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.

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.

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.

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].

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.

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.).

Acute and persistent platelet and coagulant activities in atherothrombosis.

The potential relevance of murine atherothrombosis models for understanding human disease has been debated in the past. Despite this, in the last decade, many thrombosis studies with atherogenic Apoe(-/-) mice have been performed, which provide novel insight into the molecular mechanisms by which platelet and coagulation processes accomplish acute thrombus formation after plaque disruption in vivo. Support for these mechanisms has come from whole blood flow perfusion studies over plaque material in vitro, which are also reviewed in this study. The main plaque-derived triggers for thrombus formation appear to be collagen and tissue factor, next to bioactive mediators such as prostaglandin E2. The atherothrombotic process relies on collagen- and ADP-receptor-induced platelet activation as well as on thrombin/fibrin generation via the extrinsic and intrinsic coagulation pathways. Less is known of the persistent effects of a thrombus on atherosclerosis progression, but evidence suggests roles herein of activated platelets and ongoing thrombin generation.

Desmopressin treatment improves platelet function under flow in patients with postoperative bleeding.

BACKGROUND: Patients undergoing major cardiothoracic surgery are subjected to dilution, owing to massive fluid infusion and blood component transfusion. These patients may experience bleeding perioperatively, and are frequently treated with the endothelium-activating agent desmopressin. OBJECTIVES: To investigate the effect of desmopressin administration on von Willebrand factor (VWF)-dependent coagulant and platelet functions under flow conditions. PATIENTS/METHODS: Blood from 16 patients with postoperative bleeding was obtained before and after desmopressin treatment (0.3 µg kg(-1) body weight), and assessed for coagulant properties and platelet function. Furthermore, VWF antigen levels and multimer composition were determined in both samples. RESULTS: Desmopressin treatment did not change thrombin generation in plasma or whole blood thromboelasticity. Also coagulation factor levels (other than factor VIII) and coagulation times were unchanged, suggesting that desmopressin treatment did not have a major effect on the coagulant activity. On the other hand, desmopressin treatment raised the already high plasma levels of VWF from a median of 116 IU mL(-1) (interquartile range [IQR] 102-154 IU mL(-1) ) to a median of 160 IU mL(-1) (IQR 126-187 IU mL(-1) ) (P = 0.007), owing to accumulation of the high molecular weight VWF multimers. Furthermore, desmopressin treatment caused an increase in collagen-dependent thrombus formation and platelet phosphatidylserine exposure. Markers of thrombus formation correlated with the plasma levels of VWF. In vitro control experiments confirmed a major contribution of VWF to thrombus formation and procoagulant activity under conditions of blood dilution. CONCLUSIONS: Desmopressin treatment of patients with bleeding complications after cardiothoracic surgery induces the release of high molecular weight VWF multimers, which enhance platelet activation and thrombus formation under flow conditions.

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.

Whole blood thrombin generation in Bmal1-deficient mice.

The Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20- to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437 ± 7 nM.min; mean ± SD, n=7) when compared with WT mice (ETP=220 ± 45 nM.min; mean ± SD, n=5). The peak heights also differed significantly (p=0.027). By applying SEM we found that Bmal1 deficient mice display a denser fibrin network with smaller pores compared to WT mice. In conclusion, the whole blood TG assay in mice revealed to be reproducible. As a proof-of-principle we have shown that the whole blood TG assay is capable of detecting a prothrombotic phenotype in Bmal1-KO mice.

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.

Orai1-induced store-operated Ca(2+) entry enhances phospholipase activity and modulates canonical transient receptor potential channel 6 function in murine platelets.

BACKGROUND: Orai1, the major store-operated Ca(2+) entry (SOCE) channel in platelets, is not only critical for enhancing diverse signaling pathways, but may also regulate receptor-operated Ca(2+) entry (ROCE). Dynamic coupling of the Orai1 signalosome to canonical transient receptor potential channels (TRPCs) has been suggested as an essential step in the activation of SOCE and ROCE. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms remains controversial. OBJECTIVE: We aimed to elucidate the role of Orai1 in diacylglycerol (DAG)-mediated ROCE. METHODS: Trpc6(-/-) , Orai1(-/-) and Orai1(-/-) /Trpc6(-/-) mice were generated, and their platelets were analyzed. RESULTS: Thapsigargin (TG)-induced SOCE was further reduced in Orai1(-/-) /Trpc6(-/-) platelets as compared with Orai1(-/-) platelets, thus revealing that TG-induced signaling pathways can activate TRPC6. Thapsigargin-induced SOCE leads to enhanced phospholipase C and D activity in wild-type platelets. The activity of both enzymes was significantly reduced in Orai1(-/-) platelets upon TG stimulation, whereas receptor-induced phospholipase activity was not affected. Furthermore, TG-induced and glycoprotein VI-mediated thromboxane A2 release was strongly dependent on Orai1-mediated SOCE. CONCLUSION: The regulation of TRPC6 activity can occur independently of the physical interaction with Orai1. TRPC6 operates in crosstalk with Orai1 through Orai1-induced DAG production via phospholipase activation. Orai1-induced DAG production and thromboxane release amplify the second phase of Ca(2+) signaling in platelets.

Additive roles of platelets and fibrinogen in whole-blood fibrin clot formation upon dilution as assessed by thromboelastometry.

Blood dilution after transfusion fluids leads to diminished coagulant activity monitored by rotational thromboelastometry, assessing elastic fibrin clot formation, or by thrombin generation testing. We aimed to determine the contributions of blood cells (platelets, red blood cells) and plasma factors (fibrinogen, prothrombin complex concentrate) to fibrin clot formation under conditions of haemodilution in vitro or in vivo.Whole blood or plasma diluted in vitro was supplemented with platelets, red cells, fibrinogen or prothrombin complex concentrate (PCC). Thromboelastometry was measured in whole blood as well as plasma; thrombin generation was determined in parallel. Similar tests were performed with blood from 48 patients, obtained before and after massive fluid infusion during cardiothoracic surgery.Addition of platelets or fibrinogen, in additive and independent ways, reversed the impaired fibrin clot formation (thromboelastometry) in diluted whole blood. In contrast, supplementation of red blood cells or prothrombin complex concentrate was ineffective. Platelets and fibrinogen independently restored clot formation in diluted plasma, resulting in thromboelastometry curves approaching those in whole blood. In whole blood from patients undergoing dilution during surgery, elastic clot formation was determined by both the platelet count and the fibrinogen level. Thrombin generation in diluted (patient) plasma was not changed by fibrinogen, but improved markedly by prothrombin complex concentrate. In conclusion, in dilutional coagulopathy, platelets and fibrinogen, but not red blood cells or vitamin K-dependent coagulation factors, independently determine thromboelastometry parameters measured in whole blood and plasma. Clinical decisions for transfusion based on thromboelastometry should take into account the platelet concentration.

Calcium-activated and apoptotic phospholipid scrambling induced by Ano6 can occur independently of Ano6 ion currents.

Immune cells and platelets maintain plasma membrane phospholipid asymmetry. Upon activation, this asymmetry is disrupted by phospholipid scrambling (PS), which is a major step during activation of immune cells, hemostasis and apoptosis. Anoctamin 6 (Ano6; TMEM16F) causes chloride (Cl(-)) and cation currents and is required for Ca(2+)-dependent PS. It is defective in blood cells from patients with Scott syndrome, a rare bleeding disorder. We examined if Cl(-) currents and PS are related, whether both processes are Ca(2+) dependent, and whether Ca(2+)-independent scrambling during intrinsic and extrinsic apoptosis is controlled by Ano6. Ca(2+) increase by ionomycin activated Ano6 Cl(-) currents and PS in normal lymphocytes, but not in B-lymphocytes from two different patients with Scott syndrome. Fas ligand (FasL) did not increase intracellular Ca(2+), but activated Cl(-) currents in normal but not in Scott lymphocytes. Whole-cell currents were inhibited by Cl(-) channel blockers and by siRNA knockdown of Ano6. In contrast, intrinsic mitochondrial apoptosis by ABT-737 did not induce Cl(-) currents in lymphocytes. PS was not inhibited by blockers of Ano6 or removal of Cl(-) ions. Remarkably, Ca(2+)-independent scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis was unchanged in Scott cells. We conclude that: (i) Ano6 Cl(-) currents are activated by increase in cytosolic Ca(2+), or Ca(2+) independent by stimulation of Fas receptors; (ii) Ca(2+)-dependent PS induced by Ano6 does not require Cl(-) currents; (iii) Ca(2+)-independent PS does not require Ano6; (iv) Ano6 is necessary for Ca(2+)-dependent PS, but not by increasing intracellular Ca(2+).

Platelet-based coagulation: different populations, different functions.

Platelets in a thrombus interact with (anti)coagulation factors and support blood coagulation. In the concept of cell-based control of coagulation, three different roles of platelets can be distinguished: control of thrombin generation, support of fibrin formation, and regulation of fibrin clot retraction. Here, we postulate that different populations of platelets with distinct surface properties are involved in these coagulant functions. Platelets with elevated Ca(2+) and exposed phosphatidylserine control thrombin and fibrin generation, while platelets with activated α(IIb) ß(3) regulate clot retraction. We review how coagulation factor binding depends on the platelet activation state. Furthermore, we discuss the ligands, platelet receptors and downstream intracellular signaling pathways implicated in these coagulant functions. These insights lead to an adapted model of platelet-based coagulation.

Improved EMCCD gamma camera performance by SiPM pre-localization.

High spatial resolution γ-imaging can be achieved with scintillator readout by low-noise, fast, electron-multiplying charge-coupled devices (EMCCDs). Previously we have shown that false-positive events due to EMCCD noise can be rejected by using the sum signal from silicon photomultipliers (SiPMs) mounted on the sides of the scintillator. Here we launch a next generation hybrid CCD-SiPM camera that utilizes the individual SiPM signals and maximum likelihood estimation (MLE) pre-localization of events to discriminate between true and false events in CCD frames. In addition, SiPM signals are utilized for improved energy discrimination. The performance of this hybrid detector was tested for a continuous CsI:Tl crystal at 140 keV. With a pre-localization accuracy of 1.06 mm (full-width-at-half-maximum) attained with MLE the signal-to-background ratio (SBR) was improved by a factor of 5.9, 4.0 or 2.2 compared to the EMCCD-only readout, at the cost of rejecting, respectively, 47%, 9% or 4% of the events. Combining the pre-localization and SiPM energy estimation improved the energy resolution from 50% to (19 ± 3)% while maintaining the spatial resolution at 180 µm.

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.

Microparticles from apoptotic platelets promote resident macrophage differentiation.

Platelets shed microparticles not only upon activation, but also upon ageing by an apoptosis-like process (apoptosis-induced platelet microparticles, PM(ap)). While the activation-induced microparticles have widely been studied, not much is known about the (patho)physiological consequences of PM(ap) formation. Flow cytometry and scanning electron microscopy demonstrated that PM(ap) display activated integrins and interact to form microparticle aggregates. PM(ap) were chemotactic for monocytic cells, bound to these cells, an furthermore stimulated cell adhesion and spreading on a fibronectin surface. After prolonged incubation, PM(ap) promoted cell differentiation, but inhibited proliferation. Monocyte membrane receptor analysis revealed increased expression levels of CD11b (integrin α(M)ß(2)), CD14 and CD31 (platelet endothelial cell adhesion molecule-1), and the chemokine receptors CCR5 and CXCR4, but not of CCR2. This indicated that PM(ap) polarized the cells into resident M2 monocytes. Cells treated with PM(ap) actively consumed oxidized low-density lipoprotein (oxLDL), and released matrix metalloproteinases and hydrogen peroxide. Further confirmation for the differentiation towards resident professional phagocytes came from the finding that PM(ap) stimulated the expression of the (ox)LDL receptors, CD36 and CD68, and the production of proinflammatory and immunomodulating cytokines by monocytes. In conclusion, interaction of PM(ap) with monocytic cells has an immunomodulating potential. The apoptotic microparticles polarize the cells into a resident M2 subset, and induce differentiation to resident professional phagocytes.