Anna Paola Cellai

D-DIMER CONCENTRATIONS DURING NORMAL PREGNANCY, AS MEASURED BY ELISA

In pregnant women a number of changes in blood clotting and fibrinolysis proteins have been reported so indicating the existence of a state of hypercoagulability. In addition to fibrinogen and antithrombin III (AT), D-dimer is frequently checked during pregnancy, in particular during at risk pregnancy, but the exact pattern of D-dimer modifications during uncomplicated pregnancy is not definitively described. The aim of this study was to establish the range values in three different periods of uncomplicated pregnancy (A: 1-20 wks; B: 21-30 wks; C: 31-40 wks). We measured plasma levels of D-dimer, clottable fibrinogen and AT in 108 consecutive normal pregnant women aged 16 to 42 years. In period A, the range of D-dimer values was 43-211 ng/mL, not different from controls, while fibrinogen levels were significantly higher (p < 0.05) than in matched non pregnant women. Mean D-dimer levels were higher in periods B (p < 0.05) and C (p < 0.05) vs period A. Similarly, mean fibrinogen levels were found more elevated in periods B and C vs period A (p < 0.05). A significant correlation was found between fibrinogen and D-dimer levels (p < 0.001). No differences in AT levels were found among the three periods of pregnancy. The results of this study indicate that levels of D-dimer up to 685 micrograms/L may be reached at the end of physiological pregnancy. This fact should be taken into account in the evaluation of hemostatic studies performed in uncomplicated and complicated pregnant women.

Hemostatic abnormalities in inflammatory bowel disease

Patients affected by inflammatory bowel disease (IBD) frequently suffer from thromboembolic events. Aims of this study were to investigate hemostatic system and the presence of antiphospholipid antibodies (aPL) in IBD patients. Forty-one patients affected by Crohns disease (CD) and 19 by ulcerative colitis (UC) were studied, compared to 40 healthy control subjects. Platelet count (PLT), PT, aPTT, fibrinogen (Fib), prothrombin fragment F1+2, antithrombin (AT), protein C (PC), protein S (PS), factor XIII (FXIII), plasminogen (PLG), plasminogen activator inhibitor (PA1), spontaneous platelet aggregation in platelet-rich plasma (PRP-SPA) and in whole blood (WB-SPA), and antiphospholipid antibodies (aPL) were evaluated. PLT, Fib, F1+2 and WB-SPA were significantly increased in IBD patients (p at least <0.05) both in active and inactive phases; aPL positivity was more frequent (p<0.05) and FXIII was significantly decreased (p<0.05) in comparison to control subjects. The thrombophilic state of IBD patients is not related to the degree of activity of the disease or to previous thrombotic events; aPL express the immunological alterations connected with IBD and are not the main cause of thrombotic events.

Evaluation of Clotting and Fibrinolytic Activation after Protracted Physical Exercise

The behavior of hemostatic system activation during protracted physical exercise is well known, but the duration of its modification is not yet defined. In order to evaluate the time of hemostatic system activation after prolonged strenuous endurance physical exercise (typical marathon race: 42.195 km, v=15.35 km/h; mean length of time run 2.45+/-0.15 hours) 12 well-trained long-distance male runners (mean age: 35+/-7, range 25-47 years) were investigated. Blood samples were drawn in the morning on the day before the performance, immediately after the race, and 24 hours and 48 hours after the end of run. With respect of baseline, immediately after the race, a significant decrease of fibrinogen (-25%) and significant increases of prothrombin fragment 1+2 (+633%) and thrombin-antithrombin complex (+848%) were observed. A significant acceleration of euglobulin lysis time (-41%), and rises of plasma levels of tissue plasminogen activator antigen (+361%), plasminogen activator inhibitor type 1 antigen (+235%), d-dimer (+215%), and plasma fibrinogen degradation products (+1200%) were also found. Only a slight, yet not significant, decrease in plasminogen activator inhibitor type 1 activity was observed. One day after the end of marathon different parameters were still unchanged. Forty-eight hours after the competition all parameters investigated returned to baseline values. These results indicate a persistence of clotting as well as fibrinolysis activation up to 24 hours after the end of the race.

Cardiovascular and thrombophilic risk factors for idiopathic sudden sensorineural hearing loss.

Summary.  Background: In recent years there has been a significant increase in the diagnosis of sudden sensorineural hearing loss (SSHL) in western, countries with an incidence of 20 of 100 000 people affected every year. No clear causes for this disease have been found thus far, but cochlear ischemia has been hypothesized in patients in whom an infectious episode or acoustic neurinoma have been excluded. Objectives: The aim of this case–control study was to investigate a number of acquired and inherited thrombophilic risk factors [antithrombin, protein C and S; factor V (FV) Leiden, FII polymorphism; lupus anticoagulant (LA); anticardiolipin (aCL) antibodies; fasting homocysteine (Hcy); lipoprotein(a) (Lp(a)); plasminogen activator inhibitor‐1 (PAI‐1)] in addition to cardiovascular risk factors in patients with idiopathic SSHL (ISSHL). Patients and methods: We investigated 155 patients (67 male/88 female; age: 55 (range 19–79 years) with a diagnosis of ISSHL within 30 days from the onset of symptoms, and 155 controls (67 male/88 female; age 54 (range 19–78 years). Fasting Hcy levels were significantly higher in patients than in controls [11.6 (6.7–60) μmol/L vs. 8.7 (5.0–24) μmol/L] as well as PAI‐1 levels [19 (2–95) mg/dL vs. 14.5 (4.0–87) mg/dL]. Lupus anticoagulant was present in 13 of 155 (8.4%) patients; 20 patients (12.9%) had positivity of aCL (four IgM and 16 IgG). In no patient was a deficiency of physiological clotting inhibitors antithrombin, protein C and protein S found. No significant differences between patients and controls were observed for Lp(a) plasma levels [111 (1–1146) mg/L vs. 103 (11–695) mg/L] and for the presence of FV Leiden (4.5% vs. 4.5%) and FII variant G20210A (3.8% vs. 3.2%). Results and conclusions: Independent risk factors for ISSHL at the multivariate analysis (adjusted for age, sex and the traditional cardiovascular risk factors) were the positivity of aCL: OR 5.6 (95% CI 2.0–15.3); cholesterol levels within the second and third tertiles (with respect to the first tertile): T2 = OR 4.8 (95% CI 1.9–12.6)/T3 = OR 19 (95% CI 7–50.1); PAI‐1 and Hcy levels within the third tertile (with respect to the first tertile): OR 20 (95% CI 7.8–78) and OR 4.0 (95% CI 2.0–8.1), respectively. These preliminary data suggest that hypercholesterolemia, hyperhomocysteinemia, elevated PAI‐1 levels and anticardiolipin antibodies are associated with ISSHL, so indirectly supporting the hypothesis of a vascular occlusion in the pathogenesis of the disease.

Angiotensin-converting enzyme DD genotype, angiotensin type 1 receptor CC genotype, and hyperhomocysteinemia increase first-trimester fetal-loss susceptibility

Complications of pregnancy have been found to be related with thrombophilic polymorphisms that explain about 30% of obstetric complications. We evaluated the angiotensin converting enzyme (ACE) and the angiotensin type 1 receptor (AT1R) gene polymorphisms in the renin–angiotensin system (RAS) as possible risk factors for fetal loss. Fifty-nine women with a history of three or more first-trimester fetal losses and 70 healthy women with a history of normal pregnancies were enrolled in this study. Thrombophilic factors, ACE insertion/deletion (I/D) and AT1R A1166C polymorphisms, prothrombin G20210A and factor V Leiden mutations were analyzed. At univariate and multivariate analysis, a significant association between ACE DD and AT1R CC genotype and fetal loss was observed. The effect of the ACE DD genotype on the risk of fetal loss was higher in AT1R C allele carriers. The prevalence of hyperhomocysteinemia (Hcy) (defined as baseline plasma levels higher than the 95% percentile; cut-off, 10.5 μmol/l per l) was significantly higher in women with fetal loss, and an association between Hcy and fetal loss was detected. All patients showed normal antithrombin, protein C, protein S, and plasminogen activator inhibitor-1 (PAI-1) values. The presence of one risk factor not associated with others was found in 33 out of 59 patients (56%); ACE DD genotype was the most prevalent risk factor. Our results identify new possible predictive markers for fetal loss in RAS polymorphisms and Hcy. Large-scale studies are warranted to attribute clinical relevance to these polymorphisms as risk factors for complicated pregnancies.

Culprit Factors for the Failure of Well-Conducted Warfarin Therapy to Prevent Ischemic Events in Patients With Atrial Fibrillation The Role of Homocysteine

Background and Purpose— In patients with atrial fibrillation (AF), oral anticoagulant therapy (OAT) is effective in reducing stroke and embolism. However, despite OAT, ischemic events do occur in some patients. Studies specifically addressing the identification of risk factors for ischemic events during well-conducted OAT are not available. In this study, we prospectively investigated the role of classic risk factors and homocysteine levels in the occurrence of ischemic complications in 364 AF patients on OAT. Methods— The quality of anticoagulation levels and the occurrence of bleeding and thrombotic events were recorded. Results— During follow-up (859 patient years) 21 patients had ischemic complications (rate 2.4×100 patient-years). Homocysteine plasma levels were higher in these patients than in patients without ischemic complications during OAT (P<0.01), whereas no difference was observed in relation to the quality of OAT. The presence of a history of previous ischemic events, hypertension, and homocysteine plasma levels over the 90th percentile were all associated with an increased risk of ischemic events during OAT (odds ratio [OR]=7, 4.5, and 4.7, respectively). The coexistence of these risk factors markedly increased the risk (OR=13.1; 95% CI, 3.7 to 45.7; P=0.001). Conclusion— In conclusion, our results indicate that AF patients with multiple risk factors may not be sufficiently protected by OAT, even when this is well conducted.

BLOOD CLOTTING ACTIVATION DURING NORMAL PREGNANCY

Pregnancy is considered as a hypercoagulable state and an increased incidence of thromboembolic phenomena has been reported in pregnant women. Relevant changes in the hemostatic mechanism have been reported during physiological pregnancy: briefly, increased levels of coagulation factors, enhanced thrombin generation and suppression of fibrinolysis are commonly found in women with uncomplicated pregnancy. We recently described progressive increases in fibrinogen and D-dimer plasma levels during normal pregnancy. The increase in D-dimer levels makes difficult their interpretation for the exclusion of thromboembolic phenomena in pregnancy. The behavior of prothrombin fragment 1+2 (F1+2) levels during physiological pregnancy is scarcely known. The aim of this preliminary study was to establish range values of F1+2 plasma levels for different periods of normal pregnancy.

Hypercoagulability, high tissue factor and low tissue factor pathway inhibitor levels in severe ovarian hyperstimulation syndrome: possible association with clinical outcome

&NA; During ovarian gonadotrophin stimulation for ovulation induction or in vitro fertilization, a clinical severe ovarian hyperstimulation syndrome (OHSS) may occur. Only few studies have investigated the mechanism responsible for the alterations of the haemostatic system in women affected by severe OHSS. The aim of the present study was to investigate the correlation between the magnitude of ovarian stimulation and the increase in fibrin formation and degradation in severe OHSS. Twenty‐five patients (age range 23‐43 years) who were hospitalized for severe OHSS, 25 women undergoing in vitro fertilization who did not develop OHSS (case‐control group) and 25 healthy age‐matched women (healthy control group) were investigated. On the day of admission a number of haemostatic markers, including D‐dimer, thrombin‐antithrombin complexes (TAT), prothrombin fragment 1+2 (F1 + 2), plasmin‐antiplasmin complexes (PAP), tissue factor (TF), tissue factor pathway inhibitor (TFPI) and von Willebrand factor antigen (vWF), were examined. In patients with severe OHSS, TF, D‐dimer, TAT, F1 + 2, PAP and vWF antigen plasma levels were significantly higher than those observed both in the case‐control group and in healthy controls, whereas TFPI levels were significantly lower (P < 0.005) with respect to both case‐controls and healthy controls. D‐Dimer levels were related with serum oestradiol levels and oocyte number recovered (r= 0.45, P < 0.001 and r=0.47, P < 0.001, respectively). D‐Dimer and TAT levels were significantly (P < 0.05 and P < 0.005, respectively) higher in OHSS patients with unsuccessful pregnancy outcome (D‐dimer, 226.5, 56‐1449 ng/ml; TAT, 19.8, 3.1‐82.6 μg/l) with respect to those with successful outcome of pregnancy (D‐dimer, 145, 29‐330 ng/ml; TAT, 5.0, 1.0‐19.6 μg/l). Our data indicate that a marked hypercoagulability with alterations of TF and TFPI levels is detectable in patients with severe OHSS and that it is related to the clinical outcome. Blood Coagul Fibrinolysis

D-Dimer plasma levels during normal pregnancy measured by specific ELISA

A progressive increase in D-dimer plasma levels together with an increase in fibrinogen has been previously reported during normal pregnancy. However, significantly different D-dimer levels have been observed in different assays, due to different specificity of the antibodies employed. The aim of this study was to verify the increase in fibrin degradation product levels during normal pregnancy, using a recently introduced specific D-dimer ELISA. We determined D-dimer (ELISA) and fibrinogen (clotting method) plasma levels in 63 normal pregnant women, during three different periods of pregnancy (A, 7–20 weeks; B, 21–30 weeks; C, > 30 weeks). During period A, D-dimer plasma levels (range 2–103 ng/ml) showed an insignificant increase compared with a control group of non-pregnant women (range 2–73 ng/ml). During periods B and C, we observed an increase in D-dimer level (P<0.0001) compared with period A, with a significant correlation between D-dimer levels and gestational age (P<0.0001). Period A fibrinogen levels (range 3.24–6.43 g/l) were significantly higher (P<0.0001) than in controls (range 2.31–4.71 g/l), with a further increase in periods B and C. In conclusion, we confirmed a progressive increase in plasma concentrations of fibrin degradation product during normal pregnancy, but D-dimer levels were significantly lower than those reported in the literature for other ELISAs.

Thrombophilic risk factors in patients with severe carotid atherosclerosis

Summary.  Carotid stenosis and atrial fibrillation are the strongest risk factors for ischemic stroke. Ongoing prevention efforts include the identification of novel factors that increase the risk for carotid atherosclerosis. The aim of this study was to determine the thrombophilic risk profile of patients with severe carotid stenosis by evaluating a number of genetic and metabolic risk factors [factor (F)II G20210A, factor V Leiden, MTHFR C677T polymorphisms, anticardiolipin antibodies (aCL), lipoprotein(a) (Lp(a)), and homocysteine (Hcy)]. The study population consisted of 615 patients [(410 M/205 F; median age 73 (26–94) years] with severe (> 70%) carotid stenosis, and 615 apparently healthy subjects [(410 M/205 F; age 73 (31–92) years]. On multivariate analysis, independent risk factors were elevated Hcy [odds ratio (OR) 7.6, 95% confidence interval (CI) 4.8, 11.8] and Lp(a) levels (OR 2.9, 95% CI 2.1, 3.9), the presence of aCL (OR 5.7, 95% CI 3.1, 10.4) and heterozygosity for FII G20210A polymorphism (OR 2.8, 95% CI 1.3, 5.9). In the subgroup of women, independent risk factors for severe carotid atherosclerosis were: high levels of Hcy and Lp(a) and the presence of aCL, whereas hyperhomocysteinemia, elevated Lp(a) levels, aCL, FII G20210A and MTHFR 677TT polymorphisms remained independent risk factors in the subgroup of men. The results of the present study demonstrate that the prevalence of the thrombophilic risk factors is increased in patients with severe carotid atherosclerosis.