Nicorandil associated anal ulcers: an estimate of incidence.

INTRODUCTION: Nicorandil is a commonly prescribed antianginal medication that has been found to be associated with painful anal ulceration. The incidence of this complication is unknown. We have used the best data available to us to make an estimate of this figure in a health district with a remarkably stable population of approximately 200,000 people. METHODS: using an electronic search of all letters generated from colorectal and gastroenterology clinics as well as endoscopy reports from January 2004 to November 2010, patients with anal ulceration who were taking nicorandil were identified. Other causes of ulceration were excluded by biopsy in the majority of cases. The central hospital and community pharmacy database was interrogated to estimate the number of patients who were prescribed nicorandil over a six-year period (2004-2010). RESULTS: A total of 30 patients (24 men, 6 women) with a median age of 79.5 years were identified who fulfilled the criteria of: taking nicorandil; having no other identified cause for anal ulceration; and achieving eventual healing after withdrawal of nicorandil. In the six-year period an estimated mean of 1,379 patients were prescribed nicorandil each year. The mean annual incidence of anal ulcers among nicorandil users is therefore calculated to be in the region of 0.37%. CONCLUSIONS: Anal ulceration appears to occur in approximately four in every thousand patients prescribed nicorandil each year. Prescribing physicians should explain the risk of this unpleasant complication to their patients.

Anaesthetic considerations for a child with combined Prader-Willi syndrome and mitochondrial myopathy.

We report the anaesthetic management of a child with Prader-Willi syndrome and mitochondrial myopathy for open heart surgery. We used ketamine, fentanyl, rocuronium and caudal morphine together with a propofol infusion with no untoward effects. The implications of both conditions for anaesthesia are discussed.

Reversible shear-mediated platelet dysfunction during cardiac surgery as assessed by the PFA-100 platelet function analyzer.

We undertook this investigation to assess alterations in shear-mediated platelet function during cardiac surgery and to determine the potential for the PFA-100 to predict post-operative bleeding. Platelet aggregation and PFA-100 closure times were determined in 18 adult patients at five intervals during cardiac surgery. Associations between post-operative bleeding and closure times were examined in an additional 58 patients. Statistical analysis consisted of Student's t, Wilcoxon signed rank, and Spearman correlation tests. All results are reported as mean +/- SEM. Collagen/epinephrine closure times were prolonged prior to and throughout surgery. Collagen/adenosine-5'-diphosphate (ADP) closure times were significantly prolonged by heparin administration, 141 +/- 15 s versus 115 +/- 10 s (P = 0.01), and subsequent initiation of cardiopulmonary bypass (CPB), 203 +/- 12 s (P= 0.0001); however, 15 min after protamine administration, closure times returned to near pre-operative values, 138 +/- 12 s (P = not significant). In contrast, platelet aggregation in response to ADP remained impaired in 17 of 19 patients after CPB. Neither ex vivo correction of sample hematocrits nor supplementation with Humate P affected closure times. Positive and negative predictive values for post-CPB collagen/ADP closure times to predict bleeding were 18 and 96%, respectively. These results suggest that factors both intrinsic and extrinsic to the platelet contribute to reversible shear-mediated platelet dysfunction during CPB, and that the PFA-100 may prove useful after CPB to identify patients unlikely to benefit from platelet transfusions.

Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase.

Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.

Hemostatic effects of antithrombin III supplementation during cardiac surgery: results of a prospective randomized investigation.

Failure to suppress thrombin generation during cardiac surgery promotes fibrin generation, fibrinolysis, and a consumptive coagulopathy. Acquired deficiencies of antithrombin III may play a contributory role. We hypothesized that antithrombin III supplementation to normal physiologic concentrations would decrease thrombin generation and potentially reduce peri-operative bleeding. Twenty patients undergoing coronary artery bypass graft surgery were randomized for this prospective, double-blind, placebo-controlled study. Ten patients received antithrombin III supplementation (50 U/kg) by intravenous infusion prior to incision, and 10 patients received a placebo. Blood samples were obtained pre-operatively, at 1 and 2 h following initiation of cardiopulmonary bypass (CPB), and at 1, 3, and 24 h after completion of CPB. Samples were analyzed for antithrombin III, thrombin-antithrombin III (TAT) complex, and D-dimer concentrations. Cumulative blood loss was recorded at 6 and 12 h after CPB. No statistically significant differences in patient demographics or total heparin dose administered were observed between groups. As expected, plasma antithrombin III concentrations were maintained near pre-operative values in the treatment group, but not in the placebo group. Despite this difference, no statistically significant alterations in generation of TAT complex, D-dimer, or blood loss occurred between groups. Antithrombin III supplementation to maintain normal physiologic concentrations during CPB did not alter significantly thrombin generation, fibrinolytic activity, or blood loss in adults undergoing elective cardiac surgery.

Thromboelastography as a perioperative measure of anticoagulation resulting from low molecular weight heparin: a comparison with anti-Xa concentrations.

UNLABELLED: Low molecular weight heparin (LMWH) is commonly used to prevent postoperative thromboembolism. Currently, there is no convenient test to measure the degree of anticoagulation from LMWH. This prospective study examines the relationship of thromboelastography and serum anti-Xa concentration in patients treated with enoxaparin. Twenty-four adult patients scheduled for orthopedic surgery using epidural anesthesia were enrolled. Epidural catheters were removed the morning after surgery before the commencement of subcutaneous enoxaparin 30 mg twice daily. Venous blood samples were obtained at 1) the induction of anesthesia (baseline), 2) immediately before the third dose of enoxaparin postoperatively (Day 2-trough), 3) 4 h after the third dose postoperatively (Day 2-peak), and 4) immediately before the fifth dose postoperatively (Day 3-trough). Whole blood samples were obtained for thromboelastography, activated clotting time, and anti-Xa level analyses at each of the four time intervals. At the four sample intervals, the r time (mean +/- SEM). (20 +/- 1, 25 +/- 2, 51 +/- 6, 31 +/- 3 mm) and the k time (9 +/- 0. 7, 12 +/- 1, 27 +/- 5, 14 +/- 2 mm) of the thromboelastograph were significantly correlated with the expected peak and trough levels of LMWH and serum anti-Xa levels (P: < 0.05). At the Day 3-trough, thromboelastograph r times exceeded the normal range in 6 of 25 patients (25%). Prolongation of r time and k time on postoperative Day 3 may indicate an exaggerated response to LMWH. Thromboelastography is a test that could potentially correlate with the degree of anticoagulation produced by low molecular weight heparin. IMPLICATIONS: Thromboelastography is a test that could potentially correlate with the degree of anticoagulation produced by low molecular weight heparin. The r time from the thromboelastogram correlates with serum anti-Xa concentration.

Myocardial ischemia correlates with reduced fibrinolytic activity following peripheral vascular surgery.

STUDY OBJECTIVES: To evaluate the relationship between perioperative ischemia and serial concentrations of D-dimer, which is a sensitive and specific marker of fibrinolytic activity. Myocardial ischemia and infarction are well-recognized complications of peripheral vascular surgery. We hypothesized that patients at increased risk of perioperative myocardial ischemia might be identified preoperatively by abnormal hemostatic indices. DESIGN: Prospective clinical outcomes study. SETTING: A 1,124-bed tertiary care medical center. PATIENTS: 42 ASA physical status II, III, and IV patients undergoing peripheral vascular surgery. INTERVENTIONS: Serial D-dimer concentrations were measured preoperatively, and at 24 and 72 hours postoperatively. Continuous 12-lead ST-segment monitoring (Mortara Instrument, Inc., Milwaukee, WI) was performed with the acquisition of a 12-lead ECG every 20 seconds for 72 hours. MEASUREMENTS AND MAIN RESULTS: D-dimer measurements were performed in duplicate using the Dimer Gold assay (American Diagnostica, Greenwich CT). Ischemic episodes, as defined by continuous 12-lead ST-segment monitoring, occurred in 49% of patients. There were no demographic differences between ischemic and nonischemic groups. Although baseline D-dimer concentrations were not statistically significantly different between groups, patients experiencing perioperative myocardial ischemia generated significantly less D-dimer during the perioperative period (p = 0. 014). CONCLUSIONS: PATIENTS with an impaired fibrinolytic response, as defined by reduced generation of D-dimer, experienced an increased incidence of perioperative myocardial ischemia.

Site-directed mutagenesis of the calcium-binding site of blood coagulation factor XIIIa.

Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp(438), Ala(457), Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The K(act) values for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the K(act) values were not changed by mutagenesis. However, Asp(476) and Asp(479) are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu(485) and Glu(490) are Ca(2+)-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp(476) and Asp(479) play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.

Acute inactivation of tau has no effect on dynamics of microtubules in growing axons of cultured sympathetic neurons.

Tau is a developmentally regulated microtubule (MT)-associated protein in neurons that has been implicated in neuronal morphogenesis. On the basis of test tube studies, tau has been proposed to function in axon growth by stabilizing MTs and thereby promoting MT assembly. We have tested this hypothesis by examining the effects of acute inactivation of tau on axonal MTs. Tau was inactivated by microinjecting purified antibodies against recombinant tau into neurons before they extended axons. The injected antibodies quantitatively precipitated tau into aggregates in the soma. With these conditions the neurons elaborate normal-appearing axons, and MTs extend throughout the axons and into the growth cones, but the axons and their MTs are depleted of tau. The immunodepletion of tau had no detectable effect on several parameters of the dynamics of axonal MTs. Depletion of tau also was not accompanied by a reorganization of other major MT-associated proteins or actin filaments in these neurons. Thus, neurons effectively depleted of tau can extend axons that resemble those of control cells, and the axons contain normal-appearing MT arrays with normal dynamic behavior. These observations are exactly the opposite of those expected on the basis of the hypothesis that the stability of axonal MTs is a direct function of their content of tau, indicating that tau in growing axons of cultured sympathetic neurons is not specialized to promote microtubule assembly and stability.

Standardization of prothrombin fragment 1.2 measurement: effects of preanalytical variables and calibrator selection.

BACKGROUND: Immunoassays for prothrombin fragment 1.2 (F1.2) provide a specific measure of thrombin generation and offer potential value in detecting activation of the coagulation system and monitoring anticoagulant therapy. To standardize laboratory measurements of this analyte, it is important to define factors affecting interassay variability. OBJECTIVE: To determine the potential for standardization of F1.2 measurement by examining the effects of preanalytical variables and calibrator selection on F1.2 measurement. MATERIALS AND METHODS: Using three commercially available immunoassays, interassay and intra-assay correlations for F1.2 were determined using blood samples collected into heparin, citrate, and a solution of ethylenediaminetetraacetic acid, aprotinin, and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone. In a cohort of patients, interassay correlations for F1.2 were determined using blood collected from an arterial catheter. Dose-response curves were generated for each manufacturer-supplied calibrator set by substitution into each of the previously untested competing immunoassays. RESULTS: F1.2 immunoassays with the same recommended specimen anticoagulant displayed stronger correlation than assays requiring different anticoagulants. Furthermore, a stronger interassay correlation was elicited by samples collected through an intra-arterial catheter as opposed to venipuncture. F1.2 calibrator sets differed quantitatively, with buffer-related matrix effects contributing to interassay variability. CONCLUSION: Analytical standardization of F1.2 immunoassays is possible when a common anticoagulant, blood collection method, and calibrator set are used.

Regulation of human tissue transglutaminase function by magnesium-nucleotide complexes. Identification of distinct binding sites for Mg-GTP and Mg-ATP.

Tissue transglutaminase (tTG) catalyzes a Ca(2+)-dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (Km = 38 +/- 10 microM) at a 3-fold greater steady-state affinity than with Mg-GTP (Km = 130 +/- 35 microM). In addition, Mg-ATP inhibited GTP hydrolysis (IC50 = 24 microM), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC50 values of 9, 9, and 400 microM, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.