Aspirin withdrawal in patients treated with ticagrelor presenting with non-ST elevation myocardial infarction.

Essentials Strong P2Y12 blockade may cause platelet inhibition that is only minimally enhanced by aspirin. We evaluated aspirin withdrawal on platelet reactivity in ticagrelor treated patients. Aspirin withdrawal resulted in increased platelet reactivity to arachidonic acid. Aspirin withdrawal caused little difference in adenosine diphosphate-induced platelet aggregation. SUMMARY: Background Recent studies have shown that the thromboxane A2 -dependent pathway is dependent on the ADP-P2Y12 pathway, and that strong P2Y12 receptor blockade alone causes inhibition of platelet aggregation that is minimally enhanced by aspirin. Data from the PLATO trial suggested that, among ticagrelor-treated patients, high-dose versus low-dose (< 100 mg day-1 ) aspirin is associated with an increased risk fof ischemic events. Objectives To evaluate the impact of aspirin withdrawal on platelet reactivity in acute coronary syndrome (ACS) patients treated with a potent P2Y12 blocker. Patients/Methods This was a current prospective, randomized, placebo-controlled, double-blind, cross-over study. The study population comprised 22 consecutive ACS patients who underwent percutaneous coronary intervention and were treated with aspirin (100 mg day-1 ) and ticagrelor. Thirty days post-ACS, open-label aspirin was stopped, and patients were randomized to either blinded aspirin or placebo for 2 weeks, with each patient crossing over to the other arm for an additional 2 weeks. Platelet reactivity to arachidonic acid and ADP determined with light-transmission aggregometry (LTA) and VerifyNow was evaluated at baseline, and 2 weeks and 4 weeks later. Results Aspirin withdrawal resulted in an increase in arachidonic-acid induced platelet reactivity as determined with both LTA (77.0% ± 11.3% versus 20.8% ± 4.4%) and VerifyNow (607.7 ± 10.6 aspirin reaction units [ARU] versus 408.5 ± 14.4 ARU). Platelet response to ADP, as determined with both LTA and VerifyNow, did not differ with either aspirin or placebo (32.9% ± 2.6% versus 35.8% ± 3.6%, and 33.5 ± 6.4 P2Y12 reaction units (PRU) versus 29.6 ± 5.7 PRU, respectively). Conclusions Aspirin withdrawal early post-ACS results in increased platelet reactivity in response to arachidonic acid, despite concomitant treatment with the potent P2Y12 blocker ticagrelor.

Platelet factor XI: intracellular localization and mRNA splicing following platelet activation.

BACKGROUND: The structure and function of platelet factor XI (FXI) protein and the presence of F11 mRNA in platelets are controversial. Although platelets are anucleated cells they contain spliceosome components and pre-mRNAs. Three platelet proteins have been demonstrated to be spliced upon platelet activation. OBJECTIVE: To determine whether FXI is also spliced upon activation and to discern the localization of FXI in platelets. METHODS: Localization of FXI in platelets was assessed by confocal immunofluorescence staining. ELISA, chromogenic assay and western blot analyses were used to measure antigen levels, activity levels and size of FXI in platelets, respectively. Splicing patterns of F11 mRNA were assessed in three states of platelet activation: activated platelets, resting platelets and αIIbß3-integrin activated platelets. RESULTS: Platelet FXI was exhibited in platelet granules. Activated platelets exhibited higher levels of mature F11 mRNA and protein and lower levels of F11 pre-mRNA compared to resting or αIIbß3-integrin activated platelets. CONCLUSIONS: We confirmed the presence of FXI in platelets and showed that it is localized in granules but is not restricted to the same α-granule subtype as von-Willebrand factor and p-selectin. Our study also shows that F11 is present in platelets as pre-mRNA and is spliced upon platelet activation.

Activity of Von Willebrand factor and levels of VWF-cleaving protease (ADAMTS13) in preterm and full term neonates.

Von Willebrand Factor (VWF) has a central role in primary hemostasis. Its biological activity is related to the size of VWF multimers, spontaneously binding to platelets and inducing circulating microthrombi formation. This process is down-regulated by the VWF cleaving protease ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin motif). To date, information regarding the levels of ADAMTS13 in neonates and preterm infants is scarce. Our aim was to study ADAMTS13, VWF antigen (Ag) and Ristocetin cofactor (RiCof) activity in neonates and evaluate potential correlations with perinatal complications. Our cohort consisted of 128 (48/128: born preterm) neonates, born in Sheba Medical Center and followed until hospital discharge. Control group consisted of 20 healthy adults. As expected, a significant elevation of VWF:Ag was observed in preterm and term infants compared to adults. VWF:Ag levels were highest in full term infants (Median 129.0 IQR 33.8) and lowest in adults (Median 119.0 IQR 58.5) (p<0.05), and RiCoF levels in neonates were higher than in adults. ADAMTS13 was significantly (p<0.05) higher in preterm babies in comparison to full term and adult controls. Neonates that underwent stressful conditions or experienced vascular complications such as IUGR, ROP, NEC, had lower levels of ADAMTS13 in our study. Further studies are required to validate and asses potential significance of these findings.

Primary prophylaxis for children with severe congenital factor VII deficiency - Clinical and laboratory assessment.

Severe congenital factor VII (FVII) deficiency is a rare bleeding disorder. Prophylaxis with replacement therapy has been suggested to patients, yet the most beneficial dosing regimens and therapy intervals are still to be defined. Due to the lack of evidence-based data, we hereby present our experience with long-term administration and monitoring primary prophylaxis in children with severe FVII deficiency and an extremely high bleeding risk. Four children with familial FVII deficiency, treated by prophylactic recombinant activated factor VII (rFVIIa), 15-30µg/kg/dose, given 2-3 times weekly since infancy, are discussed. Clinical follow up and monitoring laboratory assays, including thrombin generation, measured at various time points after prophylactic rFVIIa administration are presented. Among our treated patients neither FVII activity nor thrombin generation parameters (both already declined 24h post rFVIIa administration) were able to predict the impact of prophylaxis, and could not be used as surrogate markers in order to assess the most beneficial treatment frequency. However, the long clinical follow-up and comprehensive laboratory assessment performed, have shown that early primary prophylaxis as administered in our cohort was safe and effective.

Bromelain-based enzymatic debridement and minimal invasive modality (mim) care of deeply burned hands.

The objective was to critically review the data and assess the implications of NexoBrid [NexoBrid-NXB formerly Debrase Gel Dressing-DGD]a in the special field of deep hand burns. Detailed analysis of endpoints in the treatment of hand burn patients was conducted as part of a multi-center, open label, randomized, controlled two-arm study to evaluate the safety and efficacy of NXB enzymatic debridement, comparing it to the current standard of care (SOC). These results were compared to a large cohort of patients treated with NXB in a previous, single arm study. Thirty-one burned hands were treated with NXB and 41 hand burns were in the SOC group. In the NXB group, 4 out of 31 hand burns (12.9%) required some excisional debridement compared to 29 out of the 41 (70.7%) in the SOC group (p<0.0001). Mean percentage of burn wound area excised in the NXB group was 4.4 ± 13.1% compared to 52.0 ± 41.4% in the SOC group (p<0.0001). None of the NXB-treated hands required escharotomy compared to 4 out of the 41 (9.7%) in the SOC group. NXB enzymatic debridement demonstrated a statistically significant reduction in burn wound excision and auto-grafting compared to SOC, and seems to prevent the need for emergency escharotomy. a DGD is produced by MediWound and distributed under the name NexoBrid®.

Le but était de réaliser une révision attentive des données et d'évaluer la place de Nexobrid (Nexobrid-NXB, précédemment Debrase Gel Dressing-DGD) dans l'indication particulière des brûlures profondes de la main. Une analyse détaillée des objectifs dans le traitement des brûlures de la main a été conduite en partie par une étude multicentrique, ouverte, randomisée, contrôlée avec 2 groupes pour évaluer la sécurité et l'efficacité de ce débridement enzymatique par rapport aux soins habituels (Standard of Care ou SOC). Ces résultats ont été comparés à une vaste cohorte de patients traités par NXB dans une étude précédente sur un seul groupe. 31 mains brûlées furent traitées par NXB et 41 dans le groupe SOC. Dans le groupe NXB, 4 sur 31 mains brûlées soit 12,9 % nécessitèrent une excision partielle, alors que 29 sur 41 dans le groupe SOC (70,7 %) (p < 0,0001). La moyenne des zones brûlées excisées dans le groupe NXB était de 4,4 (+ ou - 13,1 %) comparée aux 52,0 (+ ou - 41,4 % du groupe SOC) (p <0,0001). Aucune des mains traitées par NXB ont nécessité une excision totale, comparée à 4 sur 41 du groupe SOC (9,7 % du groupe). Le débridement enzymatique NXB montre une réduction statistiquement significative de l'indication d'excision avec autogreffe par rapport au groupe traité classiquement et semble prévenir la nécessité d'une escarrotomie en urgence.

Burden of disease and risk factors for death among children treated for tuberculosis in Malawi.

SETTING: Tuberculosis (TB) is a leading cause of childhood death. Patient-level data on pediatric TB in Malawi that can be used to guide programmatic interventions are limited. OBJECTIVE: To describe pediatric TB case burden, disease patterns, treatment outcomes, and risk factors for death and poor outcome. DESIGN: We conducted a retrospective cohort study utilizing routine data. Odds ratios (ORs) for factors associated with poor outcome and death were calculated using generalized estimating equations. RESULTS: Children represented 8% (371/4642) of TB diagnoses. The median age was 7 years (interquartile range 2.8-11); 32.8% (113/345) were human immunodeficiency virus (HIV) infected. Of these, 54.0% were on antiretroviral therapy (ART) at the time of anti-tuberculosis treatment (ATT) initiation, 21.2% started ART during ATT, and 24.8% had no documented ART. The treatment success rate was 77.3% (11.2% cured, 66.1% completed treatment), with 22.7% experiencing poor outcomes (9.5% died, 13.2% were lost to follow-up). Being on ART at the time of ATT initiation was associated with increased odds of death compared to beginning ART during treatment (adjusted OR 2.75, 95%CI 1.27-5.96). CONCLUSION: Children represent a small proportion of diagnosed TB cases and experience poor outcomes. Higher odds of death among children already on ART raises concerns over the management of these children. Further discussion of and research into pediatric-specific strategies is required to improve case finding and outcomes.

Association between parity and fistula location in women with obstetric fistula: a multivariate regression analysis.

OBJECTIVE: To compare primiparous and multiparous women who develop obstetric fistula (OF) and to assess predictors of fistula location. DESIGN: Cross-sectional study. SETTING: Fistula Care Centre at Bwaila Hospital, Lilongwe, Malawi. POPULATION: Women with OF who presented between September 2011 and July 2014 with a complete obstetric history were eligible for the study. METHODS: Women with OF were surveyed for their obstetric history. Women were classified as multiparous if prior vaginal or caesarean delivery was reported. The location of the fistula was determined at operation: OF involving the urethra, bladder neck, and midvagina were classified as low; OF involving the vaginal apex, cervix, uterus, and ureters were classified as high. MAIN OUTCOME MEASURES: Demographic information was compared between primiparous and multiparous women using chi-squared and Mann-Whitney U-tests. Multivariate logistic regression models were implemented to assess the relationship between variables of interest and fistula location. RESULTS: During the study period, 533 women presented for repair, of which 452 (84.8%) were included in the analysis. The majority (56.6%) were multiparous when the fistula formed. Multiparous women were more likely to have laboured <1 day (62.4 versus 44.5%, P < 0.001), delivered a live-born infant (26.8 versus 17.9%, P = 0.026), and have a high fistula location (37.5 versus 11.2%, P < 0.001). Multiparity [adjusted odds ratio (aOR) = 4.55, 95% confidence interval (CI) 2.27-9.12)] and history of caesarean delivery (aOR = 4.11, 95% CI 2.45-6.89) were associated with development of a high fistula. CONCLUSIONS: Multiparity was common in our cohort, and these women were more likely to have a high fistula. Additional research is needed to understand the aetiology of high fistula including potential iatrogenic causes. TWEETABLE ABSTRACT: Multiparity and caesarean delivery were associated with a high tract fistula in our Malawian cohort.

Environmental risk factors for oesophageal cancer in Malawi: A case-control study.

AIM: There is a high burden of oesophageal cancer in Malawi with dismal outcomes. It is not known whether environmental factors are associated with oesophageal cancer. Without knowing this critical information, prevention interventions are not possible. The purpose of this analysis was to explore environmental factors associated with oesophageal cancer in the Malawian context. METHODS: A hospital-based case-control study of the association between environmental risk factors and oesophageal cancer was conducted at Kamuzu Central Hospital in Lilongwe, Malawi and Queen Elizabeth Central Hospital in Blantyre, Malawi. Ninety-six persons with squamous cell carcinoma and 180 controls were enrolled and analyzed. These two groups were compared for a range of environmental risk factors, using logistic regression models. Unadjusted and adjusted odds ratios and 95% confidence intervals (CI) were calculated. RESULTS: Firewood cooking, cigarette smoking, and use of white maize flour all had strong associations with squamous cell carcinoma of the oesophagus, with adjusted odds ratios of 12.6 (95% CI: 4.2-37.7), 5.4 (95% CI: 2.0-15.2) and 6.6 (95% CI: 2.3-19.3), respectively. CONCLUSIONS: Several modifiable risk factors were found to be strongly associated with squamous cell carcinoma. Research is needed to confirm these associations and then determine how to intervene on these modifiable risk factors in the Malawian context.

Deleterious mutation in the FYB gene is associated with congenital autosomal recessive small-platelet thrombocytopenia.

BACKGROUND: The FYB gene encodes adhesion and degranulation-promoting adaptor protein (ADAP), a hematopoietic-specific protein involved in platelet activation, cell motility and proliferation, and integrin-mediated cell adhesion. No ADAP-related diseases have been described in humans, but ADAP-deficient mice have mild thrombocytopenia and increased rebleeding from tail wounds. PATIENTS AND METHODS: We studied a previously reported family of five children from two consanguineous sibships of Arab Christian descent affected with a novel autosomal recessive bleeding disorder with small-platelet thrombocytopenia. Homozygosity mapping and exome sequencing were used to identify the genetic lesion causing the disease phenotype on chromosome 5. Bone-marrow morphology and platelet function were analyzed. Platelets were characterized by scanning electron microscopy. RESULTS: We identified a homozygous deleterious nonsense mutation, c.393G>A, in FYB. A reduced percentage of mature megakaryocytes was found in the bone marrow. Patients' platelets showed increased basal expression of P-selectin and PAC-1, and reduced increments of activation markers after stimulation with ADP, as detected by flow cytometry; they also showed reduced pseudopodium formation and the presence of trapped platelets between the fibrin fibers after thrombin addition, as observed on scanning electron microscopy. CONCLUSIONS: This is the first report of a disease caused by an FYB defect in humans, manifested by remarkable small-platelet thrombocytopenia and a significant bleeding tendency. The described phenotype shows ADAP to be important for normal platelet production, morphologic changes, and function. It is suggested that mutation analysis of this gene be included in the diagnosis of inherited thrombocytopenia.

Normal isometric strength of rotatorcuff muscles in adults.

OBJECTIVES: The most prevalent disorders of the shoulder are related to the muscles of rotator cuff. In order to develop a mechanical method for the evaluation of the rotator cuff muscles, we created a database of isometric force generation by the rotator cuff muscles in normal adult population. We hypothesised the existence of variations according to age, gender and dominancy of limb. METHODS: A total of 400 healthy adult volunteers were tested, classified into groups of 50 men and women for each decade of life. Maximal isometric force was measured at standardised positions for supraspinatus, infraspinatus and subscapularis muscles in both shoulders in every person. Torque of the force was calculated and normalised to lean body mass. The profiles of mean torque-time curves for each age and gender group were compared. RESULTS: Our data showed that men gradually gained maximal strength in the fifth decade, and showed decreased strength in the sixth. In women the maximal strength was gained in the fourth decade with gradual decline to the sixth decade of life. The dominant arm was stronger in most of the tested groups. The torque profiles of the rotator cuff muscles in men at all ages were significantly higher than that in women. CONCLUSIONS: We found previously unrecognised variations of rotator cuff muscles' isometric strength according to age, gender and dominancy in a normal population. The presented data may serve as a basis for the future studies for identification of the abnormal patterns of muscle isometric strength in patients with pathology of the rotator cuff muscles. Cite this article: Bone Joint Res 2013;2:214-19.

Womens experiences of HIV testing and counselling in the labour ward: a case of Bwaila hospital.

INTRODUCTION: HIV counseling and testing during labour can be emotional, but is important because it allows mothers and babies to receive PMTCT prophylaxis if previous identification of HIV infection has not occurred. The study explores how HIV testing and counseling during early labour affects women. METHODOLOGY: This was a qualitative exploratory study to understand women's experiences during early labor. From September to October 2009, we conducted 10 indepth interviews with women who tested for HIV during early labour. We recruited women who tested > 3 months previously and those who had never tested for HIV from the postpartum ward of Bwaila Hospital. Data were analyzed manually using the life story approach in order to examine and analyse subjective experiences of women and their constructions of the social world. Transcripts were read multiple times to understand meanings which participants attached to their experiences. We coded data according to emerging themes and subthemes. RESULTS: Ten women 20-35 years were interviewed. Eight women had unknown HIV status while two had known HIV results but re-tested to update their status. Four women were found HIV-positive while 6 were HIV-negative. The primary theme was that women appreciated and accepted HIV testing and counseling. Testing was accepted as a necessary step to protect the infant from HIV infection. Counseling was viewed as helpful for acceptance of HIV status. One key subtheme was that HIV positive women experienced disappointment about their HIV diagnosis, though this was outweighed by the knowledge that one could protect her infant. All women viewed the short time to complete the counseling and testing procedures as favourable. CONCLUSION: Labour testing is acceptable and should be promoted to enhance PMTCT services by identifying HIV positive women with unknown status. Counseling helps women to accept being found with HIV and seek appropriate services.

MADYMO simulation of children in cycle accidents: a novel approach in risk assessment.

Head injuries are a significant cause of death and injury to child cyclists both on and off the road. Current evaluations of the effectiveness of cycle helmets rely on simplified mechanical testing or the analysis of aggregated accident statistics. This paper presents a direct evaluation of helmet efficacy by using computational modelling to simulate a range of realistic accident scenarios, including loss of control, collision with static objects and vehicle impact. A 6-year-old cyclist was modelled (as a Hybrid III 6-year-old dummy), in addition to a typical children's bicycle and a vehicle using the MADYMO dynamics software package. Simulations were performed using ranges of cyclist position, cycle speed and vehicle speed with and without a helmet that meets current standards. Wearing a cycle helmet was found to reduce the probability of head injuries, reducing the average probability of fatality over the scenarios studied from 40% to 0.3%. Similarly, helmet wearing reduced the probability of neck injuries (average probability of fatality reduced from 11% to 1%). There was no evidence that helmet wearing increased the severity of brain or neck injuries caused by rotational accelerations; in fact these were slightly reduced. Similarly, there was no evidence that increased cycling speed, such as might result from helmet related risk compensation, increased the probability of head injury.

Predictors of hemorrhage volume and disability after perimesencephalic subarachnoid hemorrhage.

OBJECTIVE: The determinants of subarachnoid hemorrhage (SAH) volume and an atypical pattern of blood are not clear. Our objective was to determine if reduced platelet activity on admission and abnormal venous drainage are associated with greater SAH volume. METHODS: We prospectively identified noncomatose patients with SAH without an identifiable aneurysm. We routinely measured platelet activity on admission and recorded aspirin use. SAH volumes were calculated with a validated technique. CT angiograms were reviewed by a certified neuroradiologist for venous drainage. Patients were followed for clinical outcomes through 3 months with the modified Rankin Scale (mRS). Data are Q1-Q3. RESULTS: There were 31 patients in the cohort. Thirty (97%) underwent an angiogram on admission, and 25 (81%) an additional delayed angiogram. SAH volume was lowest with normal venous drainage bilaterally (4.4 [3.7-16.4] mL) and higher with 1 (12.9 [3.7-20.4]) or 2 (20.9 [12.5-34.6] mL, p = 0.03) discontinuous venous drainages. Patients with reduced platelet activity had more SAH on the diagnostic CT (17.5 [10.6-20.9] vs 6.1 [2.3-15.3] mL) (p = 0.046). SAH volume was greater for patients requiring drainage for hydrocephalus (16.4 [11.5-20.5] vs 5.4 [2.7-16.4] mL) (p = 0.009). Outcomes at 3 months were generally excellent (median mRS = 0, no symptoms). CONCLUSIONS: Discontinuous venous drainage and reduced platelet activity were associated with increased SAH volume and hydrocephalus. These factors may explain thick SAH and reduce the need for repeated invasive imaging in such patients.

Extracorporeal human bone-like tissue generation.

OBJECTIVES: The need for bone tissue supplementation exists in a wide range of clinical conditions involving surgical reconstruction in limbs, the spine and skull. The bone supplementation materials currently used include autografts, allografts and inorganic matrix components; but these pose potentially serious side-effects. In particular the availability of the autografts is usually limited and their harvesting causes surgical morbidity. Therefore for the purpose of supplementation of autologous bone graft, we have developed a method for autologous extracorporeal bone generation. METHODS: Human osteoblast-like cells were seeded on porous granules of tricalcium phosphate and incubated in osteogenic media while exposed to mechanical stimulation by vibration in the infrasonic range of frequencies. The generated tissue was examined microscopically following haematoxylin eosin, trichrome and immunohistochemical staining. RESULTS: Following 14 days of incubation the generated tissue showed histological characteristics of bone-like material due to the characteristic eosinophilic staining, a positive staining for collagen trichrome and a positive specific staining for osteocalcin and collagen 1. Macroscopically, this tissue appeared in aggregates of between 0.5 cm and 2 cm. CONCLUSIONS: We present evidence that the interaction of the cellular, inorganic and mechanical components in vitro can rapidly generate three-dimensional bone-like tissue that might be used as an autologous bone graft.

A mutation in the ß3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIb ß3 to an active state.

BACKGROUND: The cytoplasmic tails of α(IIb) and ß(3) regulate essential α(IIb) ß(3) functions. We previously described a variant Glanzmann thrombasthenia mutation in the ß(3) cytoplasmic tail, IVS14: -3C>G, which causes a frameshift with an extension of ß(3) by 40 residues. OBJECTIVES: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α(IIb) ß(3) from a resting state to an active state. METHODS: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild-type (WT) α(IIb) as follows: ß(3) -742stop, a truncated mutant to evaluate the effect of deleted residues; ß(3) -749stop, a truncated mutant that preserves the NPLY conserved sequence; and ß(3) -749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. RESULTS AND CONCLUSIONS: Surface expression of α(IIb) ß(3) of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti-ligand-induced-binding-site 6 [antiLIBS6] or PT25-2). Activation of the α(IIb) ß(3) mutants was only achieved when both PT25-2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the ß(3) tail is important for maintaining α(IIb) ß(3) in a resting conformation.

Point mutations regarded as missense mutations cause splicing defects in the factor XI gene.

BACKGROUND: Point mutations within exons are frequently defined as missense mutations. In the factor (F)XI gene, three point mutations, c.616C>T in exon 7, c.1060G>A in exon 10 and c.1693G>A in exon 14 were reported as missense mutations P188S, G336R and E547K, respectively, according to their exonic positions. Surprisingly, expression of the three mutations in cells yielded substantially higher FXI antigen levels than was expected from the plasma of patients bearing these mutations. OBJECTIVES: To test the possibility that the three mutations, albeit their positions within exons, cause splicing defects. METHODS AND RESULTS: Platelet mRNA analysis of a heterozygous patient revealed that the c.1693A mutation caused aberrant splicing. Platelet mRNA of a second compound heterozygote for c.616T and c.1060A mutations was undetectable suggesting its degradation. Cells transfected with a c.616T minigene favored production of an aberrantly spliced mRNA that skips exon 7. Cells transfected with a mutated minigene spanning exons 8-10 exhibited a significant decrease in the amount of normally spliced mRNA. In silico analysis revealed that the three mutations are located within sequences of exonic splicing enhancers (ESEs) that bind special proteins and are potentially important for correct splicing. Compensatory mutations created near the natural mutations corrected the putative function of ESEs thereby restoring normal splicing of exons 7 and 10. CONCLUSIONS: The present findings define a new mechanism of mutations in F11 and underscore the need to perform expression studies and mRNA analysis of point mutations before stating that they are missense mutations.

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N-terminus of blade 1 of the ß-propeller (Asn2Asp) disrupts a calcium binding site in blade 6.

BACKGROUND: Studies of Glanzmann thrombasthenia (GT)-causing mutations has generated invaluable information on the formation and function of integrin αIIbß(3). OBJECTIVE: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. METHODS AND RESULTS: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the ß-propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbß(3) was found in patients' platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT ß(3). Although the αIIbß(3) was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbß(3) crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbß(3). Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbß(3) surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbß(3), αVß(3) harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVß(3) to calcium chelation compared with αIIbß(3). CONCLUSION: The new GT causing mutation highlights the importance of calcium binding domains in the ß-propeller for intracellular trafficking of αIIbß(3). The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.

The role of activin A in the human osteoblast cell cycle: a preliminary experimental in vitro study.

Animal studies have previously shown that activin A enhances osteoblast proliferation IN VITRO and increases bone formation and bone mechanical strength IN VIVO. For the further understanding of its action in human osteoblast, we studied the pattern of a cell cycle response to the treatment with activin A. We hypothesize that activin A alters the cell cycle pattern of human osteoblast. Primary cultures of human osteoblast-like cells were treated by activin A in a biologically effective concentration (100 ng/mL). The cells in cultured samples were counted, assayed for cellular alkaline phosphatase activity and calcitonin expression, LDH activity in the medium, cellular BrdU incorporation, cell cycle cytometry and compared to untreated controls. The treated by activin A cells responded by a significant shift toward the G1 phase of the cell cycle with parallel decrease in cell death rate (lower LDH activity and less necrotic cells in cytometric analysis). The treated cells also showed a lower alkaline phosphatase activity and calcitonin expression, indicating their undifferentiated state, and didn't change their proliferation rate. The number of cells in culture increased following treatment with activin A. We show that activin A increases the net osteoblast number in culture by reducing the cell death rate without affecting the cell proliferation. These findings should be part of cellular pathways that are involved in the initial stages of bone tissue generation.

Three residues at the interface of factor XI (FXI) monomers augment covalent dimerization of FXI.

BACKGROUND: Human plasma factor XI is a homodimer, with each monomer comprising a catalytic domain and four homologous 'apple' domains. The monomers bind to each other through non-covalent bonds and through a disulfide bond between Cys321 residues in apple 4 domains. OBJECTIVE: To identify residues essential for dimerization in the FXI monomer interface. METHODS: Specificity-determining residues in apple 4 domains were sought by sequence alignment of FXI and prekallikrein apple domains in different species. Specific residues identified in apple 4 domains were mutagenized and expressed in baby hamster kidney (BHK) cells for evaluation of their effect on FXI dimerization, analyzed by non-reduced sodium dodecylsulfate polyacrylamide gel electrophoresis and size-exclusion chromatography. RESULTS: Among the 19 residues of the FXI monomer interface, Leu284, Ile290 and Tyr329 were defined as specificity-determining residues. Substitutions of these residues or pairs of residues did not affect FXI synthesis and secretion from transfected BHK cells, but did impair dimerization, despite the presence of cysteine at position 321. The double mutant 284A/290A yielded predominantly a monomer, whereas all other single or double mutants yielded monomers as well as disulfide-bonded dimers. CONCLUSIONS: The data suggest that Leu284, Ile290 and Tyr329 in the interface of FXI monomers are essential for forming non-covalently bonded dimers that facilitate formation of a disulfide-bonded stable FXI dimer.