Anemone study: prevalence of risk factors for superficial vein thrombosis in a large Italian population of blood donors.

Knowledge of the distribution of risk factors for superficial thrombosis (SVT) in low-risk population is fundamental to improve the prevention of the disease in each individual and high-risk settings of patients. Exact frequency data for the low-risk population are scarce, but could be useful for optimal use of prophylactic strategies against venous thrombosis. Blood donors represent a low-risk population, because are healthier than the general population. The objective of this study was to assess the prevalence of vein thrombosis, particularly SVT, and associated risk factors in a low-risk population such as blood donors. In this multicentre cross-sectional study, donors from six Italian blood banks responded to a self-administered questionnaire. The enrolment lasted from 1st June 2017 to 30th July 2018. History of vein thrombosis was referred by 89 (0.76%) individuals, (49 men) with an age-dependent effect. The prevalence reached 2.9% in women and 0.8% in men aged ≥ 49 years, with a significant difference only for women. After controlling for potential confounders, a significant and independent association was found between a history of vein thrombosis and age (OR: 1.03, 95%CI 1.01-1.05), varicose veins (OR: 15.8, 95%CI 7.7-32.6), plaster cast/bed rest (OR: 2.3, 95% CI 1.0-5.3) and transfusion (OR: 5.1, 95% CI 1.3-19.5). This study shows that low-risk individuals share the same risk factors for SVT as patients in secondary care. It also suggests that transfusion confers an increased risk of SVT in healthy population.

Thromboelastography parameters in Italian pregnant women: do antithrombotic drugs change reference values?

This study was carried out to explore hemostasis modifications occurring in pregnant women and thromboelastography profiles in those taking antithrombotic drugs. An exploratory study was carried out in the period from March 2017 to May 2018. Caucasian women from Southern Italy were recruited during a routine obstetric assessment. Participants were divided into four groups: T1 (gestational week <14 weeks), T2 (14-28 weeks), T3 (29-42 weeks) and T4 in the postpartum period. We investigated thromboelastography profile in 19 and 5 women administered with low-molecular-weight heparin or low-dose aspirin, respectively. "MA" value observed in the T1 group was significantly greater than that observed in the T3 and the T4 groups, while "K" in the T1 group was significantly longer than that in the T3 and the T4 groups, indicating a gradual development of a prothrombotic state (in all cases Mann-Whitney U test, p<0.05). Significant differences within "R" were observed between the T2 and the T3 and between the T3 and the T4 ("R" parameter) (Mann-Whitney U test, p<0.05). "LY30" parameter resulted to be significantly higher in the T1 group (Mann-Whitney U test, p=0.01) compared with the T4 one, indicating fibrinolysis decreases throughout pregnancy and until post partum. No significant variations were found in women administered with prophylactic doses of low-molecular-weight heparin. Significantly higher fibrinolysis (p<0.01) was observed for "LY30" parameter in women taking low-dose aspirin versus women not taking any treatments. Our data contribute to better interpret thromboelastography profile in the context of peripartum complications, which are often unpredictable and need prompt therapies.

Validation of PLASMIC score and follow-up data in a cohort of patients with suspected microangiopathies from Southern Italy.

Severe ADAMTS13 deficiency (activity < 10%) is pathognomonic of thrombotic thrombocytopenic purpura. ADAMTS13 testing is time-consuming and unavailable in many hospitals. Recently, a seven-variables score named PLASMIC score, has been developed to stratify acute patients, based on their risk of having a severe ADAMTS13 deficiency. We present the application of this score in a cohort of patients referred to our Center. From 2012 to 2017, 42 patients with suspected thrombotic microangiopathies from 6 Centers were referred to Hemostasis and Thrombosis Center of "Casa Sollievo della Sofferenza" Hospital/Research Institute for ADAMTS13 testing. For all patients, relevant medical and laboratory information were collected. To obtain the statistical measure of the discriminatory power of PLASMIC scoring system, the Area Under the Curve Receiver Operating Characteristic (AUC ROC) was calculated. We were able to calculate the PLASMIC score in 27 out of 42 patients; we found a good discrimination performance of the score with a resulting AUC value of 0.86 (95% CI 0.71-1.0; p = 0.015). All patients but one with a high risk PLASMIC score (6-7) showed a severe deficiency. Among patients belonging to the intermediate risk (PLASMIC score 5) group, 2 showed normal ADAMTS13 activity and 2 levels below 10%. In none of the patients in the low risk group (PLASMIC score 0-4), a severe ADAMTS13 deficiency was found. Present results confirm and extend previous data regarding the predictive value of the PLASMIC score. Indeed, it shows a good diagnostic performance and can be useful for decision makers to properly and promptly define the better therapeutic approach.

Human Fibrinogen: Molecular and Genetic Aspects of Congenital Disorders.

Congenital fibrinogen disorders can be quantitative (afibrinogenemia, hypofibrinogenemia) or functional (dysfibrinognemia). To date, several genetic variants have been identified in individuals with fibrinogen disorders. The complexity of the fibrinogen molecules, formed by three non-identical chains and with a trinodal organization, renders the identification of molecular causes and of clinical and biochemical phenotypes very challenging. However, the acknowledgement of the type of molecular defect is crucial for a safer therapy, which is going to improve the clinical management of these patients. In this review, some aspects concerning molecular and clinical findings available on congenital fibrinogen disorders will be discussed.

Pharmacogenetics of dabigatran etexilate interindividual variability.

INTRODUCTION: Dabigatran etexilate is given in fixed doses without coagulation monitoring for the prevention of blood clots in at risk adults. A high inter-individual variability in blood concentrations of the active metabolite of dabigatran has been reported. ABCB1 and CES1 exert an important effect in the metabolism of dabigatran etexilate and allele variants at these two loci are likely to play a pivotal role. To investigate whether screening for polymorphisms within the ABCB1 and the CES1 genes would explain a portion of the inter-individual variability in blood concentrations of the active metabolite of dabigatran. MATERIAL AND METHODS: In a cohort of patients who had atrial fibrillation and on anticoagulant prophylaxis with dabigatran etexilate, we investigated whether genotypes at rs4148738 (ABCB1), rs8192935 (CES1), and rs2244613 (CES1) loci would affect plasma dabigatran trough and peak concentrations. RESULTS AND DISCUSSION: Among 92 patients (median age: 72.0years, range: 52-92) analyzed, no clinical variable or genotype was associated with a significant difference in dabigatran peak concentrations. As for trough concentrations, in addition to creatinine clearance, and sex a significant association with the CES1 SNP rs8192935 (p=0.023) was detected. The mean adjusted plasma levels were higher among patients with the CC genotype (86.3ng/dl) than in those carrying the T allele (62.1ng/dl). No significant effect was found for the ABCB1 SNP rs4148738. The CES1 SNP rs8192935 significantly influenced the dabigatran trough concentrations and carriers of the T allele showed significantly lower concentrations than did carriers of the CC genotype.

Functional characterization of annexin A5 gene promoter allelic variants.

BACKGROUND: M1 and M2 haplotypes are defined by 4 consecutive allelic variants in regulatory regions of the annexin A5 gene and have been found to reduce promoter activity. To date, no research has been carried out to investigate differential and individual impact each of the allelic variants has on promoter activity. In the current study, we functionally characterized the M1 and M2 haplotype allelic variants (c.-467G>A, c.-448A>C, c.-422T>C, c.-373G>A). We also characterized two other allelic variants located in the same regulatory region (c.-628C>T, c.-302T>G). MATERIALS AND METHODS: Their impact on the ANXA5 promoter activity was examined using a luciferase reporter assay in BeWo cells. Electrophoretic mobility shift assay with probes centered around each polymorphism was used to examine the binding ability of the allelic variants to nuclear proteins from BeWo cells. RESULTS: Only the c.-467G>A and c.-628C>T allelic variants influenced the activity of the ANXA5 promoter, as measured by luciferase activity. Differential specific interactions with nuclear proteins were obtained for all allelic variants, except for the c.-302T>G, indicating that these polymorphisms could have an impact on the ANXA5 expression. CONCLUSIONS: We have functionally characterized allelic variants in the ANXA5 promoter, both alone and in combinations, and the results suggest that combinations of several individual variants contribute to modulate the ANXA5 transcriptional activity, most likely through binding of nuclear factors. These results provide new knowledge and insight into the mechanisms underlying the regulation of annexin A5 levels in healthy controls.

Pregnancy-related venous thrombosis: comparison between spontaneous and ART conception in an Italian cohort.

OBJECTIVE: To evaluate in an Italian cohort the incidence of venous thromboembolic events (VTE) in pregnancies after assisted reproductive technologies (ART). SETTING: Thrombosis and Haemostasis Unit at I.R.C.C.S. 'Casa Sollievo della Sofferenza', S. Giovanni Rotondo. PARTICIPANTS: A prospective cohort of 998 women advised to undergo ART was referred by local fertility clinics from April 2002 to July 2011. Follow-up information was obtained during the check-up and/or by phone interviews. In a cohort of women who consecutively gave birth (n=3339) after spontaneous conception in our Institution, information on the diagnoses of pregnancy-related venous thromboses was obtained by linkage to a patient administrative register. PRIMARY AND SECONDARY OUTCOME MEASURES: We calculated the incidence of VTE and superficial venous thrombosis in successful ART cycles and compared it with that of the general population conceiving spontaneously. RESULTS: Overall, 684 ART cycles were carried out by 234 women, who achieved a clinical pregnancy; in case of more than one successful cycle, only the first pregnancy was considered. Three vein thromboses (two VTE and one superficial vein thrombosis) were recorded. An antithrombotic prophylaxis with LMWH alone or combined with low-dose aspirin was prescribed in 23/234 (9.8%) women. In the reference cohort of 3339 women, a total of 11 vein thromboses were observed: six VTE and five SVT. The two-tailed Fisher exact test showed a trend towards statistical significance (p: 0.06, OR: 3.9, 95% CI 0.87 to 15.3). After the exclusion of superficial thromboses in both the groups, we found that the incidence of VTE in our population of women who had undergone ART was 2/234 pregnancies (8.5 ‰), whereas that in our reference population was 6/3339 (1.8 ‰) (p: 0.09). CONCLUSIONS: Our data show a slightly higher incidence of vein thromboses in pregnancies after ART than in those after natural conception.

A novel congenital dysprothrombinemia leading to defective prothrombin maturation.

INTRODUCTION: Prothrombin deficiency is a very rare disorder caused by mutations in the F2 gene that generate hypoprothrombinemia or dysprothrombinemia and is characterized by bleeding manifestations that can vary from clinically irrelevant to life-threatening. AIM: Here we characterize a patient with a novel missense mutation in F2, c.1090T/A (p.Val322Glu), that causes severe dysprothrombinemia. METHODS: Coagulation assays, prothrombin Western Blotting, FII activation by Ecarin, fibrinogen degradation products quantification and thrombin generation assay were carried out to assess prothrombin expression and function. PCR followed by direct sequencing was carried out to characterize the mutation. In silico analysis for missense variant and molecular modeling were applied to predict the mechanism that leads to dysprothrombinemia. RESULTS AND CONCLUSIONS: The homozygous patient had a markedly prolonged prothrombin time, strongly reduced FII activity (0.82%) but normal antigen levels. In the thrombin generation assay the lag time and the peak height were unmeasurable, suggesting that the Val322Glu mutation results in the inability of the mutant prothrombin to be fully activated to thrombin. In fact, prothrombin activation by ecarin was defective, with a massive accumulation of the meizothrombin intermediate. Molecular modeling and dynamic simulation studies showed that the Val322Glu mutation interferes with protein flexibility at Arg271 and Arg320. This impairs the switch of the protein from zymogen to proteinase, thus preventing the formation of thrombin. Accumulated meizothrombin, however, maintains some fibrinogen-degrading activity, as shown by the formation of FDPs, and this probably explains the patient's mild bleeding phenotype.

Liquid chromatography-tandem mass spectrometry method as the golden standard for therapeutic drug monitoring in renal transplant.

Kidney transplanted patients need immunosuppressant therapies. Therapeutic drug monitoring approaches produce the optimal clinical outcome is still under debate. This review details strength and limits of methods available: immunoassay and chromatography-based. Liquid chromatography with mass spectrometry detection is a major breakthrough in therapeutic drug monitoring of immunosuppressive agents and is considered as the method of choice in therapeutic drug monitoring (TDM) of immunosuppressants. Despite the initial high cost for the instrumentation, HPLC-MS is more cost effective than microparticle enzyme immunoassay. The main important features of LC-MS/MS methodology for immunosuppressive drugs are the shortened analysis time, an increased throughput, higher selectivity, specificity, and sensitivity, and low cost of analysis.

Outcome of patients with splanchnic venous thrombosis presenting without overt MPN: a role for the JAK2 V617F mutation re-evaluation.

INTRODUCTION: Although investigation for JAK2 V617F mutation is recommended in patients presenting with splanchnic venous thrombosis (SVT), no specific clinical advice is given to SVT patients presenting without myeloproliferative neoplasms (MPN) and JAK2 V617F mutation. In MPN-free SVT patients, to investigate the clinical outcome, the clinical impact of re-evaluation for the JAK2 V617F mutation, and relationships with the occurrence and time to diagnosis of MPN. MATERIALS AND METHODS: A cohort of non-cirrhotic SVT patients, enrolled at a single centre and prospectively analyzed. RESULTS: In 121 SVT patients prospectively followed from 1994 to 2012, a MPN was present in 28 (23.1%). Additional 13 patients (10.7%) showed only the JAK2 V617F mutation. During the follow-up, the JAK2 V617F mutation and/or MPN were identified in 8 patients (median time of development: 21 months, range 6-120), whereas 72 remained (MPN and JAK2 V617F)-free until the end of the observation. The mortality rate was higher among patients presenting with MPN and/or the JAK2 V617F mutation than in patients who developed later or remained disease-free (p=0.032). The thrombosis-free survival was lower in patients with (p=0.04) or developing later MPN and the JAK2 V617F mutation (p=0.005) than in patients (MPN and JAK2 V617F)-free. The incidence of bleeding was similar among groups. CONCLUSIONS: MPN with or without circulating positive clones for JAK2 V617F mutation can occur long after a SVT, identifying at risk patients for new thrombotic events. If confirmed in other studies, re-evaluation for JAK2 V617F mutation may be of help in early MPN detection and clinical management of SVT patients.

Obstetric complications and pregnancy-related venous thromboembolism: the effect of low-molecular-weight heparin on their prevention in carriers of factor V Leiden or prothrombin G20210A mutation.

Whether the administration of low-molecular-weight heparin (LMWH) during pregnancy is effective in preventing obstetric complications and pregnancy-related venous thromboembolism (VTE) in women who are carriers of factor V Leiden (FVL) and/or prothrombin variant G20210A (PTm) is controversial. This observational study investigated the possible efficacy of pharmacological treatment with LMWH ± aspirin (ASA) in pregnancy outcomes in 1,011 pregnancies of 416 women with thrombophilia (FVL and/or PTm). Most patients were chosen on the basis of previous obstetrical complications (36%), or because of familial or personal history of venous/arterial thromboembolism (28% and 18%, respectively); 74 patients (18%) were incidentally identified. The outcome was evaluated according to the type of treatment and of the period of pregnancy when the treatment was started. After adjustment for observation before and after diagnosis of thrombophilia, previous miscarriages and VTE, parity, age and centre, we observed that LMWH had a protective effect on miscarriages (odds ratio [OR] 0.52, 95% confidence interval [CI] 0.29-0.94) and VTE (OR 0.05, 95% CI 0.01-0.21). ASA appeared to have no effect on the prevention of obstetric complications and VTE. A nested analysis performed in 116 women with two or more obstetric complications confirmed that the highest number of live births was recorded in the group under LMWH prophylaxis (OR 0.19, 95% CI 0.05-0.75). These results suggest that LMWH prophylaxis reduces the risk of obstetric complications in carriers of FVL and/or PTm, particularly in those with previous obstetric events. Furthermore, LMWH prophylaxis reduces the risk of pregnancy-related VTE.

Sex modulation of the occurrence of jak2 v617f mutation in patients with splanchnic venous thrombosis.

BACKGROUND: The JAK2 V617F mutation is an independent risk factor for MPN and SVT. Gender-related differences in MPN distribution have been reported and, recently, variability in the JAK2 V617F allele burden between sexes has been suggested. We wondered whether gender would modulate the role of the JAK2 V617F mutation as susceptibility risk factor for SVT. MATERIALS AND METHODS: In 180 patients presenting with SVT, medical history was collected. The presence of the JAK2 V617F mutation and 46/1 haplotype was determined by polymerase chain reaction followed by TaqMan SNP genotyping assays. RESULTS: Among patients with SVT, 43 (23.9%; 95%-CI: 18.2-30.7) carried the JAK2 V617F mutation. The JAK2 V617F mutation was found more frequently in women (29/95: 30.5%; 95%-CI: 22.1-40.4) than in men (14/85: 16.5%; 95%-CI: 10.0-25.9; OR: 2.2; 95%-CI: 1.1-4.5). The distribution of 46/1 haplotype frequencies did not differ significantly between men and women. In women carrying the rs12343867 CC genotype, the frequency observed for the occurrence of the V617F mutation was significantly higher than that observed in those not carrying (60.0% [95% CI: 31.2-83.3] vs. 26.8% [95% CI: 18.4-37.4]; OR: 4.1; 95%-CI: 1.1-14.9). In men, a similar prevalence was found among carriers of the rs12343867 CC genotype (16.7% [95% CI: 3.5-46.0]) and in non carriers (16.4% [95% CI: 9.3-27.2]). The V617F allele burden was unrelated to clinical characteristics and significantly higher in carriers of the rs12343867 CC genotype. CONCLUSIONS: Present findings suggest that, in patients presenting with SVT, the JAK2 V617F mutation is frequently found in women and, possibly by interacting with the 46/1 haplotype, may represent a gender-related susceptibility allele for SVT.

Maternal thrombophilia and the risk of recurrence of preeclampsia.

OBJECTIVE: The aim of this prospective study was to determine the impact of thrombophilia on the recurrence of preeclampsia. STUDY DESIGN: In a multicenter, observational, cohort design, 172 white patients with a previous pregnancy complicated by preeclampsia were observed in the next pregnancy. They were evaluated for heritable thrombophilia (factor V Leiden and factor II G20210A mutations, protein S, protein C, and antithrombin deficiency), hyperhomocystinemia, lupus anticoagulant, and anticardiolipin antibodies. Development of preeclampsia and maternal complications and both gestational age at delivery and birthweight were recorded. RESULTS: Sixty women (34.9%) showed the presence of a thrombophilic defect. They had a higher risk for the recurrence of preeclampsia (odds ratio [OR], 2.5; 95% confidence interval [CI], 1.2-5.1), compared to patients without thrombophilia. Similar findings were observed considering only heritable thrombophilia. Thrombophilic patients were at increased risk for the occurrence of very early preterm delivery (< 32 weeks; OR, 11.6; 95% CI, 3.4-43.2). CONCLUSION: When counseling white women with a history of preeclampsia, screening for thrombophilia can be useful for preconceptional counseling and pregnancy management.

Annexin V expression in human placenta is influenced by the carriership of the common haplotype M2.

In a group of placentas from patients with preeclampsia or fetal growth restriction, reduced annexin V gene expression in those carrying the M2 haplotype was observed. This is the first "ex vivo" demonstration that annexin V gene expression in placentas is dependent on the M2 haplotype.