Activated Protein C Resistance Ratio Research Articles

Apixaban Does Not Interfere With Protein S or Activated Protein C Resistance (Factor V Leiden) Testing Using aPTT-Based Methods.

Apixaban causes a false increase in activated protein C resistance (APCR) ratios and possibly protein S activity. To investigate whether this increase can mask a diagnosis of factor V Leiden (FVL) or protein S deficiency in an actual population of patients undergoing apixaban treatment and hypercoagulation testing. During a 4.5-year period involving 58 patients, we compared the following 4 groups: heterozygous for FVL (FVL-HET)/taking apixaban, wild-type/taking apixaban, heterozygous for FVL/no apixaban, and normal APCR/no apixaban. Patients taking apixaban were also tested for protein S functional activity and free antigen (n = 40). FVL-HET patients taking apixaban had lower APCR ratios than wild-type patients (P < .001). Activated protein C resistance in FVL-HET patients taking apixaban fell more than 3 SD below the cutoff of 2.2 at which the laboratory reflexes FVL DNA testing. No cases of FVL were missed despite apixaban. In contrast to rivaroxaban, apixaban did not interfere with the assessment of protein S activity (mean activity 93.9 IU/dL, free antigen 93.1 IU/dL, P = .39). A total of 3 of 40 patients (8%) had low free protein S antigen (30, 55, and 57 IU/dL), with correspondingly similar activity results (27, 59, and 52 IU/dL, respectively). Apixaban did not cause a missed diagnosis of protein S deficiency. Despite apixaban treatment, APCR testing can distinguish FVL-HET from healthy patients, rendering indiscriminate FVL DNA testing of all patients on apixaban unnecessary. Apixaban did not affect protein S activity.

DOAC-Remove abolishes the effect of direct oral anticoagulants on activated protein C resistance testing in real-life venous thromboembolism patients.

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.

Rivaroxaban Causes Missed Diagnosis of Protein S Deficiency but Not of Activated Protein C Resistance (Factor V Leiden).

- Rivaroxaban causes a false increase in activated protein C resistance (APCR) ratios and protein S activity. - To investigate whether this increase masks a diagnosis of factor V Leiden (FVL) or protein S deficiency in a "real-world" population of patients undergoing rivaroxaban treatment and hypercoagulation testing. - During a 2.5-year period, we compared 4 groups of patients (n = 60): FVL heterozygous (FVL-HET)/taking rivaroxaban, wild-type/taking rivaroxaban, FVL-HET/no rivaroxaban, and normal APCR/no rivaroxaban. Patients taking rivaroxaban were tested for protein S functional activity and free antigen (n = 32). - The FVL-HET patients taking rivaroxaban had lower APCR ratios than wild-type patients ( P < .001). For FVL-HET patients taking rivaroxaban, mean APCR was 1.75 ± 0.12, versus 1.64 ± 0.3 in FVL-HET patients not taking rivaroxaban ( P = .005). Activated protein C resistance in FVL-HET patients fell more than 3 SDs below the cutoff of 2.2 at which the laboratory reflexes FVL DNA testing. No cases of FVL were missed despite rivaroxaban. In contrast, rivaroxaban falsely elevated functional protein S activity, regardless of the presence or absence of FVL ( P < .001). A total of 4 of 32 patients (12.5%) had low free protein S antigen (range, 58%-67%), whereas their functional protein S activity appeared normal (range 75%-130%). Rivaroxaban would have caused a missed diagnosis of all cases of protein S deficiency during the study if testing relied on the protein S activity assay alone. - Despite rivaroxaban treatment, APCR testing can distinguish FVL-HET from normal patients, rendering indiscriminate FVL DNA testing of all patients on rivaroxaban unnecessary. Free protein S should be tested in patients taking rivaroxaban to exclude hereditary protein S deficiency.

Activated protein C resistance in patients following venous thromboembolism receiving rivaroxaban versus vitamin K antagonists: assessment using Russell viper venom time-based assay.

: Activated protein C resistance (APC-R) is assessed as part of thrombophilia screening, preferably in patients not taking oral anticoagulants. Rivaroxaban is known to alter some APC-R assays. To our knowledge, there have been no reports on the effect of rivaroxaban on the Russell viper venom time (RVVT)-based APC-R assay in real-life patients. In 168 consecutive outpatients suspected of having venous thromboembolism because of thrombophilia, APC-R was determined using the RVVT-based ProC Ac R assay (Siemens, Marburg, Germany). Patients receiving rivaroxaban or vitamin K antagonists were eligible. We measured rivaroxaban concentrations using the anti-Xa Biophen DiXal assay (Hyphen Biomed, Neuville-Sur-Oise, France) and factor V Leiden using the real-time PCR. APC-R was detected in 23 (28%) patients on rivaroxaban (n = 81) administrated 2-48 h since the blood draw, 15 (28%) patients on vitamin K antagonists (n = 54), and in four (12%) patients off anticoagulation (n = 33). Compared with nonanticoagulated patients, APC-R ratios were similar in patients on rivaroxaban, without any correlation with rivaroxaban concentrations (from 0 to 303 μg/l). None of the patients on rivaroxaban were found to have false-negative or false-positive APC-R ratios. Rivaroxaban concentrations up to 300 μg/l do not affect results of the ProC Ac R RVVT-based assay, which could be recommended in patients referred to a clinic for thrombophilia screening in whom the time since the last dose of rivaroxaban is uncertain.

Maximising the diagnostic potential of APTT-based screening assays for activated protein C resistance.

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. 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. 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. 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.

The effect of dabigatran and rivarovaban on a prothrombinase-based assay for activated protein C resistance: a preliminary study in subjects heterozygous for factor V Leiden.

Oral direct inhibitors of activated factor II (FIIa), such as dabigatran, and of activated factor X (FXa), such as rivaroxaban, have the potential to interfere with the great majority of clot-based coagulation assays and some chromogenic assays, whereas these drugs would not be expected to interfere in antigen-based assays1–4. Data concerning the influence of anti-FIIa and anti-FXa on functional assays for determination of activated protein C resistance (APCr) are still rather scarce and available studies have mainly been conducted using tests based on the activated partial thromboplastin time (aPTT). Moreover the data were obtained from in vitro studies performed on plasma from normal individuals, which was spiked with variable amounts of the drugs1–4. For dabigatran there is evidence that aPTT-based APCr methods over-estimate the APCr ratio, which may lead to patients with heterozygous factor V Leiden (FVL) being misclassified as normal1–4. In contrast, for rivaroxaban there is evidence that aPTT-based APCr methods may under-estimate the APCr ratio2,3. As previously reported, in our laboratory we adopted a prothrombinase-based method to study the APCr5. Data concerning the influence of dabigatran and rivaroxaban on this assay are still rather scarce. We, therefore, report the influence of FIIa and FXa direct oral inhibitors on the APCr, evaluated with a prothrombinase assay, in a small number of FVL patients.

An Improved Algorithm for Activated Protein C Resistance and Factor V Leiden Screening

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. 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. 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. 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.

The detection of anticoagulant function in Kazakh women before and after delivery and clinical significance

Objective To investigate the anticoagulant factors that Kazakh women are prone to develop deep vein thrombosis before or after delivery.Methods The protein C,protein S,antithrombin Ⅲ (AT-Ⅲ) activity,activated protein C resistance ratio (APCR) of 36 Kazakh women cases and 39 Hans women cases before and after delivery were determined.Results The protein S (43.13±11.36,58.05±17.10) was significant changed (P＜0.01)in Kazakh women before and after delivery.The protein C (97.34±18.37,118.02±23.46) and protein S (58.05±17.10,67.97±19.22) were statistically different between Kazakh women and Han women after delivery(P＜0.05,which protein C was P＜0.01).The anticoagulant indexes of Kazak women after delivery was still within normal range.Conclusions Normal women have prothrombotic state before and after delivery,especially the Kazakh women.It may be an important factor of deep vein thrombosis-prone before and after delivery that protein C and protein S in Kazakh women have lower activity than that in Han women.The detection of anti-coagulation have some clinical significance on the prevention of the deep vein thrombosis in Kazakh women before and after delivery. Key words: Parturition; Pregnancy; Venous thrombosis/ET/ME; Protein C/ME; Protein S/ME; Antithrombin Ⅲ/ME

Prevalence of Thrombophilia In Patients with Sickle Cell Disease and Osteonecrosis

AbstractAbstract 3184 Background:Osteonecrosis (ON) of the femoral and humeral heads is frequently seen in patients with sickle cell disease (SCD). Earlier studies reported a high prevalence of thrombophilia in patients with ON. Aims:To study the prevalence of thrombophilia in patients of SCD with ON. Methods:Case records of SCD patients with ON were retrospectively reviewed for protein S, protein C, and anti-thrombin deficiency, along with activated protein C resistance (APCR). Results:A total of sixty-three patients were identified, 35 of whom were males, with a median age of 21 years (range 15to 46). Median haemoglobin, total white cell count and platelet counts were 10 g/dL (range 7.7to 13.3), 7.5 ×109/L (range 3.4 to 16.7) and 302 ×109/L (range 72 to 1101) respectively. Twenty-eight patients were on hydroxyurea. Thrombophilia testing showed that 29% (95% confidence interval: 17–40), 47% (95% CI: 29–64) and 79% (95% CI: 65–93) of the patients had low levels of functional (<60 unit/dl), total antigenic (<70 unit/dl) and free antigenic (<70 unit/dl) protein S respectively, while 21% (95% CI: 10–31) and 67% (95% CI: 46–87) had low levels of functional (<70 unit/dl) and antigenic (<70 unit/dl) protein C respectively. In addition, 14% (95% CI: 5–23) and 22% (95% CI: 0–56) of the patients had low levels of functional (<80 unit/dl) and antigenic (<80 unit/dl) anti-thrombin levels respectively. Only 2% (95% CI: 0–5) of these patients had an abnormally low APCR (APCR ratio ≤2.3). Summary/Conclusions:Patients with SCD and ON have a high prevalence of thrombophilia. These results indicate that a prospective study with more detailed thrombophilia work-up, along with confirmatory DNA analysis, as well as a study of the role of prophylactic anticoagulation in such patients is highly warranted. Disclosures:Pathare:Sultan Qaboos University: Employment, Research Funding. Alkindi:Sultan Qaboos University: Employment, Research Funding.

Markers of Coagulation Activation and Inflammation in Sickle Cell Disease and Sickle Cell Trait

It has been well-described that sickle cell disease (SCD) due to HbSS or HbSC is associated with systemic activation of coagulation. A single study has reported a similar, albeit lesser, degree of chronic hyperactivation of coagulation in subjects with sickle cell trait (SCT) (Am J Hem 2002; 69(2):89–94), and we have shown that SCT is a risk factor for venous thromboembolism (Blood 2007; 110(3):908–12). The aim of this study was to simultaneously compare markers of activation of coagulation and inflammation in SCD and SCT. Ninety Saudi Arabian individuals (40 SCD (HbSS), 20 SCT (HbAS) and 30 controls (HbAA)) were recruited. SCD patients were not in vaso-occlusive crisis at the time of sample collection. None of the subjects was taking any relevant medication (anticoagulants, hormones or hydroxyurea), nor had they received blood transfusion for at least 6 weeks. On all subjects, complete blood count (CBC), prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer antigen, protein C activity, protein S activity, antithrombin (AT) activity, fibrinogen concentration, activated protein C resistance ratio (APCR), and C-reactive protein (CRP) levels were obtained. Student's “t” test was used to compare two independent means. Multivariate analysis “ANOVA” test was done to test statistical difference between more than 2 means.Results: D-dimer and CRP were significantly higher, whereas protein C, protein S, and fibrinogen were significantly lower in SS patients compared to controls (Table). In SCT, D-dimer levels were higher, and levels of proteins C and S were significantly lower than controls. APC resistance was more pronounced in both SCD and SCT compared to controls. In summary, this study provides further evidence of hypercoagulability in SCD, but more intriguingly, confirms that significant changes in coagulation parameters may be found in SCT. We speculate that loss of asymmetry of phospholipids in the red cell membrane may occur in certain hypoxic vascular beds (such as the renal vasculature) in SCT. We are currently exploring other possible underlying mechanisms that may contribute to activation of coagulation in SCT.Table: Comparison of Markers of Inflammation and Coagulation Activation in all GroupsGroup I (SS) (n=40)Group II (AS) (n=20)Group III (AA) (n=30)CRP22.5 ± 10.9*0.3 ± 0.20.3 ± 0.1PT12.5 ± 1.6*12.2 ± 1.0**11.5 ± 0.6APTT33.3 ± 4.235.8 ± 4.6**32.9 ± 3.2D-dimer287 ± 140*278 ± 207**141 ± 65Protein C77.9 ± 20.3*97.7 ± 21.7**109.4 ± 17.2APCR0.82 ± 0.12*0.79 ± 0.12**1.05 ± 0.19Protein S54.0 ± 18.4*64.6 ± 15.4**89.7 ± 14.9AT94.4 ± 16.6*102.2 ± 7.4**109.3 ± 10.7Fibrinogen305 ± 101361 ± 146358 ± 80*P < 0.05 comparing SCD and control groups by paired t- test**P <0.05 comparing SCT and control groups by paired t- test

Effects of switching from oral to transdermal or transvaginal contraception on markers of thrombosis

Background The study was conducted to determine the impact of switching from oral to transdermal patch or vaginal ring contraception on biomarkers of thrombosis. Study Design Current healthy oral contraceptive (OC) users were randomized to switch to either a contraceptive ring (CR) or patch (CP) and underwent phlebotomy to measure surrogate biomarkers of thrombosis [sex hormone-binding globulin (SHBG), free protein S and activated protein C resistance (APC-r)] before switching, and during the fourth cycle of use of the new method. Results Of 142 reproductive age women enrolled, 120 sample pairs were available for analysis. SHBG increased significantly from baseline in CP users [mean change (95% CI), +29.9 nM (9.6–50)] but not in CR users [−1.6 (−16.6 to 13.5)]. Protein S decreased significantly from baseline in CP users [mean change −7.1% (−12.1 to −2.1)], but increased significantly in CR users [+5.3% (1.1–9.6)]. The APC-r ratio did not undergo a significant change from baseline in either group [CP +0.06 (−0.06 to 0.18), CR +0.02 (−0.10 to 0.14)]. Compared to CR users, subjects using the CP had significantly higher SHBG [187.5 (167.0–208), 146 (132.6–159.4), p=.012], significantly lower protein S [81.8 (76.8–86.8), 93.6 (89.1–98.1), p=.001] and similar APC-r ratios [2.99 (2.85–3.14), 3.09 (2.96, 3.22), p=.3] at the Cycle 4 visit. Conclusion OC users who switch to the ring exhibit beneficial changes in biomarkers of thrombosis, while those switching to the patch display a shift favoring clot formation.

Effect of transdermal and transvaginal administration of contraceptive steroids on markers of thrombosis

To determine the impact on blood markers of thrombosis of switching from an oral to a transdermal or vaginal combined hormonal contraceptive method. Multicenter, randomized, open label, comparator. Current healthy oral contraceptive (OC) users randomized to switch to either a vaginal ring (NuvaRing®) or skin patch (OrthoEvra®) contraceptive in an acceptability study were asked to participate in a substudy investigating the effect of these products on markers of thrombosis. Blood samples were obtained for Sex Hormone Binding Globulin, (SHBG), total Protein S, and Activated Protein C resistance (APC-r) before, and 4 months after switching to the new method. Following separation of serum and plasma, samples were frozen on dry ice, and stored between – 70 to –80°C until shipment to a central lab for analysis. Changes in mean levels of the markers from baseline were compared using separate repeated measures analysis for the ring and patch groups. A total of 142 reproductive age women were enrolled at three study sites. Of these, 120 completed 4 cycles and contributed both blood samples. SHBG increased significantly from baseline values in the women who switched to the Patch (mean (SD) change +29.9 nM (74.8), P=0.005) but not those who switched to the Ring (−1.6 (60.7), P=0.83). Coherent with this, Protein S decreased significantly from baseline values in the Patch (mean change −7.1% (18.3), P=0.006) but increased significantly in those who switched to the Ring (+5.3% (17.1), P=0.014) The APC-r ratio did not undergo a significant change from baseline in either group [(Ring +0.02 (0.48), P=0.7), Patch +0.06 (0.43), P=0.3)]. While there were no significant differences between groups in baseline values of SHBG, Protein S, or APC-r, after 4 cycles SHBG was significantly higher (187.5 (75.9), 146 (54.2), P=0.001) and Protein S significantly lower (81.8 (18.2), 93.6 (18.2), P=0.001) in Patch than in Ring users. The APC-r ratio was not different (2.99 (0.52) Patch, 3.09 (0.51) Ring, P=0.3). OC users who switch to the patch undergo a change in hemostatic balance (decreased Protein S, increased SHBG) consistent with a more prothrombotic state. Further epidemiological studies of thrombosis risk in patch users are warranted to determine the clinical significance.

A comparative study of the effect of continuous combined conjugated equine estrogen plus medroxyprogesterone acetate and tibolone on blood coagulability

Hormone therapy (HT) after the menopause is associated with increased risk of venous thromboembolism (VTE). Tibolone has pharmacodynamic properties different from other hormone preparations. We compared the effect of a combined HT and tibolone on the inhibition of haemostasis. Thirty-eight post-menopausal women were randomly assigned to 1.25 or 2.5 mg per day of tibolone or oral continuous combined conjugated equine estrogen plus medroxyprogesterone acetate (CEE/MPA). Inhibitors of haemostasis were measured at baseline and after 12 months. Results from the two groups of women receiving tibolone were not significantly different and, to improve the power of the study, the two groups were merged. Higher concentration of protein S (1.16 versus 1.00 IU ml(-1); P = 0.005) and higher activated protein C resistance ratio (APC-R) (4.2 versus 3.65; P = 0.04) were observed in the tibolone group than in the CEE/MPA group. Both doses of tibolone increased APC-R significantly (P < 0.01). Tissue factor pathway inhibitor (TFPI) was lower in the CEE/MPA group than in the tibolone group (67.8 versus 79.9 ng ml(-1); P = 0.03). CEE/MPA reduced the concentration of antithrombin (P = 0.002), protein S (P < 0.001) and TFPI (P < 0.001). Both preparations reduced the concentration of plasminogen activator inhibitor 1 (P < 0.05). Tibolone induces fewer pharmacological alterations on blood coagulability than CEE/MPA and has a potentially favourable effect on APC-R. This may translate into a corresponding low risk of VTE, as also indicated from the existing clinical data.

Activated protein C resistance -- in the absence of factor V Leiden -- and pregnancy.

Activated protein C (APC) resistance with or without factor V Leiden (FVL) is a major risk factor for venous thromboembolism. Many previous pregnancy studies have been focused on APC resistance caused by FVL. Very few have investigated APC resistance in the absence of FVL (APCR(FVL-)). In a prospective study of 2480 unselected gravidae, blood was drawn in early pregnancy (mean = 12 weeks of gestation). APC resistance was analyzed by an APTT-based method (Coatest) APC-resistance) and the presence of FVL was determined by PCR. The APCR(FVL-) group had similar mean APC resistance ratio as the heterozygous carriers of FVL. The analyses were carried out no earlier than 3 months after delivery when all data were recorded. Small-for-gestational age (SGA) was used as a proxy for intrauterine growth restriction. When compared with the control group, women with APCR(FVL-) had no increased risk of SGA, pre-eclampsia, first trimester fetal loss or venous thromboembolism. However, they had an increased risk of second trimester fetal loss (7.3% vs. 2.7%, P = 0.01), and a tendency of being overweight (17.3% vs. 12.6%, P = 0.19) and of delivering extremely preterm (2.8% vs. 1.0%, P = 0.11). Women with APC resistance not caused by FVL were not at increased risk for SGA, pre-eclampsia, first trimester fetal loss, or abnormal blood loss. However, they showed an increased prevalence of second trimester fetal loss.

Prevalence of hyperhomocysteinaemia, activated protein C resistance and prothrombin gene mutation in inflammatory bowel disease

Thromboembolic disease is a significant cause of morbidity and mortality in patients with inflammatory bowel disease (IBD). A hypercoagulable state exists in IBD that may involve many components of haemostasis and is closely linked to the disease pathogenesis. It has been proposed that microvascular thrombosis and infarction may trigger the underlying inflammatory process. To determine the prevalence of prothrombotic factors including hyperhomocysteinaemia, activated protein C (APC) resistance and prothrombin gene mutations as well as vitamin levels in the local IBD population. A total of 68 patients (37 men and 31 women) attending the IBD clinic were enrolled into the study. Citrated and ethylenediamine tetraacetic acid blood samples were collected from all patients as well as from 30 controls. Homocysteine levels were measured using the IMX immunoassay. APC resistance was measured using an unmodified activated partial thromboplastin time-based clotting assay. Prothrombin mutations were determined using polymerase chain reaction with the HB-gene factor II detection system. Mean homocysteine levels were significantly higher and APC resistance ratios significantly lower in IBD patients compared with controls. No significant difference was detected between patients with ulcerative colitis or Crohn's disease. There was no significant increase in the incidence of prothrombin mutation in IBD patients. IBD patients had lower vitamin B12 and higher serum folate levels than controls. High homocysteine and high serum folate may be associated with low vitamin B12 levels in IBD patients. We did not find any association between a low APC ratio and the factor V Leiden mutation or high factor VIII levels. Both hyperhomocysteinaemia and a low APC ratio may contribute to an increased risk of thromboembolic disease in IBD patients.

Hypercoagulability risk factors in children with minimal change disease and the protective role of protein-C activity

It is believed that thrombotic activity in nephrotic syndrome is due to an imbalance between procoagulant/thrombotic and anticoagulant/antithrombotic factors in plasma. The aim of this study was to investigate the hypercoagulability risk in childhood minimal change disease and to find possible protective mechanisms with respect to hemostasis. Twenty-six children with minimal change disease were enrolled in this study. All patients were evaluated during an attack and on remission. The control group consisted of 33 healthy children. During the attack period, prothrombosis parameters, total lipid, cholesterol, fibrinogen levels and platelet count increased significantly compared to levels in the remission period. This denotes that hyperviscosity increases thrombosis tendency. In the attack period, the significant increase of prothrombin fragments 1 + 2 which shows thrombin formation and thrombin-antithrombin complex which causes prothrombin activation, are an indication of increased thrombosis risk. Five patients with lupus anticoagulant present and 7 patients with, activated protein-C resistance ratios carried an increased thrombosis risk. D-dimer level of fibrinolytic factors significantly increased during the attack period. These findings emphasize the existence of thrombotic activity causing the activation of the fibrinolytic system. The significant increase in protein-C activity in these patients represents one of the protective mechanisms against thrombosis. The decrease in tissue plasminogen activator and antiplasmin indicates the protective role of fibrinolytic activity. Consequently, an increase in the protein-C activity is one of the protective mechanisms. The fibrinolytic system also plays an important role in preventing thrombotic activity in these patients.

Generation of Factor VIII Molecules Partially Resistant to Activated Protein C.

Following a vascular injury, factor VIII (FVIII) is rapidly activated by thrombin cleavage at arginine (Arg) 372, 740 and 1689. Activated FVIII, an heterotrimer composed by the association of A1, A2 and A3-C1-C2 domains, is rapidly degraded to limit thrombosis risk. Two main phenomenons that account for the disappearence of FVIIIa consist of an intrinsic dissociation of the trimer due to the loss of A2 domain, and the cleavage of the molecule by activated protein C (APC). APC cleaves FVIIIa at Arg 336 and 562. Mutant FVIII molecules were already generated, with one or two arginines substituted, and a subsequent APC cleavage diminished. Since the thrombotic potential of high plasma levels of FVIII has been described, we aimed to generate new factor VIII molecules where the APC cleavage was only modulated. The ultimate goal being to increase in vivo the FVIIIa half-life. Among the two APC cleavage sites, the sequence around the site 562 was the most conserved between species. This region was therefore chosen to be modified with a prior verification that the amino-acids to be modified were not described in any hemophilic phenotype. Subsequently, the six following mutations were realized in a BDD-FVIII cDNA: Q561N, G563A, N564D, N564A, N564Q and I566M as well as the controls R336I, R562K and R336I+R562K. The constructs were transiently expressed in BHK cells to determine the specific activities of the corresponding molecules. The mutant specific activities, as determined by one- and two-stage clotting assays, ranged from 40 to 94 % of the wild-type except for G563A that was almost inactive. CHO clones expressing FVIII molecules were obtained. The mutants and control FVIII molecules were produced, partially purified on heparin column and further analyzed. The comparative specific activities in a two-stage clotting assay were the following (+/− SD; n=6): BDD-FVIII 100 %, Q561N 105 % +/− 45, G563A 6 % +/− 6, N564D 50 % +/− 23, N564A 45 % +/− 21, N564Q 80 % +/− 26, I566M 100 % +/− 41. The one-stage clotting assay gave identical results than the two-stage assay for each mutant. The mutants were then activated by thrombin (1:1) and the occurrence of FVIII activity was monitored. A 20- to 25-fold increase in FVIII activity was measured within 2 minutes following the addition of thrombin for all mutants. The mutants, except for the inactive G563A, were then analyzed for their resistance to APC by three different assays: an APC resistance kit (Coatest, Chromogenix), an in vitro assay that measured APC sensitivity of FVIIIa and an immunoblot assay that visualized the cleavage efficiency of the A2 fragment. These three assays confirmed the APC resistance of the previously published R336I, R562K and R336I+R562K, as compared with wild-type FVIII. They also revealed that the mutants N564D, N564A and I566M behaved similarly to the wild-type FVIII whereas Q561N and N564Q mutants were partially resistant to APC. The APC resistance ratio were the following 2.3 +/− 0.3 for BDD-FVIII, 2.1 +/− 0.4 for N564Q, 1.7 +/− 0,1 for Q561N, 1.6 +/− 0.3 for R562K and 1.3 +/− 0.3 for R336I+R562K. The N564Q and Q561N mutants exhibited a profile intermediate between wild type FVIII and R562K regarding of the loss of FVIIIa activity that was confirmed on the immunoblot profile. In conclusion we have generated new factor VIII molecules that retained their full procoagulant function while possessing a reduced sensitivity to APC cleavage.

The Effect of Lupus Anticoagulant in the Second-Generation Assay for Activated Protein C Resistance

The activated protein C resistance (APCR) assay is the test of choice to screen for factor V Leiden. We evaluated the effect of lupus anticoagulant on the baseline clotting time of the second-generation APCR assay with plasma samples from 54 patients to determine whether a falsely low APCR ratio could be predicted. We also assessed whether a modification of the assay could make it more reliable in the presence of strong lupus anticoagulants. Of 54 plasma samples, 5 yielded a false-positive APCR ratio, and all 5 had a prolonged baseline clotting time. Further dilution (1:40) of the plasma samples in factor V–deficient plasma led to correction of the APCR ratio and did not affect the sensitivity of the test for factor V Leiden. Our data support that the baseline clotting time is a good predictor of a false-positive APCR test result and should be checked before calculating the ratio. The modified APCR assay reliably identified the false-positive ratios and could be used to screen for factor V Leiden in samples with strong lupus anticoagulant.

Extensive Venous and Arterial Thrombosis Associated With an Inhibitor to Activated Protein C

Activated protein C resistance (APCR) in the absence of alterations in the factor V gene has been observed during pregnancy, in patients on oral contraceptives, in the presence of antiphospholipid antibodies, and in patients with ischemic stroke. We report a 49-year-old woman with recurrent major venous and arterial thromboses who displayed pronounced APCR, yet no changes in the activated protein C (APC) cleavage sites of factor V. The APCR values determined by four different assays were similar to those obtained in plasma from a homozygote for factor V Q506. Addition of IgG isolated from the patient's serum to normal plasma lowered the APCR ratio from 2.4 to 1.6. Incubation of patient's IgG with normal APC resulted in a profound change in the mobility of APC in crossed immunoelectrophoresis. APC was also shown to bind to patient's IgG immobilized on a protein A agarose column. Factor Va inactivation by APC was inhibited by patient's IgG, but not by control IgG in the presence or absence of either phospholipids or protein S. These results provide evidence for the existence of an acquired antibody against APC in the patient's plasma, which gave rise to the APCR phenotype and was probably responsible for the major thrombotic events. We suggest that acquired APCR due to anti-APC antibodies be considered a potential cause for severe venous and arterial thromboses.

Activated protein C resistance in young African American patients with ischemic stroke.

It has been proposed that activated protein C resistance (APCR) due to the factor V Leiden (FVL) mutation may be a risk factor for stroke in young adults. However, this may not be the case for all ethnic groups due to variability in the prevalence of the FVL mutation. Case series from a university neurology clinic. Patients with an APCR ratio of 2.2 or below were tested for the FVL mutation (nine patients). Patients on warfarin were also tested for the FVL mutation (14 patients). 38 African American patients under age 55 with an arterial stroke were identified. The mean age of the patients is 43.1 years. Five percent had an APCR ratio of 2.0 or below. None of the patients with an APCR ratio of 2.2 or below or the patients directly tested for the FVL mutation had the mutant allele. Activated protein C resistance due to the FVL mutation does not appear to be a major risk factor for stroke in young African Americans. Other, as yet unidentified, mechanisms leading to activated protein C resistance may be important in a small subset of young African American stroke patients.