Increase of Neutrophil Activation Markers in Venous Thrombosis-Contribution of Circulating Activated Protein C.

Upon activation, neutrophils release their content through different mechanisms like degranulation and NETosis, thus prompting thrombosis. The natural anticoagulant activated protein C (APC) inhibits neutrophil NETosis and, consequently, this may lower the levels of neutrophil activation markers in plasma, further diminishing the thrombotic risk exerted by this anticoagulant. We aimed to describe the status of markers of neutrophil activation in plasma of patients with venous thrombosis, their association with the thrombotic risk and the potential contribution of APC. We quantified three markers of neutrophil activation (cell-free DNA, calprotectin, and myeloperoxidase) in 253 patients with venous thromboembolism (VTE) in a stable phase (192 lower extremity VTE and 61 splanchnic vein thrombosis) and in 249 healthy controls. In them, we also quantified plasma APC, soluble endothelial protein C receptor (EPCR), and soluble thrombomodulin (TM), and we genotyped two genetic regulators of APC: the EPCR gene (PROCR) haplotypes (H) and the TM gene (THBD) c.1418C>T polymorphism. We found a significant increase in plasma cell-free DNA (p < 0.0001), calprotectin (p = 0.0001) and myeloperoxidase (p = 0.005) in VTE patients compared to controls. Furthermore, all three neutrophil activation markers were associated with an increase in the thrombotic risk. Cell-free DNA and calprotectin plasma levels were significantly correlated (Spearman r = 0.28; p < 0.0001). As expected, the natural anticoagulant APC was significantly decreased in VTE patients (p < 0.0001) compared to controls, what was mediated by its genetic regulators PROCR-H1, PROCR-H3, and THBD-c.1418T, and inversely correlated with cell-free DNA levels. This is the largest case-control study that demonstrates the increase in markers of neutrophil activation in vivo in VTE patients and their association with an increased thrombotic risk. This increase could be mediated by low APC levels and its genetic regulators, which could also increase NETosis, further enhancing thrombosis and inflammation.

Factor XIII deficiency in two Spanish families with a novel variant in gene F13A1 detected by next-generation sequencing; symptoms and clinical management.

Coagulation factor XIII (FXIII) has a major role in coagulation stabilizing the haemostatic clot. FXIII deficiency is associated with an increased risk of bleeding. Severe phenotypes lead to spontaneous, traumatic and surgical bleeding. Umbilical cord bleeding is especially common, and intracranial bleeding may occur in up to one third of patients without prophylaxis. In this work, we used NGS for screening all the coding and intronic boundary regions of F13A1 and F13B genes in two families affected by severe FXIII deficiency. Outcome confirmation analysis and variant studies in related patients was done by Sanger sequencing. Two variants were found: c.34A > G (p.Arg12Gly; NM_00129.3) and c.514C > T (p.Arg172Ter; NM_00129.3), both located in the F13A1 gene. The variant p.Arg172Ter is already described in literature and was found in homozygosis in one family and in compound heterozygosis in the other family. The variant p.Arg12Gly variant has not been described previously. This variant is located in the activation peptide of the FXIII A-subunit which is highly conserved among FXIII homologs. Given the high risk of dangerous bleeding and early manifestation in severe FXIII-deficient patients, a prompt genetic confirmation is imperative. In this sense, NGS technology allows a rapid and simultaneous analysis of all regions of all the genes involved in the pathology.

Identification of 58 Mutations (26 Novel) in 94 of 109 Symptomatic Spanish Probands with Protein C Deficiency.

Presently, no data on the molecular basis of hereditary protein C (PC) deficiency in Spain is available. We analyzed the PC gene (PROC) in 109 patients with symptomatic PC deficiency and in 342 relatives by sequencing the 9 PROC exons and their flanking intron regions. In 93 probands, we found 58 different mutations (26 novel). Thirty-seven consisted of a nucleotide change, mainly missense mutations, 1 was a 6-nucleotide insertion causing the duplication of 2 amino acids, and 4 were deletions of 1, 3, 4, and 16 nucleotides. Nine mutations caused type II deficiencies, with the presence of normal antigen levels but reduced anticoagulant activity. Using a PC level of 70% as lowest normal limit, we found no mutations in 16 probands and 25 relatives with PC levels ≤ 70%. On the contrary, 4 probands and 12 relatives with PC levels > 70% carried the mutation identified in the proband. The spectrum of recurrent mutations in Spain is different from that found in the Netherlands, where the most frequent mutations were p.Gln174* and p.Arg272Cys, and is more similar to that found in France, where the most frequent were p.Arg220Gln and p.Pro210Leu. In our study, p.Val339Met (9 families), p.Tyr166Cys (7), p.Arg220Gln (6), and p.Glu58Lys (5) were the most prevalent. This study confirms the considerable heterogeneity of the genetic abnormality in PC deficiencies, and allowed genetic counseling to those individuals whose PC levels were close to the lower limit of the normal reference range.

Effective and Safe Management of Oral Anticoagulation Therapy in Patients Who Use the Internet-Accessed Telecontrol Tool SintromacWeb.

BACKGROUND: Despite the existing evidence that highlights the benefits of oral anticoagulation therapy (OAT) self-testing and self-management by patients in comparison with conventional control, significant progress is still needed in the implementation of computer-based, Internet-assisted systems for OAT within health care centers. The telecontrol tool "SintromacWeb" is a previously validated system for OAT management at home, which is currently operative and accessed by patients through a hospital Web portal. OBJECTIVE: The intent of the study was to assess the effectiveness and safety of OAT management in patients using the SintromacWeb telecontrol system in reference to control in patients using the conventional system (management at the hematology department), in terms of time in therapeutic range (TTR) of International Normalized Ratio (INR). METHODS: In this observational prospective study, patients were identified by their physician and divided in two groups according to the OAT management system that they were already using (conventional control or telecontrol with SintromacWeb). For 6 months, patients were required to visit the hematology department every time their physician considered it necessary according to usual clinical practice. Sociodemographic and clinical variables for the study were collected at first visit (baseline) and at those visits closest to 2, 4, and 6 months after first visit. RESULTS: A total of 173 patients were evaluated, 87 with conventional control and 86 with telecontrol. Follow-up time was a median of 6.3 (range 5.2-8.1) months. The average time of OAT treatment prior to enrollment was 9.2 (SD 6.4) years. Patients in the telecontrol group tested their INR a median of 21 (range 4-22) days versus a median of 35 (range 14-45) days in patients in the conventional control group (P<.001). TTR in the telecontrol group was 107 (SD 37) days versus 94 (SD 37) days in the conventional control group (an increase of 12.6%; P=.02). In all visits, the percentage of TTR was higher in the telecontrol group (at the third visit: 59% vs 48%; P=.01). Higher TTR (positive coefficient) was associated with patients under OAT telecontrol (P=.03). Under-anticoagulation (INR<1.5) and over-anticoagulation (INR>5) were observed in 34 (19.7%, 34/173) and 38 (22.0%, 38/173) patients, respectively (no differences between treatment groups). Seven thrombotic and/or bleeding events were serious, 12 were non-serious, and most of them (5 and 10, respectively) occurred in the conventional control group. CONCLUSIONS: In clinical practice, OAT management with the Internet-based tool SintromacWeb is effective and safe for those patients who are eligible for OAT telecontrol.

Association of the thrombomodulin gene c.1418C>T polymorphism with thrombomodulin levels and with venous thrombosis risk.

OBJECTIVE: To investigate the association of the THBD c.1418C>T polymorphism, which encodes for the replacement of Ala455 by Val in thrombomodulin (TM), with venous thromboembolism (VTE), plasma soluble TM, and activated protein C levels. In addition, human umbilical vein endothelial cells (HUVEC) isolated from 100 umbilical cords were used to analyze the relation between this polymorphism and THBD mRNA and TM protein expression. APPROACH AND RESULTS: The THBD c.1418C>T polymorphism was genotyped in 1173 patients with VTE and 1262 control subjects. Levels of soluble TM and activated protein C were measured in 414 patients with VTE (not on oral anticoagulants) and 451 controls. HUVECs were genotyped for the polymorphism and analyzed for THBD mRNA and TM protein expression and for the ability to enhance protein C activation by thrombin. The 1418T allele frequency was lower in patients than in controls (P<0.001), and its presence was associated with a reduced VTE risk, reduced soluble TM levels, and increased circulating activated protein C levels (P<0.001). In cultured HUVEC, the 1418T allele did not influence THBD expression but was associated with increased TM in cell lysates, increased rate of protein C activation, and reduced soluble TM levels in conditioned medium. CONCLUSIONS: The THBD 1418T allele is associated with lower soluble TM, both in plasma and in HUVEC-conditioned medium, and with an increase in functional membrane-bound TM in HUVEC, which could explain the increased activated protein C levels and the reduced VTE risk observed in individuals carrying this allele.

Effects of oral anticoagulant therapy and haplotype 1 of the endothelial protein C receptor gene on activated protein C levels.

Oral anticoagulants (OACs) reduce activated protein C (APC) plasma levels less than those of protein C (PC) in lupus erythematosus and cardiac patients. Carriers of the H1 haplotype of the endothelial PC receptor gene (PROCR) have higher APC levels than non-carriers. We aimed to confirm these results in a large group of patients treated with OACs because of venous thromboembolism (VTE) and to assess whether the effect is influenced by the PROCR H1 haplotype. We evaluated APC, PC, and factor (F)II levels in 502 VTE patients (158 with and 344 without OACs) and in 322 healthy individuals. Mean APC, PC and FII levels were significantly lower in OAC patients than in patients not taking OACs. During anticoagulant therapy, the FII/PC ratios were independent of the PC values, whereas APC/FII and APC/PC ratios significantly increased when FII and PC levels decreased. Of the 22 OAC patients carrying the H1H1genotype, 11 (50%) showed APC/PCag ≥2.0 and 10 (45%) APC/FIIag ratios ≥2.0, whereas for the 49 OAC patients non-carrying the H1 haplotype these figures were 6 (12%) and 4 (8%), respectively (p<0.001). Barium citrate adsorption of plasma from OAC patients showed that most of the circulating free and complexed APC, but only part of PCag, is fully carboxylated. In conclusion, during anticoagulant therapy VT patients have APC levels disproportionately higher than the corresponding PC levels, mainly due to the presence of the PROCR H1 haplotype. Furthermore, a sufficiently carboxylated PC Gla-domain seems to be essential for PC activation in vivo.

Implementation of SintromacWeb(R), a new internet-based tool for oral anticoagulation therapy telecontrol: Study on system consistency and patient satisfaction.

Most computer- or internet-assisted systems for oral anticoagulation therapy (OAT) telemanagement have limitations when it comes to implementation within a healthcare center. It was the objective of this study to evaluate convenience and patient satisfaction with the use of SintromacWeb, a new OAT telecontrol system, compared with the conventional control. SintromacWeb consists of a point-of-care device for patient international normalized ratio (INR) self-testing and software that allows internet mediated interaction with physicians. Patients initiated the use of SintromacWeb and were followed up during a three-month period. A score-based questionnaire was completed in three controlled visits, and data were subsequently analysed. A total of 102 patients were enrolled. At first visit, 55.7% of the patients had their INR within normal range, and 64.9% at the final visit. Internal consistency of the questionnaire was good (Cronbach's a: 0.79). Scores in the questionnaire were independent of patient's age, education level, working status and INR value. The most valued features of SintromacWeb were: fewer visits to the hospital, simplicity and convenience of the system, and time administration for control tasks (86.7%, 82.7% and 77.6% of very satisfied patients, respectively). Also, patients showed indifference or were dissatisfied with the conventional system. At the final visit, 99% of patients declared that they were satisfied with their OAT control. Moreover, all patients continued using SintromacWeb after completion of the study. In conclusion, SintromacWeb telecontrol is a new model for management of anticoagulated patients. It was highly accepted and can be used by all patients regardless of their sociodemographic characteristics.

Haplotypes of the EPCR gene, prothrombin levels, and the risk of venous thrombosis in carriers of the prothrombin G20210A mutation.

BACKGROUND: Haplotypes A1 and A3 in the endothelial protein C receptor (EPCR) gene are tagged by 4678G/C and 4600A/G respectively. We assessed whether these haplotypes modify the risk of venous thromboembolism in carriers of the prothrombin 20210A allele. DESIGN AND METHODS: We genotyped 4678G/C and 4600A/G in 246 20210A carriers: 84 venous thromboembolism propositi and 162 relatives (13 symptomatic), and in 140 relatives not carrying the 20210A variant. Prothrombin and soluble EPCR (sEPCR) levels were also measured. RESULTS: Among propositi, the mean age at first onset was lower in carriers (35 +/- 8 years) than non-carriers of the 4600G allele (44 +/- 14 years) (p = 0.004). The probability of being free of thrombosis at age 40 was lower in 20210A carriers with the EPCR 4600G allele (p = 0.015). The frequency of the 4600G allele (p=0.002) and the levels of prothrombin antigen (p = 0.002) and sEPCR (p < 0.001) were higher in the propositi than in their asymptomatic relatives. Multivariate analyses showed that the presence of the 4600G allele (OR = 2.5, 95% confidence interval 1.3-5.0), sEPCR > 147 ng/mL (2.8, 1.5-5.2) and prothrombin > 129% (3.8, 1.8-8.3) all increased the thrombotic risk. In bivariate analysis, including the 4600G allele and sEPCR > 147 ng/mL, only the latter remained associated with risk. CONCLUSIONS: These results show that in 20210A carriers the venous thromboembolism risk is influenced both by the actual prothrombin levels and by the EPCR A3 haplotype, via its effect on sEPCR levels.

Influence of lipids and obesity on haemorheological parameters in patients with deep vein thrombosis.

It is not well established whether haemorheological alterations constitute independent risk factors for deep vein thrombosis (DVT). We have determined in 149 DVT patients and in 185 control subjects the body mass index (BMI), the haemorheological profile: blood viscosity (BV), plasma viscosity (PV), fibrinogen (Fg), erythrocyte aggregation (EA), erythrocyte deformability (ED) and plasma lipids. In the crude analysis BMI, Fg, PV, EA, triglycerides (TG) and ApoB were statistically higher and HDL cholesterol (HDL-Chol) statistically lower in DVT patients than in controls. No differences in BV and ED were observed. After BMI adjustment, Fg, PV and EA remained statistically higher in DVT cases than in controls (P = 0.013; P = 0.012; P = 0.013; P = 0.028, respectively). When the risk of DVT associated with these variables (using cut-offs that corresponded to the mean plus one SD of the control group) was estimated, EA > 8.2 and PV > 1.28 mPa . s were significantly associated with DVT even further adjustment for lipids and obesity (OR = 2.78, P = 0.004; OR = 1.91, P = 0.024, respectively). However, PV did not remain statistically significant after additional adjustment for Fg. When we consider together all the analyzed variables in order to control every variable for each other, TG > 175 mg/dl (OR = 3,2, P = 0.004) and BMI > 30 kg/m(2) (OR = 3.5, P = 0.003), were also independently associated with a greater risk of DVT. Our results suggest that increased EA constitute an independent risk factor for DVT. However, when associated to hyperlipidaemia and obesity it further increases thrombotic risk.

Sialic acid is an inflammation marker associated with a history of deep vein thrombosis.

INTRODUCTION: Deep vein thrombosis (DVT) induces a systemic chronic inflammation and it has been associated with atherosclerosis. Increased levels of total sialic acid (TSA) have been shown to correlate with inflammation and atherosclerotic processes. The aim of this study was to investigate whether or not increased levels of TSA are associated with a history of DVT and with inflammation and coagulation markers, as well as with the lipid profile. MATERIALS AND METHODS: TSA, fibrinogen, C-reactive protein (CRP), fibrin D-dimer (D-dimer), prothrombin fragment 1+2 (F1+2), endogenous thrombin generation, cholesterol and triglycerides were measured in 68 patients who had suffered, in the previous 6-12 months, a first episode of idiopathic DVT, and in 68 age- and sex-matched healthy subjects. RESULTS: Levels of TSA, fibrinogen, CRP and D-dimer observed in patients were significantly higher than those detected in healthy subjects. TSA positively correlated with fibrinogen (R=0.47, p<0.01), cholesterol (R=0.46, p<0.01), triglycerides (R=0.38, p<0.01) and CRP (R=0.28, p<0.05). The logistic regression analysis confirmed that both high fibrinogen (> or =340 mg/dl) and cholesterol (> or =267 mg/dl) levels significantly and independently influence the TSA concentration. TSA levels above the 95th percentile of controls (>72 mg/dl) were detected in 33% of patients (OR=8.9; p<0.0001; 95% CI 2.4 to 31.7). CONCLUSIONS: Patients with a history of DVT had associated high levels of TSA. In these patients, TSA correlated to markers of inflammation activity and lipid profile. Thus, TSA appears to be a useful vascular inflammatory marker in idiopathic DVT.

Spontaneous renal subcapsular hematoma in an anticoagulated patient.

This is a case report of spontaneous subcapsular renal hematoma in an anticoagulated patient who was without excessive hypocoagulability and who was hemodynamically stable. Active bleeding was ruled out and a conservative treatment of discontinuing anticoagulant therapy was chosen. The patient was observed expectantly with serial abdominal computed tomography and abdominal ultrasound during her stay in the hospital. When diminution of the hematoma was detected, oral anticoagulation was resumed.

Contribution of polymorphisms in the endothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk.

Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C (APC) by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR), which is generated by metalloprotease activity, is present in plasma. The distribution of sEPCR levels in healthy populations is bimodal. Previously, we described two polymorphisms in exon 4 of the EPCR gene, 4600A/G that encodes the substitution of Ser219 by Gly in the transmembrane region of EPCR and 4678G/C in the 3'-UT region. The aim of this study was to investigate the relationship between these two polymorphisms and plasma sEPCR and APC levels and risk of venous thrombosis. We genotyped 401 healthy controls from the Spanish population and measured their plasma sEPCR and APC levels. Carriers of the 4600AG genotype had significantly higher sEPCR levels than those with the AA genotype, while the 4678CC genotype was associated, to a lesser extent, with elevated APC levels. To assess the effect of these polymorphisms on the risk of thrombosis, we genotyped 405 patients with venous thromboembolism. The frequency of the 4600AG genotype was very similar in patients and controls (p=0.975), whereas the 4678CC genotype was significantly more frequent in controls than in patients (p=0.008). In multivariate analysis, carriers of the 4678CC genotype had a decreased risk of thrombosis (OR=0.61, p=0.009). These data indicate that individuals carrying the 4600AG genotype have high sEPCR levels but do not have an increased risk of thrombosis, whereas individuals carrying the 4678CC genotype have higher APC levels and lower risk of venous thromboembolism.

Factor V Leiden and prothrombin G20210A mutations in young adults with cryptogenic ischemic stroke.

The association between factor V Leiden (FVL) and prothrombin G20210A (PT 20210) mutations and ischemic stroke remains controversial, particularly in young adults with cryptogenic stroke. Prevalence of FVL (4.1%) and PT 20210 (8.2%) mutations was assessed in 49 patients under 50 years with cryptogenic stroke and compared with controls. Odd ratio (OR) for cryptogenic stroke was 2.62 (95% CI, 0.49-13.95) for FVL and 3.75 (95% CI, 1.05-13.34) for PT 20210 and 3.28 (95% CI, 1,17-9.20) for some recognized genetic thrombophilic defect. Moreover, the OR for cryptogenic stroke in young women using oral contraceptives (OC) was 3.59 (95% CI, 1.28-10.5). When some genetic thrombophilic defect was associated with OC, the OR was much higher (OR: 14.27; 95% CI, 0.66-309.99). Our results suggest that in the Mediterranean populations the PT 20210 mutation, but not FV Leiden, is a risk factor for cryptogenic stroke in young adults. OC use is also a significant risk factor for cryptogenic stroke, which is increased in women with some genetic thrombotic risk factor.

Rheological behaviour of red blood cells in beta and deltabeta thalassemia trait.

In major and intermediate thalassemia a decrease in erythrocyte deformability and increased erythrocyte aggregability has been described, but few studies have dealt with the question of rheological red blood cell behaviour in minor beta and deltabeta thalassemia carriers, mostly in deltabeta, because it is a less common entity. To ascertain whether there are differences in red blood cell behaviour between minor thalassemia and controls and between both types of thalassemia trait beta and deltabeta, we determined erythrocyte deformability and aggregability in 30 beta and 30 deltabeta trait carriers diagnosed both with conventional methods and globin gene analysis, and in 40 age- and sex-matched controls. Erythrocyte deformability determined by means of the Rheodyn SSD showed a statistically significant lower Elongation Index (EI) at all the shear stresses tested in both thalassemic groups compared with controls (p<0.001). Minor beta thalassemia carriers showed lower EI than deltabeta carriers (p<0.001). Erythrocyte aggregability measured with the Myrenne aggregometer was significantly lower in both thalassemic groups than in controls (p<0.001), although no significant differences could be observed between both thalassemic groups. The rheological alterations found in thalassemia carriers are in part due to microcytosis, hypochromia and the morphological changes that characterize this kind of anaemia. The less altered deformability found in deltabeta carriers, is in agreement with the fact that it deals with a more benign trait.