Eugenia Biguzzi

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.

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.

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.

Low borderline plasma levels of antithrombin, protein C and protein S are risk factors for venous thromboembolism

Summary.  Background:  Inherited deficiencies of antithrombin (AT), protein C (PC) and protein S (PS) are risk factors for venous thromboembolism (VTE). They are usually defined by laboratory cut‐offs (in our setting 81, 70 and 63 IU dL−1, respectively), which give only a rough idea of the VTE risk associated with plasma levels of these proteins.

Low levels of protein Z and the risk of venous thromboembolism.

botic disease. Am J Hum Genet 2002; 70: 567–74. 11 Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 1998; 11: 1–3. 12 RennerW,KoeppelH,HoffmannC,SchallmoserK,SatngerO,Toplak H,WascherCT,PilgerE.Prothrombin G20210A, factor V Leiden, and factor XIII Val34Leu: commonmutations of blood coagulation factors and deep vein thrombosis in Austria. Thromb Res 2000; 99: 35–9. 13 Girolami A, Sartori MT, Lombardi AM, Pellati D. Rebuttal: factor XII levels, factor XII CT polymorphism and venous thrombosis: a word of caution is needed. Thromb Haemost 2004; 92: 892–3. 14 Ishii K, Oguchi S, Murata M, Mitsuyoshi Y, Takeshita E, Ito D, Tanahashi N, Fukuuchi Y, Oosumi K, Matsumoto K, Kitajima M, Yamamoto M, Watanabe G, Ikeda Y, Watanabe K. Activated factor XII levels are dependent on factor XII 46C/T genotypes and factor XII zymogen levels, and are associated with vascular risk factors in patients and healthy subjects. Blood Coagul Fibrinolysis 2000; 11: 277–84.

The JAK2 V617F mutation in patients with cerebral venous thrombosis.

Summary.  Background:  It is currently unclear whether or not cerebral venous thrombosis, such as splanchnic venous thrombosis, can be the first manifestation of an underlying myeloproliferative neoplasm.

Risk factors for postpartum hemorrhage in a cohort of 6011 Italian women.

INTRODUCTION Postpartum hemorrhage is responsible for 25% of maternal pregnancy-related deaths and it is the first cause of maternal morbidity and mortality worldwide. OBJECTIVE To define the prevalence of postpartum hemorrhage and associated risk factors after vaginal birth and to develop a risk model that improves postpartum hemorrhage prediction. PATIENTS AND METHODS All women who underwent a vaginal delivery at the Obstetric Unit of a large University hospital in Milan (Italy) between July 2007 and September 2009 were enrolled. Postpartum hemorrhage was defined as ≥ 500 mL blood loss. A nomogram tailored to predict postpartum hemorrhage was developed, summarizing the impact of each covariate on the probability of postpartum hemorrhage. RESULTS 6011 women were studied (24% had blood loss ≥ 500 mL and 4.8% ≥ 1000 mL). Nulliparity, episiotomy, retained placenta and high neonatal body weight were confirmed as risk factors for postpartum hemorrhage. The odds ratio of postpartum hemorrhage was 0.86 (95%CI 0.78-0.90) for each 1 gr/dL increase in ante-partum hemoglobin. An extensive internal validation of the developed nomogram demonstrated a good stability of the risk model. CONCLUSIONS Low ante-partum hemoglobin is a new potentially modifiable risk factor for postpartum hemorrhage. A nomogram to predict the probability of postpartum hemorrhage is now available for external validation.

Polymorphisms of the protein Z-dependent protease inhibitor (ZPI) gene and the risk of venous thromboembolism.

Letters to the Editor: Polymorphisms of the protein Z-dependent protease inhibitor (ZPI) gene and the risk of venous thromboembolism -

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.

Advances in the treatment of bleeding disorders.

Historically, the bleeding episodes in subjects with coagulation disorders were treated with substitution therapy, initially with whole blood and fresh frozen plasma, and more recently with specific factor concentrate. Currently, patients with hemophilia have the possibility of choosing different effective and safe treatments, including novel extended half‐life and alternative hemostatic drugs. The availability of novel extended half‐life products could probably overcome current prophylaxis limitations, particularly in hemophilia B patients, by reducing the frequency of injections, achieving a higher trough level, and improving the quality of life of the patients. In addition, subcutaneous administration of alternative therapeutics would simplify prophylaxis in patients with hemophilia A and B with and without inhibitors. Regarding von Willebrand disease, a recombinant von Willebrand factor was recently developed to control bleeding episodes in patients with this disease, in addition to available von Willebrand factor/factor VIII concentrates. The management of patients affected by rare bleeding disorders (RBDs) is still a challenge, owing to the limited number of specific products, which are mainly available only in countries with high resources. Some improvements have recently been achieved by the production of new recombinant factor (F) XIII A subunit‐derived and FX plasma‐derived products for the treatment of patients affected by FXIII and FX deficiency. In addition, the development of novel alternative therapeutics, such as anti‐tissue factor pathway inhibitor, ALN‐AT3, and ACE910, for patients with hemophilia might also have a role in the treatment of patients affected by RBDs.