Assessment of light transmission aggregometry on the routine coagulation analyzer Sysmex CS-2500 using CE-marked agonists from Hyphen Biomed.

Light transmission aggregometry (LTA) is still considered as the "gold standard" for platelet function assessment but, as acompletely manual technology, it is labour intensive. This challenge can be overcome by performing platelet aggregometry in anautomated method on a routine coagulation analyzer. We aimed to compare and correlate results obtained from a traditional manual LTA solution realized in our Reference Center with an optimized automated system using CE-marked agonist reagents. Platelet rich plasma from patients with suspected platelet disorders, von Willebrand disease or antiplatelet therapy have been assessed using a wide range of agonist concentrations. Results were expressed as Maximal Platelet Aggregation and correlation was analyzed using the Passing and Bablok regression test. Platelet aggregometry studies were performed in 49 samples. Maximal aggregation response with ADP (0.5-10 µM), collagen (2 mg/µL), ristocetin (1.2 mg/mL) and arachidonic acid (1 mM) agonists showed significant correlation between the two aggregometers (p< .001). We observed a more variable response using lowconcentrations of ADP (≤5 µM). Moreover, we also noted discrepancies with the low dose of ristocetin, showing excessive paradoxical agglutination with the CS-2500, suggesting that a lower ristocetin dose should be used with this system. These data show that CS-2500 has the advantages of a walk-away technology and the use of CE-marked reagents also permit the possibility of an easier certification.

Arrhythmogenic response to isoproterenol testing vs. exercise testing in arrhythmogenic right ventricular cardiomyopathy patients.

Aims: To compare the arrhythmic response to isoproterenol and exercise testing in newly diagnosed arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. Methods and results: We studied isoproterenol [continuous infusion (45 µg/min) for 3 min] and exercise testing (workload increased by 30 W every 3 min) performed in consecutive newly diagnosed ARVC patients. Both tests were evaluated with regard to the incidence of (i) polymorphic premature ventricular contractions (PVCs) and couplet(s) or (ii) sustained or non-sustained ventricular tachycardia (VT) with left bundle branch block [excluding right ventricular outflow tract VT]; and compared to a control group referred for the evaluation of PVCs without structural heart disease. Thirty-seven ARVC patients (63.5% male, age 38 ± 16 years) were included. The maximal sinus rhythm heart rate achieved during isoproterenol testing was significantly lower compared to exercise testing (149 ± 17 bpm vs. 166 ± 19 bpm, P < 0.0001). However, the incidence of polymorphic ventricular arrhythmias was much higher during isoproterenol testing compared to exercise testing [33/37 (89.2%) vs. 16/37 (43.2%), P < 0.0001]. Interestingly, isoproterenol testing was arrhythmogenic in all 15 patients in whom baseline PVCs were reduced or suppressed during exercise testing. During both isoproterenol and exercise testing, control group presented a low incidence of ventricular arrhythmias compared to ARVC patients (8.1% vs. 89.2%, P < 0.0001 and 2.7% vs. 43.2%, P < 0.0001, respectively). Conclusions: The incidence of polymorphic ventricular arrhythmias is significantly higher during isoproterenol compared to exercise testing in newly diagnosed ARVC patients, suggesting its potential utility for the diagnosis.

An adenine insertion in exon 6 of human GP6 generates a truncated protein associated with a bleeding disorder in four Chilean families.

BACKGROUND: Glycoprotein VI (GPVI), 60-65 kDa, is a major collagen receptor on platelet membranes involved in adhesive and signaling responses. Mice lacking GPVI have impaired platelet response to collagen and defective primary adhesion and subsequent thrombus formation. Complete or partial deficiency of GPVI in humans is a rare condition presenting as a mild bleeding disorder. The defect in most of the reported patients is acquired and associated with other diseases. To date, only two patients have been characterized at the molecular level who carry different compound heterozygous mutations in the GP6 gene. OBJECTIVE: To report four unrelated patients from non-consanguineous families who presented with mucocutaneous bleeding. They had absent platelet aggregation and (14) C-5-HT secretion with collagen, convulxin and collagen-related peptide. RESULTS: Flow cytometry and immunofluorescence-confocal microscopy showed an absence of GPVI in non-permeabilized platelets. All the patients had an adenine insertion in exon 6 (c.711_712insA), changing the reading frame and generating a premature 'stop codon' in site 242 of the protein. The mutation predicts the synthesis of the truncated protein before the trans-membrane domain, corresponding to a band of ≈49 kDa observed in western blots and in permeabilized platelets by immunofluorescence. Platelet mRNA from all the patients was sequenced and contained the corresponding adenine insertion. Heterozygous relatives had no pathological bleeding, normal response to collagen and convulxin and intermediate membrane expression of GPVI. CONCLUSIONS: The identification of four unrelated homozygous patients with an identical defect suggests that inherited GPVI deficiency is more frequent than previously suspected, at least in Chile.

Ankle brachial index using an automatic blood pressure device in occupational medicine: relevance in routine examination and comparison with Framingham cardio-vascular risk score.

AIM: The aim of this study was to assess the feasibility and relevance of determining ankle brachial index (ABI) using an automatic blood pressure device in subjects seen for their annual routine examination by occupational physicians and to compare the obtained ABI with the Framingham score. PATIENTS AND METHODS: Sixteen physicians randomly recruited 634 subjects in 12 departments of occupational medicine. Subjects aged between 40 and 60 years underwent a determination of ABI using an OMRON HM 722 device and the analysis of Framingham score. Other analysed variables were: sex, age, smoking habit, hypertension, diabetes, hypercholesterolemia, glycaemia, total cholesterol, HDL and LDL cholesterol and triglycerides levels. RESULTS: Mean age of the population studied was 48.1 ± 6.0 years; 73% were men, 36% were smokers, 14% had hypertension, 3.3% diabetes and 22% hypercholesterolemia. Biochemical values were glycaemia 0.90 ± 0.30 g/l, total cholesterol 2.10 ± 0.4 g/l, HDL cholesterol level 0.50 ± 0.20 g/l, LDL cholesterol level 1.30 ± 0.40 g/l, and triglycerides 1.40 ± 1.0 g/l. Mean ABI were 1.1 ± 0.1 in both legs. Mean Framingham score was 8.2 ± 5.4%. Only 20 subjects (3%) had an ABI < 0.90. No relation was found between pathological ABI and Framingham score (abnormal ABI : 9.9 ± 5.5 vs. normal ABI : 8.2 ± 5.4, NS). CONCLUSION: The determination of ABI using a simple commercially available automatic blood pressure device is feasible and easy to implement by preventive or general physicians in all kinds of routine examinations. In our opinion automatic ABI very easy and quick to determine provides, in addition to Framingham score, a simple and useful tool to detect subjects at high cardio-vascular risk.

Natural history of platelet antibody formation against αIIbß3 in a French cohort of Glanzmann thrombasthenia patients.

Treatment of the bleeding syndrome in Glanzmann thrombasthenia (GT) is often complicated by naturally occurring isoantibodies directed against the αIIbß3 integrin that cause the removal of or render ineffective transfused donor platelets. Such antibodies are produced after transfusion or pregnancy when the patient's immune system comes into contact with normal platelets. Despite many reports of anti-αIIbß3 antibodies in GT patients, there is no consensus pertaining to their frequency, their long-term evolution in the circulation, or their formation in relation to either (i) the extent of the αIIbß3 deficiency in the patient's platelets or (ii) the nature of the genetic defect (ITGA2B or ITGB3 genes). Antibody screening was performed on a large series of 24 GT patients in South-West France dividing the patients into two cohorts: (i) 16 patients with the French gypsy mutation (c.1544 + 1G>A) within ITGA2B that gives platelets totally lacking αIIbß3 and (ii) 8 patients carrying other defects of ITGA2B or ITGB3 with different expression levels of αIIbß3. Our results confirm that patients with premature termination mutations resulting in platelets lacking αIIbß3 are the most susceptible to form isoantibodies, a finding that may be useful in deciding the choice of therapy between platelet transfusion and the use of recombinant factor VIIa (FVIIa).

An acquired inhibitor to the GPVI platelet collagen receptor in a patient with lupus nephritis.

BACKGROUND: GPVI is a major platelet collagen signaling receptor. In rare cases of immune thrombocytopenic purpura (ITP), autoantibodies to GPVI result in receptor shedding. OBJECTIVES: To investigate a possible pathogenic role of plasma anti-GPVI antibody located in a woman with lupus nephritis. METHODS: Measured were (i) platelet aggregation to collagen and convulxin, (ii) platelet GPVI expression (flow cytometry and western blotting), (iii) plasma soluble GPVI (sGPVI, dual antibody ELISA), and (iv) plasma anti-GPVI antibody (ELISA using recombinant sGPVI). RESULTS: In 2006 and early 2007, the patient had a normal platelet count but a virtual absence of platelet aggregation to collagen and convulxin. Her platelets responded normally to other agonists including cross-linking ITAM-dependent FcgammaRIIA by monoclonal antibody, IV.3. Flow cytometry and western blotting showed a platelet deficiency of GPVI. Plasma sGPVI levels were undetectable whereas ELISA confirmed the presence of anti-GPVI antibody. Sequencing revealed a normal GPVI cDNA structure. The patient's plasma and the isolated IgG3 fraction activated and induced GPVI shedding from normal platelets. A deteriorating clinical condition led to increasingly strict immunosuppressive therapy. This was globally associated with a fall in plasma anti-GPVI titres, the restoration of platelet GPVI and the convulxin response, and the loss of her nephrotic syndrome. CONCLUSIONS: Our results show that this patient acquired a potent anti-GPVI IgG3 antibody with loss of GPVI and collagen-related platelet function. Further studies are required to determine whether anti-GPVI antibodies occur in other lupus patients with nephritis.

Short- and long-term risk factors for sudden death in patients with stable angina.

Sudden death is most common and often the first manifestation of coronary heart disease although its risk is difficult to predict. It has been studied mainly in patients with severe ventricular arrhythmia or recent myocardial infarction, but little is known about the different risk factors for short- and long-term risk of sudden death in patients with stable angina. To assess risk factors for sudden death in patients with stable angina and angiographically proven coronary artery disease, 319 consecutive patients were recruited prospectively and followed-up. Patients with clinical heart failure or recent myocardial infarction were excluded. Clinical, angiographic and biological variables were recorded. The association between each variable and the risk of sudden death was assessed in univariate and logistic multivariate analysis. There were 25 sudden deaths during the follow-up period (97+/-29 months). The univariate predictors in the short-term (2 years) were: peripheral arterial disease, left ventricular hypertrophy, low density lipoprotein cholesterol and ejection fraction. The independent predictors were: peripheral arterial disease (relative risk: 6.3), ejection fraction (relative risk 1.05) and low density lipoprotein (relative risk: 1.8). In the long-term (8-10 years), body mass index, coronary score, ejection fraction and fibrinogen were univariate predictors. Only body mass index (relative risk: 1. 2), ejection fraction (relative risk: 1.06) and fibrinogen (relative risk: 2) remained independent predictors. The risk factors for sudden death in stable angina were time-dependent, peripheral arterial disease appeared as the best predictor with LDL for short time, and body mass index (obesity: index >27) and fibrinogen for long time. Ejection fraction was the only time-independent predictor.

Detection of coronary stenoses by stress echocardiography using a previously implanted pacemaker for ventricular pacing: preliminary report of a new method.

BACKGROUND: The number of patients with pacemakers has been increasing and a large number of them will present with chest pain or symptoms suggesting angina pectoris. Myocardial ischemia and presence of coronary artery disease are difficult to detect and assess by noninvasive methods in patients with a pacemaker; the electrocardiogram (ECG) at rest and during exercise is usually very difficult to analyze in terms of ischemia or even presence of an acute myocardial infarction. HYPOTHESIS: To detect significant coronary stenosis in patients with previously implanted pacemakers, we tested a new stress echocardiography method using incremental ventricular pacing by already implanted pacemakers. METHODS: We studied prospectively 25 consecutive patients who underwent stress echocardiography with increasing ventricular pacing up to either 85% of the age-predicted maximal heart rate or chest pain. Positive tests were defined by new hypokinesia or worsening of a preexisting alteration in wall motion in at least two adjacent territories. All patients underwent coronary angiograms to define the presence and severity of coronary stenoses. RESULTS: Among the 25 tests, 11 (44%) were stopped for chest pain. 1 (4%) for moderate discomfort, 1 (4%) for a drop in blood pressure, and the target pacing rate was achieved in the tests of the remaining 12 patients (48%). There were no complications. Thirteen patients had significant stenoses. In 10 cases, stress echocardiography was a true positive test with respect to coronary angiography. There were 11 true negative, 1 false positive, and 3 false negative tests. The sensitivity was 77%, specificity was 90%, the positive predictive value was 91%, and the negative predictive value 79%. The accuracy was 84%. CONCLUSIONS: This new stress echocardiography method appears feasible, easy, safe, and effective for detection of significant coronary stenoses in patients with pacemakers.

In vivo phosphorylation of the purine/cytosine permease from the plasma membrane of the yeast Saccharomyces cerevisiae.

The purine/cytosine permease, encoded by the FCY2 gene, is a carrier located in the plasma membrane of the yeast Saccharomyces cerevisiae. Polyclonal antibodies were raised against two peptides that corresponded to the sub-N-terminal and C-terminal sequences of the putative protein deduced from the FCY2 gene. Immunoprecipitation experiments performed with protein extracts labelled in vivo with 35S showed that purine/cytosine permease is specifically detected as a broad and diffuse band. The apparent molecular mass of this protein was 45-50 kDa. By means of in vivo pulse/chase 35S-labelling experiments, we observed a slight increase in the apparent molecular mass of purine/cytosine permease during the chase. This shift in electrophoretic mobility of the protein suggested a post-translational modification. This molecular mass increase was eliminated by alkaline phosphatase treatment of the immunoprecipitate, which strongly suggested phosphorylation of the carrier. This proposal was confirmed by in vivo [32P]P(i) labelling and immunoprecipitation of purine/cytosine permease with purified anti-(sub-N-terminal peptide) IgG or anti-(C-terminal peptide) IgG. Phosphoamino acid analysis indicated that phosphorylation occurred on seryl residues of purine/cytosine permease. By means of thermosensitive secretory-pathway-mutant strains, we demonstrated that purine/cytosine permease phosphorylation occurred either between the Golgi apparatus and the plasma membrane or in the plasma membrane itself.

Nucleotide receptor P2u partially mediates ATP-induced cell cycle progression of aortic smooth muscle cells.

mRNA of the P2u purinoceptor (or nucleotide receptor) is detected both by polymerase chain reaction or Northern blot analyses in cultured aortic smooth muscle cells. When added to the culture medium of these cells, UTP, a specific ligand of the P2u receptor, induces an increased expression of both immediate-early and delayed-early cell cycle-dependent genes. This induction demonstrates similar features (kinetics, concentration dependence) to those obtained after stimulation of aortic smooth cells by exogenous ATP, a common ligand for most P2 purinoceptors. In contrast, 2-methylthioATP, a preferential ligand for P2y purinoceptors, induces only a significant increase of immediate-early genes but not of delayed-early genes. Moreover, the 2-methylthioATP-induced responses (c-fos mRNA increase, free intracellular calcium transient) are lower than those induced by ATP or UTP and are complementary to those of UTP. These results demonstrate that functional P2u receptors are present on cultured aortic smooth muscle cells and suggest that the bulk of responses induced by extracellular ATP on cell cycle progression are mediated via P2u purinoceptors, a hypothesis confirmed by cytofluorometric studies. Since some ATP- or UTP-induced genes code for chemotactic proteins (monocyte chemoattractant protein-1 and osteopontin), this study suggests that these nucleotides may contribute to vascular or blood cell migration and proliferation and consequently to the genesis of arterial diseases.