Elena M. Faioni

Identification of differentially expressed genes in coronary atherosclerotic plaques from patients with stable or unstable angina by cDNA array analysis.

Summary.  The composition of atherosclerotic plaques is a crucial factor in determining rupture, thrombosis and clinical events. In this study, we analyzed gene expression in coronary plaques from patients with stable or unstable angina using gene arrays. Total RNA was extracted from eight plaques collected by therapeutic directional coronary atherectomy. cDNA probes, generated by amplification, were hybridized to nylon arrays containing 482 genes. Here we report the results for the inflammation, adhesion and hemostasis subsets. Many genes not previously associated with atherosclerosis, such as the lymphocyte adhesion molecule MadCAM, were expressed in the plaques. anova analysis showed higher tissue factor (TF) expression in unstable angina samples. Five genes were expressed at lower levels in unstable angina samples: anticoagulant protein S, cyclooxygenase (COX)‐1, interleukin (IL)‐7 and chemokines monocyte chemotactic protein (MCP)‐1 and ‐2. Gene arrays provide a new approach to study plaque composition and identify candidate markers of plaque instability.

Why does ticagrelor induce dyspnea

In studies that compared the reversible P2Y12 inhibitor ticagrelor with the irreversible inhibitor clopidogrel, dyspnea was observed more frequently among ticagrelor-treated patients than among clopidogrel-treated patients. Because dyspnea was not associated with acidosis, pulmonary or cardiac dysfunction, alterations in the mechanisms and pathways of the sensation of dyspnea may be involved in its pathogenesis. It has been hypothesised that the sensation of dyspnea in ticagrelor-treated patients is triggered by adenosine, because ticagrelor inhibits its clearance, thereby increasing its concentration in the circulation. However, dipyridamole, a much stronger inhibitor of adenosine clearance than ticagrelor, usually does not cause dyspnea. We hypothesise that inhibition of P2Y12 on sensory neurons increases the sensation of dyspnea, particularly when reversible inhibitors are used. We base our hypothesis on the following considerations: 1) cangrelor and elinogrel, which, like ticagrelor, are reversible P2Y12 inhibitors, also increase the incidence of dyspnea; 2) it is biologically plausible that inhibition of P2Y12 on sensory neurons increases the sensation of dyspnea; 3) inhibition of P2Y12 on platelets (which do not have a nucleus) by clopidogrel is permanent, despite the once daily administration and the short plasma half-life of the inhibitor; 4) in contrast, inhibition of P2Y12 on neurons by clopidogrel may be temporary and transient, because neurons have a nucleus and can therefore rapidly replace the inhibited receptors with newly synthetised ones; 5) inhibition of P2Y12 on neurons by reversible inhibitors is permanent, because the plasma drug concentration is maintained high by repeated dosing, in order to ensure permanent inhibition of platelet P2Y12.

Expression of endothelial protein C receptor and thrombomodulin in the intestinal tissue of patients with inflammatory bowel disease

Objective:Inflammatory bowel diseases are characterized by disorders of immunity, thrombosis of large vessels, and microthrombosis of mucosal vessels. The expression of endothelial protein C receptor (EPCR) and thrombomodulin—two receptors of the protein C pathway involved in thrombin scavenging and inflammation—was studied in intestinal resection specimens or mucosal biopsies from patients with inflammatory bowel disease and from controls. The soluble forms of the receptors in plasma were measured. Data Source:This study involved patients from two large university hospitals. After surgery or biopsy, tissue samples were either frozen or fixed in formalin and embedded in paraffin. Sections for immunohistochemistry examination were cut and tested with the specific antibodies to EPCR and thrombomodulin. RNA was extracted from frozen tissue for amplification via reverse-transcriptase polymerase chain reaction. Normal intestinal and diverticulitis tissue was used as a control. Resection samples from 36 patients with ulcerative colitis, 38 with Crohn’s disease, 38 with colonic cancer, and 32 with diverticulitis were studied by immunohistochemistry, and frozen sections from the same patients were studied by immunofluorescence. Twelve biopsy specimens of adjacent intestinal areas from six patients with inflammatory bowel disease were included in the study for reverse-transcriptase polymerase chain reaction. Soluble receptors were measured in the plasma of 52 inflammatory bowel disease patients and 52 controls. Data Summary:EPCR and thrombomodulin were expressed on the mucosal endothelium of controls, and the intensity of the signal decreased in inflammatory bowel disease patients. EPCR was expressed by dendritic-like cells in controls, which also stained positive for CD21. The EPCR+/CD21+ dendritic-like cells were not as commonly observed in sections from ulcerative colitis patients as they were in sections from control patients (12.0 ± 3.6 cells per high-power field vs. 23.8 ± 10.4 cells per high-power field, p = .03), and this decrease was less evident in sections from Crohn’s disease patients. Levels of messenger RNA for EPCR paralleled protein expression. Soluble thrombomodulin and EPCR levels were both higher in patients than in controls: 41.5 vs. 26.0 ng/mL (p <.0001) and 141 vs. 130 ng/mL (p <.05), respectively. Conclusions:EPCR expression on dendritic-like cells that bear the key complement receptor CD21 suggests a role for EPCR in innate immunity. The reduced expression of thrombomodulin and EPCR in the mucosal vessels in inflammatory bowel disease impairs protein C activation, favoring microthrombosis.

Screening for thrombophilia and antithrombotic prophylaxis in pregnancy: Guidelines of the Italian Society for Haemostasis and Thrombosis (SISET)

The term thrombophilia describes an increased tendency to develop thrombosis and many laboratory markers with different strengths of association with thrombosis have been identified. The main causes of maternal mortality and morbidity in developed countries is venous thromboembolism (VTE) and obstetric complications. During pregnancy and puerperium the risk for VTE increases due to hemostatic imbalance towards a prothrombotic state, and it is further increased in women carriers of thrombophilia; recent studies have also demonstrated an association between thrombophilia and obstetric complications. These complications are, therefore, considered potentially preventable with the prophylactic administration of anticoagulant drugs, although their efficacy is not proven by data from randomized controlled trials. After a systematic comprehensive literature review and using a rigorous methodology, the expert panel formulated recommendations regarding the usefulness of screening for thrombophilia in pregnancy to identify high-risk women and for the management of antithrombotic prophyalxis. When evidence is lacking, consensus-based recommendations are provided.

Mutations in the thrombomodulin and endothelial protein C receptor genes in women with late fetal loss

Late fetal loss can be associated with placental insufficiency and coagulation defects. Thrombomodulin (TM) and the endothelial protein C receptor (EPCR) are glycoprotein receptors expressed mainly on the endothelial surface of blood vessels and also in the placenta; they both play a key physiological role in the protein C anticoagulant pathway. Defects in these proteins might play an important role in the pathogenesis of late fetal loss. We performed a case–control study in 95 women with unexplained late fetal loss (> 20 weeks), to elucidate whether TM or EPCR gene mutations were associated with an increased risk for this complication of pregnancy. The control group comprised 236 women who gave birth to at least one healthy baby and had no history of late fetal death or obstetrical complications. The entire TM and EPCR genes, including the promoter region, were screened. In total, five mutations were identified in the TM gene in 95 patients and three in 236 control subjects, and two mutations were identified in the EPCR gene in 95 patients and one in 236 control subjects. The relative risk for late fetal loss when having a mutation in the TM or EPCR gene was estimated by an odds ratio of 4·0 (95% CI 1·1–14·9). In conclusion, identified mutations in the TM and EPCR genes of women with unexplained fetal loss are more prevalent compared with women with no obstetrical complications.

Determination of vitamin K1 in plasma by solid phase extraction and HPLC with fluorescence detection.

We describe a procedure for quantification of vitamin K(1) in human plasma by HPLC. Samples, enriched with a vitamin K derivative as internal standard, were deproteinized, purified on polymeric RP-SPE cartridges and injected into HPLC equipped with a post-column on-line zinc metal reactor and a fluorometric detector. Median level in blood donors (n=87) was 1.967 nmol/L (0.93-4.01, 5th-95th percentiles), with a significant correlation between plasma levels and age (r=0.276, p=0.00958) and a lower (not significant) value in women than in men. This method, easy-to-handle and with a high throughput, can be used to identify covert states of vitamin K intake deficiency in patients thus at risk of alterations in blood clotting or bone mineralization.

ProCMD: a database and 3D web resource for protein C mutants

BackgroundActivated Protein C (ProC) is an anticoagulant plasma serine protease which also plays an important role in controlling inflammation and cell proliferation. Several mutations of the gene are associated with phenotypic functional deficiency of protein C, and with the risk of developing venous thrombosis. Structure prediction and computational analysis of the mutants have proven to be a valuable aid in understanding the molecular aspects of clinical thrombophilia.ResultsWe have built a specialized relational database and a search tool for natural mutants of protein C. It contains 195 entries that include 182 missense and 13 stop mutations. A menu driven search engine allows the user to retrieve stored information for each variant, that include genetic as well as structural data and a multiple alignment highlighting the substituted position. Molecular models of variants can be visualized with interactive tools; PDB coordinates of the models are also available for further analysis. Furthermore, an automatic modelling interface allows the user to generate multiple alignments and 3D models of new variants.ConclusionProCMD is an up-to-date interactive mutant database that integrates phenotypical descriptions with functional and structural data obtained by computational approaches. It will be useful in the research and clinical fields to help elucidate the chain of events leading from a molecular defect to the related disease. It is available for academics at the URL http://www.itb.cnr.it/procmd/.

Reduced free protein S levels in patients with inflammatory bowel disease: prevalence, clinical relevance, and role of anti-protein S antibodies.

We evaluated free plasma levels of protein S, a natural anticoagulant factor, the prevalence of anti-protein S antibodies, a possible cause of protein S deficiency, and their correlation with anti-phospholipid antibodies in 53 patients with inflammatory bowel disease (IBD) and 53 age- and sex-matched controls. Mean free plasma protein S levels (± sd) were significantly lower in IBD patients (0.98 ± 0.32 IU/ml) than in controls (1.06 ± 0.28 IU/ml) (P < 0.05); only one patient showed protein S deficiency. Specific antibodies to protein S were found in four IBD patients (7.5%) and in one control (1.9%) (P = NS). Five IBD patients (9.4%) and none of the controls showed anti-phospholipid antibodies (P < 0.06). No correlation was found between free protein S levels and anti-protein S antibodies or between anti-protein S and anti-phospholipid antibodies. In conclusion, free plasma protein S levels are slightly but significantly decreased in IBD patients. The prevalence of anti-protein S and anti-phospholipid antibodies is increased in IBD patients. Anti-protein S antibodies do not appear to determine low protein S levels or to overlap with or belong to anti-phospholipid antibodies.

Mutations in the thrombomodulin gene are rare in patients with severe thrombophilia

Summary. Because thrombomodulin plays a key role in the protein C pathway, we evaluated the contribution of thrombomodulin gene mutations to venous thrombosis. We examined 38 patients with recurrent, documented thrombotic events at a young age and a positive family history. Twelve individuals with low levels of soluble thrombomodulin in plasma were also studied. Finally, the allelic frequency of the Ala455Val polymorphism was estimated in 192 patients with at least one thrombotic event and in 369 age‐ and sex‐matched asymptomatic controls. Two mutations were identified; G/A−201, in a severely thrombophilic patient and G/T 1456, in a patient with low soluble thrombomodulin levels. The first mutation has been reported by some, but not others, to be associated with moderately reduced levels of thrombomodulin. The second was identified previously in a patient with low soluble thrombomodulin, but expression studies failed to show functional changes in the mutant. Thrombomodulin gene mutations thus appear to be rare even in highly selected thrombophilic patients, and possibly functionally irrelevant. The allelic frequency of the Ala455Val polymorphism was identical in patients and controls. Considering the lack of a phenotype and the costly screening procedure, we recommend that thrombomodulin defects be sought only for research purposes.

Resistance to activated protein C in unselected patients with arterial and venous thrombosis

Four hundred and ninety‐three consecutive patients referred for arterial or venous thrombosis were screened for congenital and acquired abnormalities of blood coagulation predisposing to thrombosis, and were compared to 341 age‐ and sex‐matched controls. The aim of the study was to determine the prevalence and clinical characteristics of resistance to activated protein C (APC), a defect shown to have different prevalences in different ethnic groups and to be associated with an increased risk of thrombosis. Seventy‐three (15%) patients had both APC resistance and the 1691 G to A Factor V gene mutation, compared to 6/341 (2%) controls. Seven patients had antithrombin deficiency (1.4%), 11 had protein C deficiency (2.2%), and 4 had protein S deficiency (0.8%). The relative risk of thrombosis in APC‐resistant patients was 9.4. Resistance to APC was associated mainly with venous thrombosis, the most frequent being deep‐vein thrombosis of the lower limbs. Fifty‐eight percent of APC‐resistant patients had an associated risk factor at the first thrombotic event: pregnancy and oral contraceptive intake were associated with the first thrombotic episode in 35% and 30% of women, respectively. APC resistance is the most frequent defect of blood coagulation in the general population and in the unselected thrombotic population studied by us. Am. J. Hematol. 55:59‐64, 1997.