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Background: Factor (F)V is pivotal in both procoagulant and anticoagulant mechanisms. The present report describes a novel F5 mutation in a FV-deficient patient (FV activity, 6 IU/dL; FV antigen, 32 IU/dL) complicated by recurrent deep vein thrombosis. The patient demonstrated activated protein C resistance (APCR) with compound heterozygous mutations consisting of FV-Y1961C (FV Kanazawa ) and FV-1982_1983del., Objectives: To clarify thrombotic mechanisms associated with this FV abnormality., Methods and Results: Levels of FV-1982_1983del were below the detection sensitivity in our expression experiments using human embryonic kidney 293T cells, and analyses were targeted, therefore, on the FV-Y1961C mutation. Activated partial thromboplastin time-based clotting assays demonstrated that FV-Y1961C exhibited APCR and that the reduced activated protein C (APC) susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/protein S-catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor-induced procoagulant function. These characteristics were similar to those of FV-W1920R (FV Nara ) and FV-A2086D (FV Besançon )., Conclusion: We identified a compound heterozygous FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FV Kanazawa ) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function but also impaired inhibition of tissue factor-induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of competing interests V.P. received lecture fees from Werfen Group, Milan, Italy. All other authors have no conflicts to declare.

Introduction: Activated protein C (APC) resistance is a major risk factor of venous thrombosis which may be acquired by hormonal therapy or other causes. The FibWave, a sensitive global clot-based assay design to analyze the coagulation kinetics in plasma, may be a good candidate to assess this prothrombotic state. This study aims to assess the suitability of the FibWave to differentiate the coagulation kinetics of women on oral contraceptives., Materials and Methods: Fifty-four healthy volunteers were divided into 5 groups: men [n = 13], women not using hormonal contraception [n = 12], women using second [n = 12] or third generation [n = 12] combined oral contraceptives, and women using progestin only contraceptive [n = 5]. Patients with coagulation abnormalities were also assessed [n = 8]. The APC resistance was assessed on the FibWave using exogenous APC or Protac, and on the Calibrated Automated Thrombogram using the ETP-based APC resistance assay., Results: Either in presence or in absence of APC or Protac, the FibWave was able to detect a hypercoagulable state in plasma samples. All combined oral contraceptives showed a lower FW-Max 1 , FW-Max 2, and FW-Min 2 percentage of inhibition and a lower FW-Ttpeak ratio than the other groups. The sensitivity of the FibWave was similar to the one of the ETP-based APC resistance assay., Conclusion: The FibWave is able to differentiate APC resistance levels observed in women on combined oral contraceptive. The FW-Max 1 , FW-Max 2, and to a lesser degree FW-Min 2 were identified as the most sensitive parameters with a similar performance to the ETP-based APC resistance assay., (© 2020 John Wiley & Sons Ltd.)

Objectives: Risk factors for thromboembolism in SLE are poorly understood. We hypothesized a possible role for protein C, based on its dual activity in inflammation and haemostasis and on the evidence of an association between acquired activated protein C (APC) resistance (APCR) and high-avidity anti-protein C antibodies (anti-PC) with a severe thrombotic phenotype in venous thrombosis APS patients., Methods: In a cross-sectional study of 156 SLE patients, the presence and avidity of IgG anti-PC was established by in house-ELISA, and APCR to exogenous recombinant human APC (rhAPC) and Protac (which activates endogenous protein C) was assessed by thrombin generation-based assays. Associations with aPL profile, thrombotic history and disease activity (BILAG and SLEDAI-2K) were also established., Results: Anti-PC were detected in 54.5% of patients and APCR in 59%. Anti-PC positivity was associated with APCR to both rhAPC (P <0.0001) and Protac (P =0.0001). High-avidity anti-PC, detected in 26.3% of SLE patients, were associated with APCR in patients with thrombosis only (P <0.05), and with the development of thrombosis over time (range: 0-52 years; P =0.014). High-avidity anti-PC levels correlated with SLEDAI-2K (P =0.033) and total BILAG (P =0.019); SLEDAI-2K correlated inversely with APCR to Protac (P =0.004)., Conclusion: Anti-PC occur in patients with SLE, independently of aPL profile, and are associated with APCR. High-avidity anti-PC are associated with thrombosis and with active disease and might prove a novel marker to monitor the risk of thrombosis and disease progression in SLE., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background Investigation of hemostasis is problematic when patients are on anticoagulant therapy. Rivaroxaban especially causes substantial interference, extending many clot-based tests, thereby leading to false positive or negative events. In particular, rivaroxaban affects some assays for activated protein C resistance (APCR). Methods We assessed, in an international setting, cross laboratory (n = 31) testing using four samples to evaluate rivaroxaban induced interference in APCR testing, and whether this interference could be neutralised. The samples comprised: (A) pool of normal plasma (APCR-negative control); (B) this normal pool spiked with rivaroxaban (200 ng/mL) to create rivaroxaban-induced interference (potential 'false' positive APCR event sample); (C) the rivaroxaban sample subsequently treated with a commercial direct oral anticoagulant 'DOAC-neutraliser' (DOAC Stop), or (D) treated with andexanet alfa (200 μg/mL). Testing was performed blind to sample type. Results The rivaroxaban-spiked sample generated false positive APCR results for some, but unexpectedly not most APCR-tests. The sample treated with DOAC Stop evidenced a correction in the rivaroxaban-affected APCR assays, and did not otherwise adversely affect the rivaroxaban 'unaffected' APCR assays. The andexanet alfa-treated sample did not evidence correction of the false positive APCR, and instead unexpectedly exacerbated false positive APCR status with many tests. Conclusions DOAC Stop was able to neutralise any APCR interference induced by rivaroxaban. In contrast, andexanet alfa did not negate such interference, and instead unexpectedly created more false-positive APCR events.

We aimed to review the interfering effect of DOACs on tests for haemostatic function and then to discuss overcoming these with activated carbon (AC) products, thereby eliminating DOAC issues from test plasmas. Recent relevant articles were reviewed and are discussed. Laboratory tests for DOACs, lupus anticoagulant, factor assays and APC Resistance were carried out in such publications with and without an AC product on various instruments using reagents approved for diagnostic use in well-regulated clinical laboratories. All reports on this plasma pre-treatment by AC products agree that they extract DOACs from plasma samples with minimal effect on underlying clotting tests. The specific extraction of DOACs significantly reduced false positive lupus anticoagulant detection and provided more reliable results in clotting factor assays, APC resistance and other thrombophilia tests. Dabigatran and edoxaban seem to be adsorbed more thoroughly by AC from plasmas than rivaroxaban and apixaban. In summary, most of the AC products reviewed here appear to remove DOACs from test plasmas without significantly affecting underlying clotting tests and permit correct diagnosis of various haemostatic conditions despite the initial presence of DOACs. The application of such agents as a sample pre-treatment to overcome the effects of DOACs for routine coagulation testing is supported by the emerging literature., (© 2020 John Wiley & Sons Ltd.)

Background Direct oral anticoagulants (DOACs) may cause false results of activated protein C resistance (APC-R) ratio. DOAC-Remove, a new reagent based on activated carbon, has been designed to eliminate the interference of DOACs on coagulation assays. The aim of the study was to investigate whether the use of DOAC-Remove enables to determine APC-R in patients treated with DOACs. Methods We assessed 74 venous thromboembolism (VTE) patients, including 25 on rivaroxaban, 25 on apixaban and 24 taking dabigatran. APC-R was determined using the Russell Viper Venom Time (RVVT)-based clotting test. APC-R and DOAC concentrations were tested at baseline and following DOAC-Remove. Thrombophilia, including factor V Leiden (FVL) mutation was tested. Results FVL mutation was found in 20 (27%) patients. The APC-R ratio at baseline was measurable in 43 patients (58.1%), including 20 (80%) on rivaroxaban, 19 (76%) on apixaban and four (16.7%) on dabigatran. In patients with measurable APC-R at baseline, the ratio >2.9 was found in 23 patients (53.5%). In 16 (37.2%) subjects APC-R ratio <1.8 suggested FVL mutation which was genetically confirmed. Four (9.3%) FVL carriers on dabigatran showed negative/equivocal APC-R results. In 11 (14.9%) patients taking rivaroxaban or apixaban, in whom blood was collected 2-5 h since the last dose, we observed unmeasurable APC-R. DOAC-Remove almost completely eliminated all plasma DOACs. After addition of DOAC-Remove all APC-R ratios were measurable. In four FVL carriers on dabigatran with false negative APC-R, DOAC-Remove resulted in APC-R ratios <1.8. Conclusions DOAC-Remove effectively reduces DOACs concentration in plasma, which enables FVL testing using APC-R.

Studies on last genetic and epigenetic predisposition to APS are summarized. It is well known that genetic predisposition is in HLA system (DR4 and DRw53) and that lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are both associated with the same HLA antigens. Other genes, outside the MHC, give their contribution to the development of this autoimmune syndrome, such as IRF5, STAT4 and those related to inherited thrombophilia - factor V Leiden and G20210A prothrombin polymorphisms. Finally, post-transcriptional modifications of anti-beta2GPI antibodies could be implicated too. The most important discovery of last years is that altered microRNAs' expression is linked to autoimmunity, thrombosis, early atherosclerosis, and oxidative stress in APS., (Copyright © 2019 Elsevier B.V. All rights reserved.)

: Postmenopausal hormone therapy increases the risk of venous thrombosis. Sex hormone binding globulin (SHBG) is a suggested marker of 'total estrogenicity'. The study objective was to evaluate the impact of hormone therapy on SHBG and the association with coagulation variables. The study populations comprised 202 healthy postmenopausal women randomized to treatment with low-dose or conventional-dose hormone therapy, tibolone or raloxifene (RET-study) and 140 women with a history of venous thrombosis randomized to conventional-dose hormone therapy or placebo (EVTET-study). SHBG was determined in serum collected at baseline and after 12 weeks. In healthy women, conventional-dose hormone therapy increased SHBG with mean 9.7 (95% confidence interval 4.8-14.5) nmol/l, low-dose hormone therapy by mean 5.9 (0.4-11.5) nmol/l, raloxifene by mean 7.2 (3.9-10.4) nmol/l, while tibolone reduced SHBG with mean -25.1 (-29.9 to -20.4) nmol/l. SHBG correlated with protein S, tissue factor pathway inhibitor (TFPI) and protein C at baseline, and with protein S and TFPI after 12 weeks, but the change in SHBG from baseline to 12 weeks was only associated with the change in activated protein C (APC) resistance. In women with a history of venous thrombosis, the mean increase in SHBG was 13.6 (8.4-18.9) nmol/l in the conventional-dose hormone therapy group, with no change in the placebo group. Baseline SHBG was higher among women who developed recurrent venous thrombosis on conventional-dose hormone therapy. SHBG correlated with several coagulation inhibitors, but the change in SHBG induced by postmenopausal hormone therapy was only associated with the change in APC resistance.

Introduction: To investigate the thrombotic tendency in patients with systemic lupus erythematosus (SLE) by evaluating congenital and acquired abnormalities with an increased risk of thrombosis., Patients and Methods: A total of 53 patients with SLE were included in the study. Fifty-three healthy controls paired by age and sex were assessed. Anticardiolipin antibodies (aCL), anti β2 glycoprotein (aβ2GP), lupus anticoagulant (LAC), protein C (PC), protein S (PS), antithrombin (AT), acquired activated protein C, and homocysteinemia were evaluated. Comparisons for categorical variables were analyzed by Chi 2 and student tests., Results: The patients were all female and had a mean age of 30.6 years (16/58). The healthy controls were all female and their mean age was 30.8 years (17/56). Five patients (9.4%) developed venous thrombosis during the 24 months of follow-up. The antiphospholipid antibodies were positive in 17 patients (32.1%) and negative in all healthy controls (P=0.01). PS deficiency was noted in 17 patients (32.1%) and in 5 controls (P=0.004). Hyperhomocysteinemia was noted in 16 patients (30.2%) versus 3 controls (5.6%) (P=0.002). Test for PC deficiency and acquired activated protein C showed no significant difference between the two groups. No AT deficiency was found in the patients. The study of clinical and biological correlations based on the presence and absence of thrombophilic parameters concluded to a significant association between Protein C deficit and thrombosis (P=0.02) and acquired activated protein C resistance and thrombosis (P=0.04). There was no significant association between the APL and thrombosis., Conclusion: Thrombophilic abnormalities were significantly more frequent in lupus patients than in healthy controls. Thrombotic events were significantly associated with PC deficit and acquired protein C resistance. There was no correlation between antiphospholipid antibodies and thrombosis., (Copyright © 2018. Published by Elsevier Masson SAS.)

Objective: Combined oral contraceptives induce a reversible hypercoagulable state with an enhanced risk of venous thromboembolism, but the underlying mechanism(s) remain unclear. Subjects on combined oral contraceptives also demonstrate a characteristic resistance to APC (activated protein C) in the thrombin generation assay. Here, we report the potential role of plasma factor IXa (FIXa) as a mechanism for hormone-induced systemic hypercoagulability., Approach and Results: A novel assay was used to determine FIXa activity in plasma samples from volunteer blood donors. Plasma from 36 premenopausal females on hormonal contraception and 35 not on hormonal contraception, 35 postmenopausal females, and 10 males were analyzed for FIXa activity, total PS (protein S), total tissue factor pathway inhibitor (TFPI), and TFPI-α antigen. Premenopausal females on hormonal contraception demonstrated significantly increased FIXa activity and decreased TFPI-α compared with the other groups. Remarkably, FIXa values were not normally distributed in the hormonal contraception group, but skewed toward the high end. Plasma FIXa activity inversely correlated with both TFPI-α and total PS antigen. Ex vivo determination of TF-dependent FIX activation in FV-deficient plasma demonstrated that inhibitory anti-TFPI antibodies enhanced FIXa generation by 2- to 3-fold, whereas addition of 75 nmol/L PS reduced FIXa generation by ≈2-fold. Further, increasing FIXa concentration enhanced APC resistance during TF-triggered plasma thrombin generation., Conclusions: Elevation of plasma FIXa activity in association with reductions in TFPI-α and PS is a potential mechanism for systemic hypercoagulability and resistance to APC in premenopausal females on hormonal contraception., (© 2017 American Heart Association, Inc.)

Background: The aim of this study was to evaluate ex vivo and in vitro interference of a direct factor IIa inhibitor, dabigatran, on a prothrombinase-based assay to detect activated protein C resistance., Materials and Methods: An ex vivo study was performed in six heterozygous factor V Leiden carriers and 12 normal subjects without the factor V Leiden mutation who were treated with dabigatran. An in vitro study was also performed considering 12 plasma samples (six from normal subjects and six from heterozygous factor V Leiden carriers) spiked with dabigatran. The dabigatran concentration was evaluated using a diluted thrombin time assay, activated protein C resistance was evaluated using a prothrombinase-based assay., Results: In both the ex vivo and in vitro studies dabigatran interfered significantly with activated protein C resistance ratios observed in normal subjects and in factor V Leiden heterozygous carriers., Discussion: The results reported in this paper seem to confirm that dabigatran is able to interfere with the Penthafarm prothrombinase-based assay used to study activated protein C resistance, significantly increasing observed ratios. This effect appears to be present already at low concentrations of dabigatran (6 ng/mL) and affects both normal subjects and heterozygous carriers of factor V Leiden. In this group of patients, dabigatran, at concentrations in the therapeutic range (100-200 ng/mL), could markedly increase the activated protein C resistance ratio, bringing it up to within the reference range for normal subjects, thus potentially leading to misclassification of patients.

Essentials Protein S is a cofactor of activated protein C (APC) and tissue factor pathway inhibitor (TFPI). There are no assays to quantify separate APC and TFPI cofactor activities of protein S in plasma. We developed assays to measure the APC- and TFPI-cofactor activities of protein S in plasma. The assays were sensitive to protein S deficiency, and not affected by the Factor V Leiden mutation., Summary: Background Protein S plays an important role in the down-regulation of coagulation as cofactor for activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Aim To develop functional assays to quantify the APC- and TFPI-cofactor activities of protein S in plasma. Methods APC- and TFPI-cofactor activities of protein S in plasma were measured using calibrated automated thrombography in protein S-depleted plasma supplemented with a small amount of sample plasma either in the presence of anti-TFPI antibodies and APC (APC-cofactor activity) or at excess full-length TFPI without APC (TFPI-cofactor activity). Total and free protein S levels in plasma were measured by ELISAs. Results Average APC-cofactor activities of protein S were 113%, 108% and 89% in plasma from normal individuals (n = 15), FV Leiden heterozygotes (n = 14) and FV Leiden homozygotes (n = 7), respectively, whereas the average APC-cofactor activity of protein S in plasma from heterozygous protein S-deficient individuals (n = 21) was significantly lower (55%). Similar trends were observed for the TFPI-cofactor activity of protein S, with averages of 109%, 115% and 124% in plasma from individuals with normal protein S levels and different FV Leiden genotypes, and 64% in plasma from protein S-deficient patients. APC-cofactor activities of protein S correlated significantly with free and total protein S antigen levels, whereas TFPI-cofactor activities correlated less with protein S antigen levels. Conclusion We have developed functional protein S assays that measure both the APC- and TFPI-cofactor activities of protein S in plasma, which are hardly if at all affected by the FV Leiden mutation., (© 2017 International Society on Thrombosis and Haemostasis.)

Background: In pregnancy, interpretation of results from coagulation parameters can be difficult because of the procoagulant physiological changes. The aim of this study was to describe the course of 5 coagulation parameters (thrombophilia markers) in healthy pregnancies, and to estimate and compare the within-subject biological variation (CV I ) of these parameters in healthy pregnant and nonpregnant women., Methods: Blood samples were obtained every 4th week during pregnancy and 3 samples after delivery in 20 healthy women and every 4th week during 40 weeks in 19 healthy nonpregnant women. Protein C (PC), antithrombin (AT), protein S free (PS free), protein S activity (PS activity), and activated protein C resistance (with factor V-depleted plasma) (APCR) were analyzed. Before the calculation of CV I , results were transformed into multiples of the median (MoM) and natural logarithm of MoM (lnMoM) to adjust for the physiological changes during pregnancy., Results: During pregnancy, PC results showed large variability, AT decreased slightly, and PS free and PS activity decreased significantly. Both activated partial thromboplastin time tests used to calculate APCR decreased, and the APCR ratio was constant. The CV I (lnMoM) in pregnancy were for PC 8.4%, for AT 3.8%, for PS free 11.5%, for PS activity 9.3%, and for APCR 0.5%, and similar to corresponding results in nonpregnant women., Conclusions: Transformation of coagulation parameters in healthy pregnancies to lnMoM is a tool to establish a kind of steady state. Although there is a physiological change in PC, AT, and PS free and PS activity during pregnancy, the CV I was comparable with the CV I of nonpregnant women., (© 2016 American Association for Clinical Chemistry.)

Thrombophilia due to activated protein C resistance (Leiden mutation) is the most common inherited thrombophilic disorder with 5% incidence in whites. Renal transplant of these patients entails a risk of vascular thrombosis soon after the transplant; and acute rejection episodes and graft loss within the first year. We present a case of a successful living-related renal transplant in man with a recent history of repeat episodes of vascular access thrombosis attributed to inherited thrombophilia (heterozygosity for factor V mutation Q506 and homozygosity for mutation T677 for methylene-tetrahydrofolate reductase). Transplant recipient was administered anticoagulation therapy with low molecular weight heparin pre- and postoperatively. No thrombotic or hemorrhagic events occurred posttransplant. A high suspicion of thrombophilic disorders in patients with end-stage renal disease with vascular access thrombotic events should be screened further to prevent failure of a subsequent renal transplant. Inherited thrombophilic disorders may not exclude living-related kidney transplant provided that anticoagulation therapy is admin-istered perioperatively.

Activated protein C resistance (APCR) describes a hemostatic disorder characterized by a poor anticoagulant response to activated protein C (APC). This results in an increased risk of venous thrombosis, including deep vein thrombosis and pulmonary embolism. Protein C is a natural anticoagulant that is synthesized in the liver and is activated to APC via proteolysis. APC then degrades Factors Va and VIIIa. APCR describes the reduced inability of APC to cleave Factors Va and VIIIa, which therefore promotes increased thrombin generation and potentially leads to a prothrombotic state. APCR may be hereditary or acquired. The most common hereditary defect is due to mutations in Factor V, predominantly the Factor V Leiden [FVL] mutation-a G1691A missense mutation at Arginine 506 that results in its replacement by a glutamine [R506Q] and the abolition of an APC inactivation cleavage site in Factor Va. Laboratory testing for APCR may be undertaken by a variety of methods, but this chapter describes an automated procedure using a commercial Russell Viper Venom-based clotting assay, and using CS-5100 and STA-R analyzers.

In 2010-2012, the North American Specialized Coagulation Laboratory Association (NASCOLA) distributed 12 proficiency testing challenges to evaluate laboratory testing for protein S (PS). Results were analysed to assess the performance of PS activity, PS free antigen, and PS total antigen testing. Statistical analysis was performed on the numeric results and qualitative classification submitted for each method. There were 2,106 total results: 716 results from PS activity assays, 833 results from PS free antigen assays, and 557 results from PS total antigen assays. The three assay types performed well in the classification of five normal samples and nine abnormal samples, although certain PS activity methods were more likely to classify normal samples as abnormal and one PS total antigen assay was more likely to classify abnormal samples as normal. PS activity methods were affected by interfering substances such as heterozygous or homozygous factor V Leiden mutation (underestimation) and the anticoagulant drug rivaroxaban (overestimation). In conclusion, NASCOLA laboratories using a variety of PS assays performed well in the classification of clearly normal and abnormal samples. Laboratories performing PS activity assays should be aware of potential interferences in samples positive for FV Leiden or containing certain anticoagulant medications.

Background: Risk management is a set of actions to recognize or identify risks, errors and their consequences and to take the steps to counter it. The aim of our study was to apply FMECA (Failure Mode, Effects and Criticality Analysis) to the Activated Protein C resistance (APCR) test in order to detect and avoid mistakes in this process., Methods: We created a team and the process was divided in phases and sub phases. For each phase we calculated the probability of occurrence (O) of an error, the detectability score (D) and the severity (S). The product of these three indexes yields the RPN (Risk Priority Number). Phases with a higher RPN need corrective actions with a higher priority., Results: The calculation of RPN showed that more than 20 activities have a score higher than 150 and need important preventive actions; 8 have a score between 100 and 150. Only 23 actions obtained an acceptable score lower than 100., Conclusions: This was one of the first experience of application of FMECA analysis to a laboratory process, and the first one which applies this technique to the identification of the factor V Leiden, and our results confirm that FMECA could be a simple, powerful and useful tool in risk management and helps to identify quickly the criticality in a laboratory process.

Objective: To examine whether tissue factor pathway inhibitor or acquired activated protein C (APC) resistance influences the increased risk of coronary heart disease (CHD) due to estrogen plus progestin therapy., Approach and Results: Prospective nested case-control study of 205 cases of CHD and 481 matched controls in the Women's Health Initiative randomized trial of estrogen plus progestin therapy. After multivariable covariate adjustment, both baseline tissue factor pathway activity (P=0.01) and APC resistance (P=0.004) were associated positively with CHD risk. Baseline tissue factor pathway activity and APC resistance singly or jointly did not significantly modify the effect of estrogen plus progestin on CHD risk. Compared with placebo, estrogen plus progestin decreased tissue factor pathway inhibitor activity and increased APC resistance but these changes did not seem to modify or mediate the effect of estrogen plus progestin on CHD risk., Conclusions: Tissue factor pathway inhibitor activity and APC resistance are related to CHD risk in women, but may not explain the increased CHD risk due to estrogen plus progestin therapy. The data from this study do not support the clinical use of measuring these hemostatic factors to help stratify risk before hormone therapy., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00000611., (© 2015 American Heart Association, Inc.)

Factor V Leiden (FVLeiden ) is a common hereditary thrombophilia that causes activated protein C (APC) resistance. This review describes many of the most fascinating features of FVLeiden , including background features, mechanisms of hypercoagulability, the founder mutation concept, the "FVLeiden paradox," synergistic interaction with other thrombotic risk factors, the intertwined relationship between FVLeiden and APC resistance testing, and other, uncommon mutations implicated in causing APC resistance. In addition, there are several conditions where laboratory tests for APC resistance and FVLeiden are or can be discrepant, including lupus anticoagulants, anticoagulants such as direct thrombin inhibitors (dabigatran, argatroban, and bivalirudin) and rivaroxaban, as well as pseudohomozygous, pseudo-wildtype, liver transplant, and bone marrow transplant patients. The laboratory test error rate for FVLeiden is also presented., (© 2015 Wiley Periodicals, Inc.)

Background: The early diagnosis of inherited thrombophilia in children is challenging because of the rarity and hemostatic maturation., Methods: We explored protein C (PC), protein S (PS), and antithrombin (AT) deficiencies in 306 thromboembolic patients aged ≤20 y using the screening of plasma activity and genetic analysis., Results: Reduced activities were determined in 122 patients (40%). Low PC patients were most frequently found in the lowest age group (0-2 y, 45%), while low PS or low AT patients were found in the highest age group (16-20 y; PS: 30% and AT: 20%). Genetic study was completed in 62 patients having no other causes of thromboembolism. Mutations were determined in 18 patients (8 PC, 8 PS, and 2 AT genes). Six of eight patients with PC gene mutation were found in age 0-2 y (75%), while six of eight patients with PS gene mutation were in 7-20 y. Two AT gene-mutated patients were older than 4 y. Four PC-deficient and two PS-deficient patients carried compound heterozygous mutations. All but one PC gene-mutated patient suffered from intracranial thromboembolism, while PS/AT gene-mutated patients mostly developed extracranial venous thromboembolism., Conclusion: Stroke in low PC infants and deep vein thrombosis in low PS/AT school age children could be targeted for genetic screening of pediatric thrombophilias.

Introduction: Activated protein C resistance (APC-R) due to FV Leiden is the most common hereditary thrombophilia. Rarer FV mutations can also confer APC-R, and acquired APC-R is encountered in a number of conditions. APC-R screening with clotting tests is common, yet they are prone to interferences and elevated baseline clotting times can invalidate testing., Methods: APTT-based classic APC-R (CAPC-R) screening, and modified screening (MAPC-R) employing dilution in FV-deficient plasma were performed on an automated analyser. Baseline clotting times and APC-R ratios of 1340 patients being screened for hereditary and acquired thrombophilia were assessed for analytical and diagnostic validity., Results: Most patients (1117/1340) had normal baseline clotting times, and in 270 of these cases, this was despite the presence of a lupus anticoagulant (LA). FV Leiden was genetically confirmed in all patients with reduced CAPC-R and MAPC-R ratios. A subgroup with normal CAPC-R but reduced MAPC-R also identified FV Leiden, but also other patients with minimally reduced MAPC-R ratios not due to FV Leiden. Reduced CAPC-R and normal modified APC-R identified possible acquired APC-R in 49 patients. LA-positive patients with elevated baseline clotting times did not affect distinction between APC-R and normality, although therapeutic anticoagulation did invalidate CAPC-R, and occasionally MAPC-R too., Conclusions: Many departments only screen with MAPC-R to detect just FV mutations. Concurrent performance of CAPC-R and MAPC-R increases diagnostic capability by detecting acquired APC-R. Elevated baseline clotting times can invalidate APC-R ratios, although prolongation by LA alone may not., (© 2015 John Wiley & Sons Ltd.)

Objectives: Preeclampsia complicates 2-8% of all pregnancies. Studies on the association of preeclampsia with thrombophilia are conflicting. Clinical heterogeneity of the disease may be one of the explanations. The present study addresses the question whether different phenotypes of preeclampsia are associated with thrombophilia factors. Study design We planned a retrospective cohort study. From 1985 until 2010 women with preeclampsia were offered postpartum screening for the following thrombophilia factors: anti-phospholipid antibodies, APC-resistance, protein C deficiency and protein S deficiency, hyperhomocysteineamia, factor V Leiden and Prothrombin gene mutation. Hospital records were used to obtain information on phenotypes of the preeclampsia and placental histology., Results: We identified 844 women with singleton pregnancies who were screened for thrombophilia factors. HELLP complicated 49% of pregnancies; Fetal growth restriction complicated 61% of pregnancies. Early delivery (<34th week) occurred in 71% of pregnancies. Any thrombophilia factor was present in 29% of the women. Severe preeclampsia was associated with protein S deficiency (p=0.01). Fetal growth restriction was associated with anti-phospholipid antibodies (p<0.01). Early onset preeclampsia was associated with anti-phospholipid antibodies (p=0.01). Extensive placental infarction (>10%) was associated with anti-phospholipid antibodies (p<0.01). Low placental weight (<5th percentile) was associated with hyperhomocysteineamia (p=0.03). No other associations were observed., Conclusions: Early onset preeclampsia, especially if complicated by fetal growth restriction, are associated with anti-phospholipid antibodies. Other phenotypes of preeclampsia, especially HELLP syndrome, were not associated with thrombophilia. We advise only to test for anti-phospholipid antibodies after early onset preeclampsia, especially if complicated by fetal growth restriction. We suggest enough evidence is presented to justify no further studies are needed., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The anticoagulant effect of dabigatran can be approximated by its prolongation of routine coagulation assays. Consequently, dabigatran also interferes with thrombophilia screening or with diagnosing hemostasis disorders that have developed after the initiation of anticoagulant treatment, such as vitamin K deficiency or acquired hemophilia A., Objectives: This study was carried out to determine whether idarucizumab, a humanized antibody fragment that binds dabigatran, could fully neutralize dabigatran in routine diagnostic coagulation assays conducted in vitro, thereby preventing false-positive or false-negative diagnostic readouts., Methods: Preliminary experiments identified coagulation assays that were sensitive to dabigatran, and identified a concentration of idarucizumab that neutralized the effects of dabigatran. These assays were then carried out with patient and control plasma samples spiked with dabigatran, with or without a molar excess of idarucizumab., Results: Dabigatran altered the prothrombin time, activated partial thromboplastin time and thrombin time, and the measurement of intrinsic and extrinsic factor levels. Screening and confirmation tests used for lupus anticoagulant detection were prolonged by dabigatran, falsely suggesting the presence of lupus anticoagulant. Conversely, the addition of dabigatran falsely corrected an abnormal activated protein C resistance ratio. Addition of idarucizumab completely normalized these measurements, and allowed the correct identification of normal and abnormal samples with these assays., Conclusions: In vitro addition of idarucizumab to plasma samples containing dabigatran fully neutralizes the drug, and facilitates the use of routine coagulation assays to allow the diagnosis of hemostasis disorders that may be concurrently present in patients taking dabigatran., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: The high allelic frequency of the prothrombotic Leiden polymorphism in human blood coagulation factor V (FV) has been speculated to reflect positive selection during evolution. Heterozygous Leiden carriers enrolled in the placebo arm of the PROWESS sepsis trial and heterozygous Leiden mice challenged with endotoxin both showed reduced mortality, whereas homozygous Leiden mice were not protected from lethal endotoxemia. Follow-up analyses of clinical outcomes and of mouse models of infection with various pathogens remained inconclusive., Objective: To establish whether activated protein C resistance of FV Leiden modifies the outcome of bacterial infection in murine sepsis models., Methods: Homozygous and heterozygous FV Leiden mice were subjected to gram-positive (S. aureus) or gram-negative (Y. pestis; E. coli) septic peritonitis or polymicrobial, focal septic peritonitis induced by cecal ligation and puncture. The effect of FV Leiden on 7-day survival and bacterial dissemination was assessed. Outcomes were compared with the sepsis survival of mice with genetically impaired hemostasis (hemophilia A, thrombocytopenia, thrombin receptor PAR4 [protease activated receptor 4] deficiency, endothelial protein C receptor [ProcR/EPCR] deficiency)., Results: Heterozygous, but not homozygous, Leiden mice were protected from lethal infection with highly virulent S. aureus and Y. pestis strains. FV Leiden did not affect the outcome of sepsis induced by cecal ligation and puncture, staphylokinase-deficient S. aureus, Pla-deficient Y. pestis, or E. coli. Thrombocytopenia, deficiency of PAR1 or PAR4 did not affect S. aureus sepsis survival, whereas hemophilia A increased mortality. ProcR deficiency selectively abolished the survival advantage of heterozygous Leiden mice., Conclusions: In mice, heterozygous FV Leiden carriers are protected from sepsis mortality after infection with clinically relevant human bacterial pathogens., (© 2015 International Society on Thrombosis and Haemostasis.)

To assess the impact of preanalytical variables of time and temperature on prothrombin time (PT), activated partial thromboplastin time (aPTT), dilute Russell viper venom time (DRVVT), activated protein C resistance (APCR), and d-dimer, samples from 23 healthy individuals and 18 patients having coagulopathy with known abnormal PT and aPTT were collected. Plasma from each individual was separately pooled and aliquoted; the first 2 aliquots were stored at room temperature then analyzed at 2 hours (baseline) and 4 hours postcollection. The remaining aliquots were stored at -20°C and thawed for analysis at 48 hours, 1, and 2 weeks. In both healthy participants and participants with coagulopathy, PT, aPTT, APCR, DRVVT, and D-dimer had no significant changes at 4 and 48 hours, and 1 and 2 weeks postcollection compared to baseline, or the changes were less than 10%. The results indicate PT, aPTT, DRVVT, APCR, and d-dimer can be stored for 2 weeks at -20°C without compromising clinical interpretation in both healthy individuals and individuals with coagulopathy. Increasing storage time will facilitate sample processing from off-site clinics., (© The Author(s) 2014.)

Background: Patients with cancer are at an increased risk for venous thromboembolism (VTE). The risk varies markedly in different patient populations. Factor V (FV) Leiden is the most common genetic risk factor for VTE, and the impact of FV Leiden on cancer-associated thrombosis is not yet fully elucidated., Objective: To study the impact of FV Leiden on the risk of thrombosis in cancer patients., Methods: In the prospective observational Vienna Cancer And Thrombosis Study (CATS), 982 patients were included and were followed until occurrence of VTE or death, for a maximum period of 2 years. FV Leiden was determined by genotyping at inclusion. Main outcome measures were symptomatic or lethal objectively confirmed VTE., Results: Of the 982 patients, FV Leiden was diagnosed in 72 (7.3%, 70 were heterozygous and 2 were homozygous). Ten of 72 (13.9%) patients with FV Leiden developed VTE, whereas this was the case in 69 of 910 (7.6%) patients without FV Leiden. In multivariate analysis that included age, sex, different tumor types, tumor stage, newly diagnosed vs. recurrence of disease, and the treatment modalities, the hazard ratio was 2.0 (95% confidence interval 1.0-4.0). In Kaplan-Meier analysis, the probability for development of VTE was 13% in those with and 5.7% in those without FV Leiden after 6 months; after 1 year, the corresponding risks were 15% and 7.3%., Conclusions: FV Leiden is a genetically determined and thus disease-independent parameter, which is associated with VTE in cancer patients and could therefore be used for individual risk assignment., (© 2014 International Society on Thrombosis and Haemostasis.)

Activated protein C resistance assays can detect factor V Leiden with high accuracy, depending on the method used. Factor Xa inhibitors such as rivaroxaban and direct thrombin inhibitors including dabigatran, argatroban, and bivalirudin can cause falsely normal results. Lupus anticoagulants can cause incorrect results in most current assays. Assays that include dilution into factor V-deficient plasma are needed to avoid interference from factor deficiencies or elevations, which can arise from a wide variety of conditions such as warfarin, liver dysfunction, or pregnancy. The pros and cons of the currently available assays are discussed., (© 2014 Wiley Periodicals, Inc.)

Thrombophilia is a heritable blood disease characterized by an increased tendency to form abnormal blood clots that can block blood vessels. In obstetrics and gynecology, it has been shown by a number of reports that a proportion of recurrent miscarriages involve thrombophilia-related mutations, in particular, Factor V G1691A, prothrombin G20210A, and MTHFR C677T and A1298C. In this study, we examined the frequency of these four mutations in 113 female Turkish patients who had prior complications in pregnancy, using the DiagCor GenoFlow Thrombophilia Array Test kit. Heterozygous MTHFR C677T and A1298C mutations were detected in 46% of the patients, and among these patients, 60% of them carried double heterozygous mutations. In contrast, the heterozygous Factor V G1691A and prothrombin G20210A were detected only in a smaller number of patients, respectively, 13% and 3%. The GenoFlow kit demonstrated 100% concordance with results from Sanger sequencing, which can be translated into sensitivity and specificity both at 100% within this series of patients.

Background: Antiphospholipid antibodies may interfere with the anticoagulant activity of activated protein C (APC) to induce acquired APC resistance (APCr)., Aims: To investigate the frequency and characteristics of APCr by using recombinant human APC (rhAPC) and endogenous protein C activation in antiphospholipid syndrome (APS)., Methods: APCr was assessed in APS and non-APS venous thromboembolism (VTE) patients on warfarin and normal controls with rhAPC or Protac by thrombin generation. IgG anti-protein C and anti-protein S antibodies and avidity were assessed by ELISA., Results: APS patients showed greater resistance to both rhAPC and Protac than non-APS patients and normal controls (median normalized endogenous thrombin potential inhibition): APS patients with rhAPC, 81.3% (95% confidence interval [CI] 75.2-88.3%; non-APS patients with rhAPC, 97.7% (95% CI 93.6-101.8%; APS patients with Protac, 66.0% (95% CI 59.5-72.6%); and non-APS patients with Protac, 80.7 (95% CI 74.2-87.2%). APS patients also had a higher frequency and higher levels of anti-protein C antibodies, with 60% (15/25) high-avidity antibodies. High-avidity anti-protein C antibodies were associated with greater APCr and with a severe thrombotic phenotype (defined as the development of recurrent VTE while patients were receiving therapeutic anticoagulation or both venous and arterial thrombosis). Twelve of 15 (80%) patients with high-avidity anti-protein C antibodies were classified as APS category I., Conclusion: Thrombotic APS patients showed greater APCr to both rhAPC and activation of endogenous protein C by Protac. High-avidity anti-protein C antibodies, associated with greater APCr, may provide a marker for a severe thrombotic phenotype in APS. However, in patients with category I APS, it remains to be established whether anti-protein C or anti-β2 -glycoprotein I antibodies are responsible for APCr., (© 2014 International Society on Thrombosis and Haemostasis.)

Activated protein C (APC) resistance, often associated with the factor V (FV) Leiden mutation, is the most common risk factor for venous thrombosis. We observed increased APC resistance in carriers of fibrinogen γ gene (FGG) haplotype 2, which is associated with reduced levels of the alternatively spliced fibrinogen γ' chain. This finding prompted us to study the effects of fibrinogen and its γ' chain on APC resistance. Fibrinogen, and particularly the γA/γ' isoform, improved the response of plasma to added APC in the thrombin generation-based assay. Similarly, a synthetic peptide mimicking the C-terminus of the fibrinogen γ' chain, which binds thrombin and inhibits its activities, greatly increased the APC sensitivity of normal and FV Leiden plasma, likely due to its ability to inhibit thrombin-mediated activation of FV and FVIII. Although the fibrinogen γ' peptide also inhibited protein C activation by the thrombin/thrombomodulin complex, it still increased the sensitivity of plasma to endogenously formed APC when thrombin generation was measured in the presence of soluble thrombomodulin. We conclude that fibrinogen, and particularly fibrinogen γ', increases plasma APC sensitivity. The fibrinogen γ' peptide might form the basis for pharmacologic interventions to counteract APC resistance., (© 2014 by The American Society of Hematology.)

Introduction: Use of combined hormonal contraceptives is associated with a three- to eight-fold increased risk of venous thrombosis compared with non-use. The thrombotic risk depends on the estrogen dose as well as the progestogen type. Use of hormonal contraceptives leads to resistance to activated protein C (APC), which may serve as marker for the risk of venous thrombosis. Hyperthyroidism is also associated with an increased risk of venous thrombosis, due to increased free Thyroxine (FT4) levels which cause a hypercoagulable state., Materials and Methods: The objective of this study was to evaluate the effects of hormonal contraceptives on levels of FT4, thyroid stimulating hormone (TSH) and thyroxine binding globulin (TBG), and to investigate the effects on APC resistance per contraceptive group. We measured FT4, TBG and TSH levels and APC resistance in 231 users of oral contraceptives., Results: Users of the most thrombogenic hormonal contraceptives, i.e. containing desogestrel, cyproterone acetate or drospirenone, had higher TBG levels than users of less thrombogenic hormonal contraceptives, i.e. the levonorgestrel-containing intrauterine device. TSH levels were not significantly elevated and FT4 levels did not change. TBG levels were also associated with APC resistance., Conclusion: Use of hormonal contraceptives lead to elevated TBG levels, slightly elevated TSH levels and unchanged FT4 levels without causing a hyperthyroid state. Thus, the increased thrombotic risk during the use of hormonal contraceptives cannot be explained by a hyperthyroid state caused by use of these hormonal contraceptives., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hypercoagulability is a well-documented and prominent risk factor for venous thromboembolism. The role of thrombophilia in arterial thrombotic events is less well defined. A 52-year-old male patient with multiple atherogenic risk factors was admitted for non-healing pedal ulcer and absent distal pulses. Based on the clinical presentation, Doppler ultrasound and angiography findings, the patient underwent elective in situ bypass arterial reconstruction. The saphenous vein graft was of satisfactory quality and the procedure went routinely. Acute graft thrombosis on postoperative day 0 was recognized immediately and prompted an emergent surgical revision. No technical errors or anatomical/mechanical causes for failed reconstruction were found and the graft was successfully thrombectomized using a Fogarty balloon-catheter. Graft rethrombosis, however, ensued after several hours. Considering the absence of threatening limb ischemia and the idiopathic recurrent thrombosis, raising suspicion of prothrombotic state, conservative treatment was pursued. Postoperative thrombophilia testing proved positive for activated protein C resistance, mandating introduction of chronic oral anticoagulation. Six months later, the operated extremity is viable. Inexplicable vascular graft thrombosis, particularly if early and recurrent, should raise suspicion of underlying thrombophilia. If confirmed by laboratory testing, long-term secondary antithrombotic prophylaxis may be required.

Mesenteric venous thrombosis (MVT) is a rare but life threatening form of bowel ischemia. It is implicated in 6%-9% of all cases of acute mesenteric ischemia. The proportion of patients with primary (or idiopathic) MVT varies from 0% to 49%, with a decrease in frequency secondary to more recent availability of newer investigations for hypercoagulability. The presence of factor V Leiden (FVL) and prothrombin G20210A mutations (PGM) have been well documented in these cases. However, there have been scarce case reports describing MVT in heterozygotes of both these mutations occurring simultaneously and its implications on long term management. Our case describes acute MVT in a previously asymptomatic young patient with no prior history of venous thromboembolism. The patient was found to be heterozygous for FVL and PGM and treated with lifelong anticoagulation with warfarin (goal international normalized ratio: 2-3) and avoidance of hormonal contraceptives.

Objective: Acquiring new information to allow prediction of the development of diseases associated with impaired coagulation. Design effective preventive measures most serious diseases (TEN) in the fields of gynecology and obstetrics. For pregnant women with preeclampsia, hypertension compared with women with normal pregnancies could lead to increased thrombin generation due to the synergistic effect of thrombotic risk factors. Based on the results and found statistically significant differences between the groups among pregnant can select for a higher risk of developing deep vein thrombosis. This risk group could then greatly benefit from more stringent follow-up and possible preventive treatment prophylactic doses of LMWH in reducing maternal and perinatal morbidity and mortality., Design: Prospective study., Setting: Department of Obstetrics and Gynecology, University Hospital Olomouc., Methods: In early pregnancy - during pregnancy standard samples (up to the end of the first trimester) patients venous blood was sampled and they completed information questionnaire. A second sampling was carried out between 24 to 28 week, the third sample and between 36th to 40th week. Obtained blood samples were subsequently processed in the coagulation laboratory Hemato-Oncology Clinic and Olomouc. The blood samples were investigated protein C and S, antithrombin, FVIII level, FII, Leiden, and plasma endothelial microparticles, and lupus anticoagulant and APC resistance standardized methodologies. Thrombin generation was determined thrombin generation test. Thrombin generation was measured fully automatically using a kit (Technothrombin TGA, Technoclone, Vienna, Austria) and analyzer Ceveron Alpha (Technoclone, Vienna, Austria) with fully automatic analysis software. As the main parameter is evaluated by the maximum thrombin generation, at the same time, however, was also detected in the total amount of thrombin and the time until the beginning of the formation of thrombin., Results: In the period 2008-2011 were analyzed blood samples of 303 healthy pregnant women. 215 women, ie 71% were nuliparas, 60 women, ie 19.8% were primiparas, 28 women, 9,2% were secundiparas. The average age of pregnant women was 28.6 years(± 3.8 years). The average maternal weight at the beginning of pregnancy was 63.6 kg (± 7.8 kg). Of the 303 women in 18 (6%) developed slight to moderate degree of preeclampsia or HELLP syndrome with varying severity of clinical manifestations. 20 mothers (6.6%) gave birth prematurely terminated before 37 week of pregnancy. 3 pregnancies (0.9%) were discontinued due to genetic indication for fetal birth defect. The complete study protocol (sampling in all three trimesters) thus completed 280 pregnancies. Of the three evaluated, parameter Lag time, ETP and peak we observed significant differences when comparing physiological pregnancies and pregnancies with preeclampsia (Table 3 and Figure 5-7), the statistical level of p < 0.01. In pregnancies with chronic hypertension, these differences were not significant. Comparison of 18 pregnancies, in which the III. trimester developed preeclampsia with other pregnant with physiological pregnancy did not show statistically significant differences in I. and II. trimester. The results suggest the activation of coagulation through the late stages of pregnancy. Results are influenced by strong clinical variability of disease. In severe and early preeclampsia this activation and significant differences begin much earlier., Conclusion: We demonstrated significantly higher activation of thrombin generation in women with preeclampsia [10]. Changes in preeclampsia are characterized by increased generation of thrombin in plasma. This fact may explain the partial success of the clinical use of aspirin in preeclampsia. In the third trimester, during the manifestation of the disease, patients with preeclampsia have significantly higher ETP compared to patients with a normal pregnancies. Pregnant women with chronic hypertension also show a slight increase in the activation of thrombin. However, these results are not statistically significant. Examination of coagulation in the first and second trimester in women who later developed preeclampsia, showed no statistically significant differences and thus can not be used in this case as predictive, but only as a diagnostic test.

Objectives: The current case-control study was aimed to determine the prevalence and the clinical significance of inherited thrombophilia - factor V Leiden and G20210A prothrombin polymorphisms - in patients with antiphospholipid syndrome (APS)., Methods: 100 patients with APS (77 with primary APS and 23 with systemic lupus erythematosus [SLE]-APS), and 100 patients with first lower extremity deep venous thrombosis (DVT), and 200 healthy individuals as a control groups were analysed. Patients and control group were tested for factor V Leiden and prothrombin G20210A gene polymorphism., Results: Factor V Leiden variant was found in 1% of APS patients, in 3% of healthy individuals (p=0.49), and 16% of patients with first DVT (p<0.0005). Prothrombin gene polymorphism was found in 6% of APS patients and in 2.5% of healthy subjects (p=0.21), and 13% of patients with DVT (p=0.14). In primary APS patients, factor V Leiden was present in 1.3% (1/77) and prothrombin gene polymorphism in 6.5% (5/77). No patient with SLE-APS had factor V Leiden and prothrombin gene variant was present in only one patient (4.3%). Patients with prothrombin polymorphism had higher prevalence of venous thrombosis, with no statistical significance (80% vs. 47.9%, p=0.35). There were no differences in the prevalence of recurrent thrombosis before or after APS diagnosis in patients with or without prothrombin gene polymorphism., Conclusions: Factor V Leiden and G20210A prothrombin variant seem to play no role in either the development of APS or in the type of involved vessel, with no increased risk of re-thrombosis during follow-up.

To prospectively evaluate the potential influence of resistance to activated protein C (APC-resistance) on the initial inflammatory response, amputation rate and survival during 10 years of follow-up in patients with critical limb ischemia (CLI). Two hundred and fifty-six consecutive CLI patients were analyzed for APC-ratio, the Factor V Leiden mutation and inflammatory mediators and then prospectively followed for 10 years. Inflammatory mediators, amputation rate, morbidity and mortality were compared between patients with and without APC resistance. Of the 256 CLI patients, 35 (14 %) were heterozygotes and 2 (1 %) homozygotes for the Factor V gene mutation, whereas 219 (86 %) patients were non-APC resistant. No significant differences were found between APC resistant and non-APC resistant patients regarding inflammatory mediators. Non-APC resistant patients more often had infrainguinal atherosclerosis (172 [79 %] vs 22 [59 %]; p = 0.017). Amputation rate at 1 year did not differ. Furthermore, there were no significant differences between groups regarding 1-, 3-, 5-, or 10-year survival. APC resistance in patients with CLI was not related to inflammatory activity, and had no impact on limb salvage or rate of amputation or long-term mortality. APC-resistant CLI-patients less frequently had infrainguinal arteriosclerosis, however.

The process of human blood clotting involves a complex interaction of continuous-time/continuous-state processes and discrete-event/discrete-state phenomena, where the former comprise the various chemical rate equations and the latter comprise both threshold-limited behaviors and binary states (presence/absence of a chemical). Whereas previous blood-clotting models used only continuous dynamics and perforce addressed only portions of the coagulation cascade, we capture both continuous and discrete aspects by modeling it as a hybrid dynamical system. The model was implemented as a hybrid Petri net, a graphical modeling language that extends ordinary Petri nets to cover continuous quantities and continuous-time flows. The primary focus is simulation: (1) fidelity to the clinical data in terms of clotting-factor concentrations and elapsed time; (2) reproduction of known clotting pathologies; and (3) fine-grained predictions which may be used to refine clinical understanding of blood clotting. Next we examine sensitivity to rate-constant perturbation. Finally, we propose a method for titrating between reliance on the model and on prior clinical knowledge. For simplicity, we confine these last two analyses to a critical purely-continuous subsystem of the model.

Objectives: To evaluate the performance of a Russell viper venom-based activated protein C resistance (APCR) screening test relative to DNA analysis for the factor V Leiden mutation., Methods: We evaluated the concordance between Pefakit APCR screening results and DNA analysis for 435 patients homozygous (n = 11), heterozygous (n = 310), or wild-type (n =114) for the G1691A allele., Results: Using receiver operating characteristic analysis, we found that a cutoff of 1.89 for the APCR ratio yields a sensitivity and specificity of 99.1%. In patients with discrepant genotype-phenotype correlation, their APCR may provide a more clinically relevant result., Conclusions: We compared several strategies for employing reflex testing and found that performing initial APCR screening followed by confirmatory molecular analysis on a subset of cases in the borderline regions between the diagnostic groups can reduce unnecessary testing by approximately 80% without compromising diagnostic accuracy.

Background: The use of combined oral contraceptives is associated with a 3- to 6-fold increased risk of venous thrombosis. This increased risk depends on the estrogen dose as well as the progestogen type of combined oral contraceptives. Thrombin generation-based activated protein C resistance (APC resistance) and sex hormone-binding globulin (SHBG) levels predict the thrombotic risk of a combined hormonal contraceptive. Recently, a four-phasic oral contraceptive containing dienogest (DNG) and estradiol valerate (E2V) has been marketed. The aim of this study was to evaluate the thrombotic risk of the DNG/E2V oral contraceptive by comparing APC resistance by measuring normalized APC sensitivity ratios (nAPCsr) and SHBG levels in users of oral contraceptives containing dienogest and estradiol valerate (DNG/E2V) and oral contraceptives containing levonorgestrel and ethinyl estradiol (LNG/EE)., Methods: We conducted a single-center, randomized, open label, parallel-group study in 74 women using DNG/E2V or LNG/EE, and measured nAPCsr and SHBG levels in every phase of the regimen of DNG/E2V., Results: During the pill cycle SHBG levels did not differ between DNG/E2V users and LNG/EE users. nAPCsr levels were overall slightly lower in DNG/E2V users than in LNG/EE users, mean difference -0.44 (95% CI, -1.04 to 0.17) for day 2, -0.20 (95% CI, -0.76 to 0.37) for day 7, -0.27 (95% CI, -0.81 to 0.28) for day 24 and -0.34 (95% CI, -0.91 to 0.24) for day 26., Conclusion: No statistical significant differences in nAPCsr and SHBG levels were found between users of the oral contraceptive containing DNG/E2V and LNG/EE, suggesting a comparable thrombotic risk., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Factor V Leiden (FVL) has been associated with ischemic stroke in children but not in adults. Although the FVL mutation is associated with increased risk for venous thrombosis, its association with ischemic stroke in young adults remains uncertain. Therefore, we examined the association between FVL and ischemic stroke in participants of the Genetics of Early Onset Stroke (GEOS) study., Methods: A population-based case control study identified 354 women and 476 men 15 to 49 years of age with first-ever ischemic stroke and 907 controls. Participant-specific data included vascular risk factors, FVL genotype and, for cases, the ischemic stroke subtype by modified Trial of ORG 10172 in Acute Stroke criteria. Logistic regression was used to calculate odds ratios for the entire population and for subgroups stratified by risk factors and ischemic stroke subtype., Results: The frequency of the FVL mutation was similar between ischemic stroke patients (3.6%; 95% confidence interval [CI] 2.5%-5.1%) and nonstroke controls (3.8%; 95% CI 2.7%-5.2%). This frequency did not change significantly when cases were restricted to patients with stroke of undetermined etiology (4.1%; 95% CI 2.6%-6.4%)., Conclusions: Among young adults, we found no evidence for an association between FVL and either all ischemic stroke or the subgroup with stroke of undetermined etiology., (Published by Elsevier Inc.)