Franca Franchi

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.

Asymptomatic homozygous protein C deficiency

We report a family in which 2 homozygotes with similarly very low protein C levels have different clinical symptoms. One had recurrent venous thrombosis starting at the age of 28 years, the other is still asymptomatic at 38 years despite exposure to thrombotic risk factors. Our review of 13 additional cases reveals a highly variable phenotypic expression of homozygous protein C deficiency, which can be subdivided into two groups. In the first group are 8 kindreds in which homozygotes presented at birth with unmeasurable protein C levels and life-threatening thrombosis and 1 kindred in which homozygotes are characterized by very low levels of protein C but delayed onset (10 months of age) of thrombosis. In the second group are 4 kindreds characterized by very low, but measurable, protein C levels in homozygotes who survived beyond the neonatal period into adulthood with histories of moderately severe thrombosis. The present case demonstrates that protein C levels lower than 10% are compatible with a negative history for thrombosis, not only in the neonatal period but also during adulthood, and suggests that in some homozygotes other factors need to interact for full clinical penetrance of the defect.

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.

Plasma Levels of Activated Protein C in Healthy Subjects and Patients With Previous Venous Thromboembolism Relationships With Plasma Homocysteine Levels

The proteolytic enzyme activated protein C (APC) is a normal plasma component, indicating that protein C (PC) is continuously activated in vivo. High concentrations of homocysteine (Hcy) inhibit the activation of PC in vitro; this effect may account for the high risk for thrombosis in patients with hyperhomocysteinemia (HyperHcy). We measured the plasma levels of APC in 128 patients with previous venous thromboembolism (VTE) and in 98 age- and sex-matched healthy controls and correlated them with the plasma levels of total Hcy (tHcy) measured before and after an oral methionine loading (PML). Forty-eight patients had HyperHcy and 80 had normal levels of tHcy. No subject was known to have any of the congenital or acquired thrombophilic states at the time of the study. Because the plasma levels of APC and PC were correlated in healthy controls, the APC/PC ratios were also analyzed. Plasma APC levels and APC/PC ratios were significantly higher in VTE patients than in controls (P=0.03 and 0.0004, respectively). Most of the increase in APC levels and APC/PC ratios were attributable to patients with HyperHcy. Patients with normal tHcy had intermediate values, which did not differ significantly from those of healthy controls. There was no correlation between the plasma levels of tHcy or its PML increments and APC or APC/PC ratios in controls. The fasting plasma levels of APC and APC/PC ratios of 10 controls did not increase 4 hours PML, despite a 2-fold increase in tHcy. This study indicates that APC plasma levels are sensitive markers of activation of the hemostatic system in vivo and that Hcy does not interfere with the activation of PC in vivo.

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.

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.

Elevated prepartum fibrinogen levels are not associated with a reduced risk of postpartum hemorrhage.

To cite this article: Peyvandi F, Biguzzi E, Franchi F, Bucciarelli P, Acaia B, Zaina B, Musallam KM. Elevated prepartum fibrinogen levels are not associated with a reduced risk of postpartum hemorrhage. J Thromb Haemost 2012; 10: 1451–3.