 22 degrees ). Medialization, lateralization or ascension greater than 10mm from the center of rotation was not observed on the postoperative films. At the last follow-up, no measurable mobilization or migration could be identified on plain X-rays. Radiolucent lines, condensations and bony defects around the cup, when visible postoperatively, were not found on the last follow-up X-rays. There were two cemented femoral pieces that developed a radiolucent line in the nonspecific metaphyseal area. There were no cases of granuloma and no cam effect. Three patients underwent revision for femoral loosening, fracture of the femur below the prosthesis and hematogenous infection. There were no cases of dislocation. DISCUSSION: Changing the design of the implant to modify its volume, material and primary fixation has eliminated the early mobilizations and inguinal pain described for the original Bousquet cup. These options have not had any deleterious effect on prosthesis stability. The question of long-term wear remains an important problem and requires optimization: a neck as thin as possible, optimized surfacing, elimination of laser marks, extraction leads and head skirts.

Mitogen-activated protein kinases in hemostasis and thrombosis.

The mitogen-activated protein (MAP) kinases ERK2, p38 and JNK1 are present in platelets and are activated by various stimuli, such as thrombin, collagen, von Willebrand factor (VWF) and ADP. Until recently, MAP kinases were only studied in the conventional model of agonist-induced platelet aggregation mediated by fibrinogen and integrin alphaIIbbeta3. However, this approach is likely to be too limited for a physiological understanding of platelet MAP kinases and their signaling pathways. Recent studies with varying blood-flow conditions and animal models of thrombosis have provided deeper insight into the role of MAP kinases in thrombus formation and the dependence of these kinases on shear conditions. This review summarizes and discusses the physiological functions of these kinases in hemostasis and thrombosis as revealed by various technical approaches.

Abrupt oxygen decrease influences thrombosis and bleeding in stenosed and endothelium-injured rabbit carotid arteries.

BACKGROUND AND OBJECTIVE: Blood oxygen concentration decrease may be associated with haemostatic impairments. We aimed to study the effect of oxygen decrease in a rabbit model of thrombosis and bleeding. METHODS: A total of 44 rabbits were anaesthetized, ventilated and monitored for blood pressure, blood arterial gas, temperature and carotid blood flow. The Folts model was used: a stenosis (75%) and an injury were carried out on the carotid artery, inducing thrombosis. Blood flow decreased as thrombus size increased until the pressure gradient was such that the thrombus was released and local arterial blood flow was suddenly restored. This is known as a cyclic flow reduction. After counting baseline cyclic flow reductions during a 20-min period (P1), rabbits were randomized blindly to one of three groups: hyperoxic, FiO2=100%; normoxic, FiO2 was decreased to obtain a PaO2 between 80 and 120 mmHg; hypoxic, PaO2 < 80 mmHg. Then CFRs were recorded over a second 20-min period (P2). At the end of the experiment, a hepatosplenic section was done and the amount of blood loss was recorded. After each period, the following parameters were measured: blood gas, ear-immersion bleeding time, haemoglobin, platelet count, prothrombin time, activated partial thromboplastin time and fibrinogen. RESULTS: Oxygen decrease during hypoxic and normoxic periods was associated with a decrease in cyclic flow reductions. Bleeding time increased in the hypoxic group unless hepatosplenic bleeding remained stable. A slight increase in activated partial thromboplastin time was observed in the normoxic and hypoxic groups. CONCLUSION: An abrupt decrease in oxygen administration was responsible for an antithrombotic effect. Increase in bleeding time occurred during hypoxia. No clinically relevant variation of any haemostasis parameters was observed.

[Results of the Evora dual mobility socket: five years follow-up]. / Résultat à cinq ans de la cupule à double mobilité Evora.

PURPOSE OF THE STUDY: Dislocation is a well-known complication of total hip arthroplasty. The risk can be reduced to one or two cases per thousand using a double mobility cup. The survival rate achieved with the Bousquet implant is 95% at 10 years. The complications with this implant are early mobilization and inguinal pain. An overly large cup and insufficient primary and secondary fixation can be implicated. The design of the original implant was later modified to limit these early complications. The purpose of this work was to check the validity of the changes made. PATIENTS AND METHODS: The chromium-cobalt moulded cup was used. The outer surface of this cup presents large geometric striations and is coated with hydroxyapatite. The cup has the shape of a half sphere of 180 degrees and a posterior wall prolongation measuring 6.5mm. Three mechanisms were used for the primary fixation: an asymmetrical growth ring, four anchorage stems, and a superior screw. Two hundred cups were implanted in 194 patients. The femoral piece was a Charnley stainless steel implant (n=139), a titanium SEM implant (n=59) or another implant (n=12). Cement was used for 193 implantations. The series included 97 women and 103 men with osteoarthritis (n=180), necrosis (n=16), surgery for fracture and primary arthroplasty (n=9). The Harris and Postel-Merle-d'Aubigné scores were noted. Eight radiographic criteria were analyzed to assess the position of the cup and the radiological course of the interface. RESULTS: Mean time to surgery was six years. Mean age at surgery was 70 years (range: 32-91) and varied depending on the operators from 67 to 73 years. At last follow-up: 17 patients had died, eight were lost to follow-up and five were bedridden. Three patients underwent revision surgery. Thus, this analysis included 170 prostheses followed for more than five years (mean: six years, range: 5-7 years). The Harris score improved from 48 to 92 and the Postel-Merle-d'Aubigné score from 2/5/4 to 5.8/5.9/5.5 (range: 4-6/5-6/1-6). None of the patients complained of anterior pain during hip flexion against resistance. Cup inclination was 46 degrees on average (range: 62-22 degrees ). Medialization, lateralization or ascension greater than 10mm of the centre of rotation was not observed on the postoperative films. At last follow-up, no measurable mobilization or migration could be identified on plain X-rays. Lucent lines, condensations and bony defects around the cup, when visible postoperatively, were not found on the last follow-up X-rays. There were two cemented femoral pieces, which developed a lucent line in the nonspecific metaphyseal area. There were no cases of granuloma and no cam effect. Three patients underwent revision for femoral loosening, fracture of the femur below the prosthesis, and hematogenous infection. There were no cases of dislocation. DISCUSSION: Changing the design of the implant to modify its volume, material and primary fixation has eliminated the early mobilizations and inguinal pain described for the original Bousquet cup. These options have not had any deleterious effect on prosthesis stability. The question of long-term wear remains an important problem and requires optimization: a neck as thin as possible, optimized surfacing, elimination of laser marks, extraction leads, head skirts.

Probing platelet factor 4 alpha-granule targeting.

The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

Fibrin clot retraction by human platelets correlates with alpha(IIb)beta(3) integrin-dependent protein tyrosine dephosphorylation.

We have analyzed tyrosine phosphorylation associated with retraction of the fibrin clot by washed platelets in purified fibrinogen. Retraction was dependent on integrin alpha(IIb)beta(3), based on absence of retraction of alpha(IIb)beta(3)-deficient thrombasthenic platelets. However, only a subset of alpha(IIb)beta(3)-blocking antibodies or peptides were able to inhibit retraction, suggesting a differential engagement of alpha(IIb)beta(3) in fibrin clot retraction versus aggregation. Immunoblotting demonstrated a phosphorylated protein pattern comparable with aggregation at early time points. However, as opposed to aggregation, tyrosine phosphorylation decreased rapidly in parallel to retraction (up to 60 min). Dephosphorylation was alpha(IIb)beta(3)-dependent, since it was blocked by alpha(IIb)beta(3)-specific inhibitors and was absent in thrombasthenic platelets. Inhibition of platelet clot retraction by phenyl-arsine oxide and peroxovanadate, suggested a role for tyrosine phosphatases. Cytochalasin D and E (5 microm) blocked fibrin clot retraction and tyrosine dephosphorylation, suggesting regulation by actin cytoskeleton assembly. Tyrosine phosphatase activities were found associated with clot retraction using the "in-gel" tyrosine phosphatase assay; however, none were alpha(IIb)beta(3)-dependent. An 85-kDa protein and to a lesser degree "Src" showed the closest dose-dependent correlation between inhibition of tyrosine dephosphorylation and inhibition of retraction. We thus postulate that alpha(IIb)beta(3) engagement in fibrin clot retraction drives, in an actin cytoskeleton-dependent manner, the interaction of tyrosine phosphatases and of the tyrosine-phosphorylated substrates 85-kDa protein and Src, the dephosphorylation of which regulates the force generation and/or transmission required for full contraction of the fibrin matrix.

Identification of a GATA-overlapping sequence within the enhancer of the murine GPIIb promoter that induces transcriptional deregulation in human K562 cells.

The human and the murine glycoprotein platelet IIb (GPIIb) promoters are megakaryocyte specific in human and murine cell systems, respectively. Here we show that the murine promoter is, however, highly active when transfected in K562 human cells in which the human promoter is almost inactive. A murine promoter, in which the enhancer element was replaced by the human, retrieves its megakaryocytic specificity in human cell lines. The human and murine GATA-binding sites located in the enhancer region display slight sequence divergence next to the consensus GATA core sequence. Gel shift experiments show that, although the murine and the human GATA sequences both bind GATA-1, the murine sequence alone forms an additional complex (B) not detected with the human sequence. When the murine GATA-containing region is replaced by the human in the context of the murine GPIIb promoter, megakaryocyte specificity is restored in the human cell lines. A G nucleotide 3 to GATA appears crucial because its substitution abrogates B but not GATA-1 binding and restores megakaryocyte specificity to the murine promoter. Conversely, substitution of the human GATA-1 binding sequence by its murine homologue that binds both GATA-1 and complex B induces an abnormal activity for the human promoter in K562 cells. Altogether, our data suggest that limited changes in the GATA-containing enhancer of the GPIIb promoter can induce the recruitment of accessory proteins that could be involved in alteration of a megakaryocyte-restricted gene activation program. (Blood. 2000;96:1348-1357)

Regulation of c-jun-NH2 terminal kinase and extracellular-signal regulated kinase in human platelets.

Platelets are an interesting model for studying the relationship betwen adhesion and mitogen-activated protein (MAP) kinase activation. We have recently shown that in platelets, ERK2 was activated by thrombin and downregulated by alpha(IIb)beta(3) integrin engagement. Here we focused our attention on the c-Jun NH2-terminal kinases (JNKs) and their activation in conditions of platelet aggregation. We found that JNK1 was present in human platelets and was activated after thrombin induction. JNK1 phosphorylation was detected with low concentrations of thrombin (0. 02 U/mL) and after 1 minute of thrombin-induced platelet aggregation. JNK1 activation was increased (fivefold) when fibrinogen binding to alpha(IIb)beta(3) integrin was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide or (Fab')(2) fragments of a monoclonal antibody specific for alpha(IIb)beta(3), demonstrating that, like ERK2, alpha(IIb)beta(3) integrin engagement negatively regulates JNK1 activation. Comparison of JNK1 activation by thrombin in stirred and unstirred platelets in the presence of RGDS peptide showed a positive regulation by stirring itself, independently of alpha(IIb)beta(3) integrin engagement, which was confirmed in a thrombasthenic patient lacking platelet alpha(IIb)beta(3). The same positive regulation by stirring was found for ERK2. These results suggest that MAP kinases (JNK1 and ERK2) are activated positively by thrombin and stirring. In conclusion, we found that JNK1 is present in platelets and can be activated after thrombin induction. Moreover, this is the first report showing that two different MAP kinases (ERK2 and JNK1) are regulated negatively by alpha(IIb)beta(3) engagement and positively by mechanical forces in platelets.

Tyrosine phosphorylation of cortactin associated with Syk accompanies thromboxane analogue-induced platelet shape change.

Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7- 3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2. 1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F(2)alpha. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal. Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA(2)-induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA(2) high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.

Abnormal tyrosine phosphorylation linked to a defective interaction between ADP and its receptor on platelets.

ADP, a primary stimulus of platelets, binds to one or more populations of receptors on the platelet surface. These receptors are linked to discrete activation pathways. Both G proteins and tyrosine kinases have been implicated in the cellular responses to this agonist. We have studied a patient with a congenital abnormality of ADP-induced platelet aggregation in an effort to gain information on the signalling pathways used by ADP. Immunoblotting with a broadly reactive rabbit antibody recognizing the GTP-binding domain of G protein alpha-subunits, and with rabbit antibodies specific for Gialpha1-3, and Galpha12 all showed normal reactivity when tested against the patient's platelets. The phosphorylation of proteins was studied using an anti-phosphotyrosine MoAb (4G10) and platelets stimulated in a platelet aggregometer with ADP, a thromboxane A2 mimetic (IBOP), TRAP-14-mer peptide and alpha-thrombin. With normal platelets, a time-dependent phosphorylation of several bands in the 60 to 130 kDa mol. wt. range was observed with all agonists. For the patient, minimal aggregation and little or no phosphorylation of proteins of 80-85 kDa (cortactin), 100-105 kDa and 125-130 kDa were seen in response to ADP. The aggregation and phosphorylation responses were slightly modified in the presence of low doses of thrombin but were normal with high doses. Aggregation and tyrosine phosphorylation were virtually absent with IBOP, a finding reproduced when normal platelets were incubated with IBOP and the CP/CPK ADP scavenging system, thereby underlining the role of ADP in the response to IBOP. Our results show that the ADP receptor pathway deficient in the patient is linked to a selective tyrosine phosphorylation response.

Negative regulation of mitogen-activated protein kinase activation by integrin alphaIIbbeta3 in platelets.

Activation of the mitogen-activated protein (MAP) kinase pathway in nucleated cells is dependent on both growth factor receptors and integrins engaged in cell adhesion. Human platelets are an interesting model for studying cell adhesion and the involvement of integrin engagement on extracellular signal-regulated kinase (ERK) activation, independently from the nuclear-DNA signal pathway. Maximal phosphorylation and activity of ERK2 occurred late during thrombin-induced platelet aggregation (90 s and later), an alphaIIbbeta3 integrin-dependent event. Surprisingly, alphaIIbbeta3 inhibition by the RGDS ligand peptide, or (Fab')2 fragments of the AP-2 monoclonal antibody, resulted in a 2-fold enhancement in ERK2 phosphorylation and activity. A similar 2-fold enhancement of ERK2 activation was observed in thrombasthenic platelets which are defective in alphaIIbbeta3 and do not aggregate. This suggests that ERK2 activation in thrombin-induced platelet aggregation is dependent on thrombin rather than on alphaIIbbeta3 and is down-regulated by alphaIIbbeta3 engaged in ligand (fibrinogen) binding and/or aggregation. Finally, in the absence of stirring which allows fibrinogen binding to alphaIIbbeta3 but prevents aggregation, ERK2 was again overactivated. This overactivation appears to be consecutive to inhibition of aggregation itself and to alphaIIbbeta3 ligand binding. We conclude that in platelets, alphaIIbbeta3 engaged in aggregation down-regulates thrombin-induced ERK2 activation. To our knowledge, this is the first report of a down-regulation of the MAP kinase pathway by integrin engagement.

Reassessment of protein tyrosine phosphorylation in thrombasthenic platelets: evidence that phosphorylation of cortactin and a 64-kD protein is dependent on thrombin activation and integrin alphaIIb beta3.

Tyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin alphaIIb beta3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack alphaIIb beta3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% alphaIIb beta3, functional), and variant type (50% alphaIIb beta3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional alphaIIb beta3 in initiating protein tyrosine phosphorylation. We noted that in addition to the characteristic absence of the 100 to 105 kD doublet, a 77 to 80 kD doublet and to a lesser extent a 64-kD band, exhibited low phosphorylation kinetics, but with normal initial phosphorylation rates (up to 60 seconds). Similar results were obtained by inhibition of thrombin aggregation of control platelets by alphaIIb beta3 antagonists (the RGDS peptide or the monoclonal antibody 10E5), or in the absence of stirring (fibrinogen binding, but no aggregation). These results suggest that tyrosine phosphorylation of the 77 to 80 kD doublet, identified by immunoprecipitation as the cytoskeletal protein cortactin, and the 64 kD band are dependent both on thrombin activation during early steps and on the late steps of alphaIIb beta3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.

Hematopoietic differentiation of embryonic stem cells: an in vitro model to study gene regulation during megakaryocytopoiesis.

We are interested in the regulation of the tissue specificity of the megakaryocyte-specific platelet glycoprotein IIb gene. The murine embryonic stem (ES) cells are able to differentiate into erythroid, mast and granulomonocytic cells in appropriate culture conditions. Our goal is to optimize the production of myeloid cells including megakaryocytes (MKs) by ES cells. We have found that coculture with MS-5 stromal cells and the presence of a cocktail of hematopoietic growth factors (HGFs) [stem cell factor, interleukin 3 (IL-3), IL-6, IL-11, G-CSF and erythropoietin] had a high synergistic activity on differentiation of ES cells into pure and MK-containing myeloid colonies from day 12 embryoid bodies. Thrombopoietin increased the number of MKs only when added to the HGF cocktail in the presence of MS-5 cells. Interestingly, many MKs exhibited a "hairy" appearance evocative of pseudopodial proplatelet formation. Expression of genes specific for the megakaryocytic lineage, GPIIb, PF4, mpl and GPIIIa, was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) during differentiation of ES cells, and their relative time course was evaluated. This demonstrates that optimized culture conditions for the differentiation of ES cells into the MK lineage provide a useful tool for the study of the regulation of expression of genes during megakaryocytopoiesis.