Screening for adolescent idiopathic scoliosis is more accurate when performed by healthcare professionals compared to untrained parents: a diagnostic accuracy study.

BACKGROUND: Many countries ended their professional scoliosis screening due to ongoing controversies. Discontinuation resulted in a shift of screening responsibility from trained healthcare professionals to untrained parents. PURPOSE: To compare the diagnostic accuracy of picture-based scoliosis screening between parents and healthcare professionals. METHODS: In this cross-sectional diagnostic accuracy study, parents and healthcare professionals assessed pictures of 28 children (20 AIS & 8 non-scoliosis). Each child had two photographs (standing position & full-flexion during forward-bending test) that were presented simultaneously. Lumbar and thoracic curves were represented with a range in severity (10 to > 40°). The assessors had to answer whether they detected an abnormality that ought to be referred to a specialist. Measures of accuracy were calculated for both groups and various curve severities. RESULTS: All pictures were assessed by 101 parents and 122 healthcare professionals. The sensitivity for detecting scoliosis was significantly lower in untrained parents (63.8%, [95% CI: 61.7-65.9%]) compared to healthcare professionals (73.4%, [95% CI: 71.6-75.2%]; p < 0.001), while the specificity was not significantly different (63.6%, [95% CI: 60.2-66.9%] vs. 65.3%, [95% CI: 62.2-68.3%]; p = 0.49). Healthcare professionals consistently recognized the gibbus as a warning sign when referring patients, while untrained parents highlighted various regions, including the spine, gibbus and scapula regions. CONCLUSION: The sensitivity of screening for scoliosis was significantly lower when it was performed by parents, while the false-positive rate was similar to healthcare professionals. The window of opportunity for conservative treatment may be missed when parents rather than professionals are responsible for screening.

Mesh Versus Patch Repair for Epigastric and Umbilical Hernia (MORPHEUS Trial); One-Year Results of a Randomized Controlled Trial.

DESIGN: This trial is a randomized controlled, patient-blinded, multicentre, superiority trial. METHODS: All patients ≥18 years with a single, symptomatic and primary umbilical or epigastric hernia (<2 fingers) qualified for participation in the study. Flat polypropylene mesh repair was compared to patch repair (PROCEED® Ventral Patch) (PVP). The objective of this trial was to identify a superior method for umbilical and epigastric hernia repair in terms of complication rates. RESULTS: A total of 352 patients were randomized in this trial; 348 patients received the intervention (n = 177 PVP vs. n = 171 mesh). No peri-operative complications occurred. PVP placement was significantly faster compared to mesh placement (30 min, SD 11 vs. 35 min, SD 11) and was scored as an easier procedure. At 1-month follow-up, 76 patients suffered any kind of complication. There was no significant difference in the proportion of complications (24.9% for PVP and 18.7% for mesh, p = 0.195). A significant difference was seen in re-operation rate within 1 month, significantly less early re-operations in the mesh group (0.0 vs. 2.8%, p = 0.027). After 1-year follow-up, no significant differences are seen in recurrence rates (n = 13, 7.8% PVP vs. n = 5, 3.3% mesh, p = 0.08). CONCLUSIONS: Both mesh and PVP had a comparable amount of reported complications. There was a significantly higher incidence of early re-operations due to early complications in the PVP group. No differences were seen in infection rates and the need for antibiotic treatment. No significant difference was seen in the recurrence rates. REGISTRATION: This trial was registered in the Dutch Trail Registry (NTR) NTR2514NL33995.060.10. [12].

Long-term outcome of low perianal fistulas treated by fistulotomy: a multicenter study.

PURPOSE: Fistulotomy is considered to be the golden standard for the treatment of low perianal fistula but might have more influence on continence status than believed. This study was performed to evaluate the healing rate after a fistulotomy and to show results for continence status. METHODS: A retrospective database study was performed in one university medical center and its six affiliated hospitals. All patients treated with a fistulotomy for a low perianal fistula were identified. Healing and recurrence of the fistula were identified. Questionnaires on continence status and quality of life were mailed to all patients. RESULTS: In total, 537 patients were identified. The primary etiology of the fistulas was cryptoglandular (66.5%). Recurrence was seen in 88 patients (16.4%) resulting in a primary healing rate of 83.6%. After secondary treatment for the recurrence, another 40 patients healed. This resulted in a secondary healing rate of 90.3%. The Kaplan-Meier analysis showed that at 5 years, the healing rate was 0.81 (95% confidence interval (95% CI) 0.71-0.85). The mean Vaizey score was 4.67 (SD 4.80). Major incontinence, defined as a Vaizey score of >6, was seen in 95 (28.0%) patients. Only 26.3% of the patients had a perfect continence status (Vaizey score 0). Quality of life was not different from the general population. CONCLUSIONS: Fistulotomy seems to be associated with a healing rate of 0.81 (95% CI 0.71-0.85) after 5 years. However, major incontinence is still reported by 26.8% of patients and only 26.3% of patients had a perfect continence status.

Rectovaginal fistula: a new technique and preliminary results using collagen matrix biomesh.

BACKGROUND: A rectovaginal fistula (RVF) is a debilitating condition that is difficult to treat. Many available techniques are invasive and involve extensive surgery. A local procedure with good closure rates would be preferable as a first step in the treatment of RVF. The aim of this study was the development of a local technique for the closure of RVF with good closure rates to prevent the use of more invasive procedures. METHODS: This was a pilot study. Patients with RVF who had undergone multiple operations in the pelvic area, local radiotherapy, chemotherapy or had been diagnosed with Crohn's disease were included in the study. All had a history of surgery for RVF. A cross-linked collagen matrix biomesh was placed in the rectovaginal septum using a transperineal or a transvaginal approach. The main outcome measure in this study was the closure rate reported as absence of the fistula at 1 year. RESULTS: Twelve patients were included in the study. Absence of fistula at 1 year was 0.64 (95 % confidence interval 0.30-0.85). Three patients (25.0 %) developed a recurrence, two were reoperated on with a gracilis flap transposition, and one was treated with laparoscopic ligation. In one patient (8.3 %), the fistula failed to close within 3 months after the mesh placement. CONCLUSIONS: Our technique shows promising results. A local and simple technique with acceptable closure and morbidity rates, like our local repair with biomesh, would be ideal as a first step in treating RVFs. Long-term results are needed.

Perioperative dilutional coagulopathy treated with fresh frozen plasma and fibrinogen concentrate: a prospective randomized intervention trial.

BACKGROUND AND OBJECTIVES: Treatment of dilutional coagulopathy by transfusing fresh frozen plasma (FFP) remains sub-optimal. We hypothesized that partial replacement of transfused FFP by fibrinogen concentrate results in improved coagulant activity and haemostasis. This was tested in a controlled clinical intervention trial with patients experiencing massive bleeding during major surgery. METHODS: Patients undergoing major elective surgery were treated according to current protocols. When transfusion with FFP was required, patients were randomized as follows: group A received 4 units FFP and group B received 2 units FFP plus 2 g fibrinogen concentrate. Blood samples were taken before and after the intervention. Analysts were blinded to the treatment type. RESULTS: Group A (B) consisted of 21 (22) patients, in 16 (17) of whom bleeding stopped after intervention. Plasma fibrinogen increased significantly more in group B (0·57 g/l) than in group A (0·05 g/l). However, levels of prothrombin and factors VIII, IX and X increased more in group A than in group B. Rotational thromboelastometry (ROTEM) of whole blood and plasma revealed improved fibrin clot formation in group B but not in group A. Thrombin generation [calibrated automated thrombogram (CAT)] in plasma increased more in group A. Principal parameters determining whole-blood thromboelastometry were the fibrinogen level and platelet count. In vitro addition of fibrinogen and prothrombin complex concentrate to pre-intervention samples restored both ROTEM and CAT parameters. CONCLUSIONS: Partial replacement of transfused FFP by fibrinogen increases fibrin clot formation at the expense of less improved thrombin generation. Coagulation factors other than fibrinogen alone are required for full restoration of haemostasis.

Tumour-stroma ratio to predict pathological response to neo-adjuvant treatment in rectal cancer.

INTRODUCTION: Management of rectal cancer has advanced, with an increasing use of neoadjuvant chemoradiotherapy (nCRT). This opens options for organ preserving treatment for those with a major response to nCRT. However, the degree of clinical response, based on MRI and post-treatment biopsies, only poorly matches the degree of actual pathological response. In order to select patients with major pathological response without surgical resection, it is of importance to define tumour markers predicting the degree of pathological response to nCRT. The intra-tumoural tumour-stroma ratio (TSR) might be this marker. METHODS: TSR in pre-treatment biopsies was estimated according to the method described by van Pelt et al. The degree of pathological response was assessed on the tumour resection according to tumour regression grading (TRG) by Mandard. The primary endpoint of this study was the difference in pathological response to nCRT between TSR-high and TSR-low groups. RESULTS: We found that 26.2% of patients with major response was classified as TSR-high, while 73.8% of patients were classified as TSR-low. A high TSR in pre-treatment biopsies was associated with a lower chance of major-response to nCRT (OR = 0.37, 95%CI; 0.19-0.73), p = 0.004), independent of tumour stage and time between nCRT and surgery. CONCLUSION: In rectal cancer, TSR in pre-treatment biopsies predicts pathologic response to nCRT, with a high TSR bringing twice the risk of poor to no response compared to low TSR. In future, assessment of TSR may fulfil a role in a therapeutic algorithm identifying patients who will or will not respond to nCRT prior to treatment initiation.

Mesenchymal/epithelial induction mediates olfactory pathway formation.

In the olfactory pathway, as in the limbs, branchial arches, and heart, mesenchymal/epithelial induction, mediated by retinoic acid (RA), FGF8, sonic hedgehog (shh), and the BMPs, defines patterning, morphogenesis, and differentiation. Neuronal differentiation in the olfactory epithelium and directed growth of axons in the nascent olfactory nerve depend critically upon this inductive interaction. When RA, FGF8, shh, or BMP signaling is disrupted, distinct aspects of olfactory pathway patterning and differentiation are compromised. Thus, a cellular and molecular mechanism that facilitates musculoskeletal and vascular development elsewhere in the embryo has been adapted to guide the differentiation of the olfactory pathway in the developing forebrain.

The making of a maggot: patterning the Drosophila embryonic epidermis.

Cell fates are instructed by signals emitted from specialized cell populations called organizers. The study of epidermal patterning in Drosophila is contributing novel insights concerning the establishment and action of such organizers. Juxtaposed rows of cells express either the wingless or hedgehog signaling molecules and thereby act as organizers of segment pattern. These signals mediate a mutually re-enforcing interaction between the two rows of cells to sustain organizer function. In a distinct and subsequent phase, wingless and hedgehog act to specify the fates of cells.

Acquired platelet antagonism: off-target antiplatelet effects of malignancy treatment with tyrosine kinase inhibitors.

Platelets can contribute to tumor progression and metastasis. Cancer patients are at increased risk of thrombosis, and advanced stages of cancer are associated with thrombocytosis or increased platelet reactivity. Tyrosine kinase inhibitors (TKIs) are widely used as a targeted strategy for cancer treatment, with the aim of prolonging progression-free survival of the patients. Because of their broad kinase target spectrum, most TKIs inevitably have off-target effects. Platelets rely on tyrosine kinase activity for their activation. Frequently observed side effects are lowering of platelet count and inhibition of platelet functions, whether or not accompanied by an increased bleeding risk. In this review, we aim to give insights into: (i) 38 TKIs that are currently used for the treatment of different types of cancer, either on the market or in clinical trials; (ii) how distinct TKIs can inhibit activation mechanisms in platelets; and (iii) the clinical consequences of the antiplatelet effects of TKI treatment. For several TKIs, the knowledge on affinity for their targets does not align with the published effects on platelets and reported bleeding events. This review should raise awareness of the potential antiplatelet effects of several TKIs, which will be enhanced in the presence of antithrombotic drugs.

Atherothrombosis and Thromboembolism: Position Paper from the Second Maastricht Consensus Conference on Thrombosis.

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia-reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.

Acquiring basic endoscopy skills by training on the GI Mentor II.

BACKGROUND: Achieving proficiency in flexible endoscopy requires a great amount of practice. Virtual reality (VR) simulators could provide an effective alternative for clinical training. This study aimed to gain insight into the proficiency curve for basic endoscope navigation skills with training on the GI Mentor II. METHODS: For this study, 30 novice endoscopists performed four preset training sessions. In each session, they performed one EndoBubble task and managed multiple VR colonoscopy cases (two in first session and three in subsequent sessions). Virtual reality colonoscopy I-3 was repeatedly performed as the last VR colonoscopy in each session. The assignment for the VR colonoscopies was to visualize the cecum as quickly as possible without causing patient discomfort. Five expert endoscopists also performed the training sessions. Additionally, the performance of the novices was compared with the performance of 20 experienced and 40 expert endoscopists. RESULTS: The novices progressed significantly, particularly in the time required to accomplish the tasks (p < 0.05, Friedman's analysis of variance [ANOVA], p < 0.05, Wilcoxon signed ranks). The experts did not improve significantly, except in the percentage of time the patient was in excessive pain. For all the runs, the performance of the novices differed significantly from that of both the experienced and the expert endoscopists (p < 0.05, Mann-Whitney U). The performance of the novices in the latter runs differed less from those of both the experienced and the expert endoscopists. CONCLUSIONS: The study findings demonstrate that training in both VR colonoscopy and EndoBubble tasks on the GI Mentor II improves the basic endoscope navigation skills of novice endoscopists significantly.

Therapeutic modulation of DMD splicing by blocking exonic splicing enhancer sites with antisense oligonucleotides.

Antisense oligonucleotides (AONs) can be used to correct the disrupted reading frame of Duchenne muscular dystophy patients (DMD). We have a collection of 121 AONs, of which 79 are effective in inducing the specific skipping of 38 out of the 79 different DMD exons. All AONs are located within exons and were hypothesized to act by steric hindrance of serine-arginine rich (SR) protein binding to exonic splicing enhancer (ESE) sites. Indeed, retrospective in silico analysis of effective versus ineffective AONs revealed that the efficacy of AONs is correlated to the presence of putative ESE sites (as predicted by the ESEfinder and RESCUE-ESE software). ESE predicting software programs are thus valuable tools for the optimization of exon-internal antisense target sequences.

Advantages of advanced laparoscopic systems.

BACKGROUND: Conventional laparoscopy offers great benefits to our patients, but suffers from major technical drawbacks. Advanced laparoscopic systems are being developed addressing some of these drawbacks. METHODS: We performed a training-box based study, performing laparoscopic tasks using conventional laparoscopy and advanced laparoscopic systems in order to assess the influence of these technical drawbacks in order to predict where the biggest advantages of newly developed surgical systems can be expected. RESULTS: The most significant technical drawbacks were two-dimensional vision, disturbed eye-hand target axis and (possibly to a lesser extent) the rigid instruments with a limited five degrees of freedom. CONCLUSION: Major advances in advanced laparoscopy might only be expected using console-based robot-arm manipulated systems like the daVinci surgical system, or a combination of a high-quality 3-dimensional vision system, restoration of the eye-hand-target axis and the use of an advanced handheld instrument offering seven degrees of freedom such as the Radius surgical system.

PO-19 - Platelet (PLT) adhesion under flow condition in essential thrombocythemia (ET) and polycythemia vera (PV) is variably influenced according to patient mutational status.

INTRODUCTION: The myeloproliferative neoplasms ET and PV are characterized by a high incidence of both arterial and venous thrombosis, and/or microcirculatory disturbances. Three somatic mutations, i.e. JAK2-V617F, Calreticulin (CalR) and MPL, commonly found in these diseases, correlate with different thrombotic risk levels. AIM: To analyze the influence of JAK2-V617F, CalR and MPL mutations on PLT adhesion, evaluated by a dynamic method under flow conditions in a group of patients with ET and PV. MATERIALS AND METHODS: 86 patients, i.e. 51 ET (19 M/32 F; age range 32-86 years) and 35PV (22 M/13 F; 41-83 yrs.), and 24 healthy controls (13 M/11 F; 28-61 yrs.) were enrolled upon informed consent. For the adhesion assay, peripheral venous whole blood was perfused over collagen for 4' at a 1,000 s-1 shear rate. PLTs were then stained with an anti-P-selectin-FITC antibody to evaluate PLT activation, and annexin V-AlexaFluor647 to detect procoagulant phosphatidylserine expression. Then, images of adherent PLTs in random fields were taken using phase contrast and fluorescence imaging by EVOS® fluorescence microscope. Results are mean±SEM of the % area covered by PLTs, or as the % of adherent PLTs positive for P-selectin or phosphatidylserine. Main hematological parameters and mutational status were recorded. RESULTS: PLT adhesion was significantly (p<0.01) greater in ET (44.6±1.6%) and PV patients (49.0±1.9%) compared to controls (37.9±1.7%). In ET, PLT adhesion was highest in JAK2-V617F mutation carriers (n=23), followed by CalR-positive (n=16) and triple negative subjects (n=9), and lowest in the MPL-positive patients (n=3). In PV, no difference in PLT adhesion was observed between JAK2-V617F heterozygous and homozygous subjects. P-selectin expression by adherent PLTs was not statistically different between patients and controls. Differently, phosphatidylserine expression on adherent PLTs was significantly reduced (p<0.01) in both ET and PV compared to healthy subjects. In ET patients, a significant (p<0.05) correlation was found between PLT adhesion and PLT count in JAK2-V617F and CalR-positive mutation carriers. Multivariate regression analysis adjusted for age and sex, confirmed PLT count as a significant determinant of PLT adhesion in JAK2-V617F positive patients only. CONCLUSIONS: ET and PV platelets show an increased adhesion to collagen in vitro, particularly in those carrying the JAK2-V617F mutation. A prospective study is ongoing to evaluate the predictive value of our PLT thrombus formation dynamic model for the thrombotic risk in ET and PV patients. ACKNOWLEDGEMENT: Project funded by "AIRC-IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research" (A.I.R.C.).

OC-08 - Multiple functional defects in platelets from thrombocytopenic cancer patients undergoing chemotherapy.

INTRODUCTION: Severe thrombocytopenia (≤50×10(9) platelets/L) is often the consequence of hematological malignancies and intensive chemotherapy. The risk of clinically significant bleeding is increased in these patients, despite the use of prophylactic platelet transfusions. The fact that there is no clear correlation between the platelet count and the risk of hemorrhage, suggests that there are other contributing factors. The contribution of impairments in platelet and coagulant function remains poorly understood. AIM: In patients with chemotherapy-induced thrombocytopenia due to hematological malignancies, we evaluate platelet and coagulant functions and determine the effects of platelet transfusion. Ultimately, we can identify specific hemostatic factors that aid in the prediction of bleeding. MATERIALS AND METHODS: In total 58 patients were included and blood was collected before and, if indicated (≤10×10(9) platelets/L), 1 hour after transfusion with platelet concentrate. Platelet function was assessed using flow cytometry by determining: 1) integrin αIIbß3 activation (PAC-1 antibody), 2) P-selectin expression (anti-P-selectin antibody), 3) phosphatidylserine exposure (Annexin-V) and 4) intracellular calcium (Fluo-4 AM). Factor levels were determined in plasma. Thrombus and fibrin formation was assessed by perfusion of whole blood over a collagen-tissue factor surface at a shear rate of 1,000 s-1. RESULTS: Platelets from the thrombocytopenic patients before transfusion showed markedly reduced integrin αIIbß3 activation and P-selectin expression in response to thrombin, collagen-related peptide and ADP, compared to healthy donor platelets. Also, agonist-induced intracellular calcium fluxes were greatly reduced. However, calcium fluxes with thapsigargin, a SERCA pump inhibitor, were similar in patient and control platelets, suggesting a normal calcium store content in the patient platelets. Furthermore, phosphatidylserine exposure was increased in unstimulated patient platelets compared to control platelets (8.2 vs. 1.8%, p<0.0001). Coagulation factor levels were within the normal range, with the exception of von Willebrand factor and fibrinogen levels, which were elevated. Platelet transfusion partly recovered the platelet integrin αIIbß3 activation and P-selectin expression induced by agonists. Platelet deposition (6.7 vs. 1.7%, p<0.0001) and fibrin formation (7.6 vs. 0.9%, p=0.0005) under flow conditions were substantially improved after platelet transfusion. CONCLUSIONS: Platelets from cancer patients undergoing chemotherapy appear to display impaired functional responses to activating stimuli. Platelet transfusion partly restores these functional defects, resulting in improved thrombus and fibrin formation.

Membrane fluidity of non-activated and activated human blood platelets.

The steady-state fluorescence anisotropy of membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or its 4'-trimethylammonio derivative, TMA-DPH, is generally considered a measure for the lipid order and, hence, inversely related to membrane fluidity. We now report that anisotropy values of DPH- and TMA-DPH-labeled human platelets are considerably influenced by experimental conditions like the platelet concentration, which do not affect membrane fluidity. Activation of platelets with thrombin increases, but activation with ionomycin decreases anisotropy values with both labels. Such anisotropy changes are not detected in platelet membranes or platelet lipids, when isolated after activation of the intact platelets. We present evidence that the anisotropy changes of intact platelets are not a consequence of modified lipid composition (e.g., as would be induced by phospholipase A2 activity) but are, at least partially, caused by changed optical properties of the cell suspension. Measurement of membrane fluidity of platelets by fluorescence polarization is severely hindered by a high turbidity of the platelet suspension and also by changes in the turbidity and platelet morphology during the activation process.

Effects of dietary fatty acids on signal transduction and membrane cholesterol content in rat platelets.

Previously, we have reported that dietary fatty acids can modify the thromboxane A2-dependent activation of rat platelets. Here, we present evidence that this dietary effect is part of a more general effect on platelet signal transduction, putatively involving structural changes in the platelet membranes. Four experiments were performed, where Wistar rats were fed with a high-fat diet enriched in either saturated, n-6 polyunsaturated or n-3 polyunsaturated fatty acids, or with a low-fat diet enriched in n-6 polyunsaturated fatty acids. The type of diet hardly influenced mean number of double bonds in the major platelet phospholipids. Platelet membranes from the rats fed with the saturated-fat diet had phospholipids with relatively high levels of arachidonate, but were low in cholesterol/phospholipid ratio. When compared to this diet group, platelets from other groups had an arachidonate content that was 21 to 47% lower and a cholesterol/phospholipid ratio 3 to 5% higher. The saturated-fat diet resulted in platelets that, in general, were less responsive to agonists than the platelets from other groups: with thrombin, collagen and thromboxane A2 analogue U46619, both early (shape change and phospholipase C-dependent rise in [Ca2+]i) and late (exocytosis and aggregation) responses were relatively low. However, platelet activation evoked by ADP was not influenced by diet type. When the cholesterol content of rat platelets was modified in vitro, it appeared that the early and late responses to thrombin and U46619 increased with the cholesterol/phospholipid ratio. Taken together, these results suggest that in rat platelets (i) the membrane cholesterol/phospholipid ratio can be modulated by a diet rich in saturated fatty acids, explaining, at least in part, the dietary effect on phospholipase C-mediated platelet activation, and (ii) relatively small changes in cholesterol content can have a more profound effect on platelet activation than substantial changes in arachidonate level.

Function of intracellular [Ca2+]i in exocytosis and transbilayer movement in human platelets surface-labeled with the fluorescent probe 1-(4-trimethylammonio)phenyl)-6-phenyl-1,3,5-hexatriene.

Ellipsometry indicated that 1-(4-(trimethylammonio)phenyl-6-phenylhexa-1,3,5-triene (TMA-DPH) bound to platelets in a reversible and saturable way. Accordingly, the fluorescence intensity (F) of a suspension of TMA-DPH-labeled platelets was described as a quantity, determined by the amount of TMA-DPH bound to the platelet surface. Most platelet activators elevated F to a degree that correlate well with the secretion of serotonin evoked by these activators. The increase in F levels reflected the increase in outer membrane surface area following exocytosis. However, activators that evoked prolonged (> 2.5 min) and strong (> 600 nM) elevations of cytosolic [Ca2+]i increased F to levels that were much higher than expected from the maximal increase in surface area due to exocytosis. This high increase in F was caused by inward transbilayer movement of TMA-DPH over the plasma membrane and the subsequent labeling of cytosolic membrane sides. The kinetics of exocytosis and changes in cytosolic [Ca2+]i were studied by stopped-flow mixing of platelets with agonist. Thrombin-induced exocytosis had a delay of only 3 s, which was shortened when external CaCl2 or ADP was present. This correlated well with a faster rise in [Ca2+]i in the presence of CaCl2 or ADP, indicating that exocytosis was linked in time to elevation of [Ca2+]i. By itself, ADP was unable to evoke exocytosis and it elicited a [Ca2+]i transient of much shorter duration than thrombin, but with similar maximum. We concluded that both exocytosis and transbilayer movement were associated with elevation of [Ca2+]i: exocytosis required a moderate, relatively prolonged rise and transbilayer movement was accompanied by a stronger rise of even longer duration. Influx of external Ca2+ was essential for transbilayer movement, but not for exocytosis.

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets.

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.

Effects of U73122 and U73343 on human platelet calcium signalling and protein tyrosine phosphorylation.

We have investigated the actions of the PLC inhibitor, U73122, and its close analogue, U73343, which does not inhibit PLC, in Fura-2-loaded human platelets. Rises in [Ca2+]i evoked by thrombin and collagen, and the TxA2-dependent rise in [Ca2+]i evoked by thapsigargin, were abolished by U73122, indicating that it inhibits the activity of both beta and gamma isoforms of PLC. The supposed control compound U73343, was found to inhibit TxA2 formation; it therefore partially inhibited the rise in [Ca2+]i evoked by low concentrations of thrombin, by thapsigargin or by collagen. U73343 had a greater effect than aspirin on the action of collagen, indicating an action on the TxA2-independent component of the signal, via PLC gamma-U73343 lowered TxA2 production by inhibiting the activation of cPLA2, probably at a tyrosine phosphorylation step. U73343 seems to inhibit only the tyrosine kinases involved in the activation of PLC gamma and the generation of TxA2. In contrast, U73122 increased tyrosine phosphorylation of platelet proteins, perhaps by inhibiting receptor independent tyrosine phosphatases, but inhibited all further tyrosine phosphorylation on addition of thrombin or other agonists.