Laboratory Testing for Activated Protein C Resistance (APCR): An Update.

Activated protein C resistance (APCR) reflects a hemostatic state defined by a reduced ability of activated protein C (APC) to affect an anticoagulant response. This state of hemostatic imbalance is characterized by a heightened risk of venous thromboembolism. Protein C is an endogenous anticoagulant that is produced by the hepatocytes and undergoes proteolysis-mediated activation to APC. APC in turn degrades activated Factors V and VIII. APCR describes a state of resistance by activated Factors V and VIII to APC-mediated cleavage of these factors, thereby promoting amplified thrombin production and a potentially procoagulant state. This resistance of APC may be inherited or acquired. Mutations in Factor V are responsible for the most frequent form hereditary APCR. The predominant mutation, a G1691A missense mutation at Arginine 506, the so-called Factor V Leiden [FVL], causes a deletion of an APC-targeted cleavage site in Factor Va, thereby rendering it resistant to inactivation by APC. There are a variety of laboratory assays for APCR, but this chapter focuses on a procedure using a commercially available clotting assay that utilizes a snake venom and ACL TOP analyzers.

Laboratory assessment of Activated Protein C Resistance/Factor V-Leiden and performance characteristics of a new quantitative assay.

Activated Protein C Resistance is mainly associated to a factor V mutation (RQ506), which induces a deficient inactivation of activated factor V by activated protein C, and is associated to an increased risk of venous and arterial thrombosis in affected individuals, caused by the prolonged activated factor V survival. Its prevalence is mainly in Caucasians (about 5%), and this mutation is absent in Africans and Asians. Presence of Factor V-Leiden is usually evidenced with clotting methods, using a two-step APTT assay performed without or with APC: prolongation of blood coagulation time is decreased if this factor is present. The R506Q Factor V-Leiden mutation is now usually characterized using molecular biology, and this technique tends to become the first intention assay for characterization of patients. Both techniques are qualitative, and allow classifying tested individuals as heterozygotes or homozygotes for the mutation, when present. A new quantitative assay for Factor V-Leiden, using a one-step clotting method, has been developed, and designed with highly purified human coagulation proteins. Clotting is triggered with human Factor Xa, in presence of calcium and phospholipids (mixture which favours APC action over clotting process). Diluted tested plasma, is supplemented with a clotting mixture containing human fibrinogen, prothrombin, and protein S at a constant concentration. APC is added, and clotting is initiated with calcium. Calibration is performed with a pool of plasmas from patients carrying the R506Q Factor V mutation, and its mixtures with normal plasma. Homozygous patients have clotting times of about <40sec; heterozygous patients have clotting times of about 40-60sec and normal individuals yield clotting times >70sec. Factor V-Leiden concentration is usually >75% in homozygous patients, 30-60% in heterozygous patients and below 5% in normal. The assay is insensitive to clotting factor deficiencies (II, VII, VIII: C, IX, X), dicoumarol or heparin therapies, and has no interference with lupus anticoagulant (LA). This new assay for Factor V-Leiden can be easily used in any coagulation laboratory, is performed as a single test, and is quantitative. This assay has a high robustness, is accurate and presents a good intra- (<3%) and inter-assay (<5%) variability. It contributes solving most of the laboratory issues faced when testing factor V-Leiden. Quantitation of Factor V-L could contribute to a better assessment of thrombotic risk in affected patients, as this complication is first associated to and caused by the presence of a defined amount of FVa.

Laboratory Testing for Activated Protein C Resistance (APCR).

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.

Effect of dabigatran on a prothrombinase-based assay for detecting activated protein C resistance: an ex vivo and in vitro study in normal subjects and factor V Leiden carriers.

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.

Identification and functional characterization of a novel F5 mutation (Ala512Val, FVB onn ) associated with activated protein C resistance.

UNLABELLED: Essentials Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. A novel missense mutation (Ala512Val - FVBonn ) was characterized in vitro and in silico. FVBonn is a new cause of APC resistance and venous thrombosis. FVBonn expresses additionally enhanced procoagulant activity in the absence of APC. SUMMARY: Background Activated protein C (APC) resistance is a prevalent risk factor for venous thrombosis. This phenotype is most commonly associated with the factor V Arg506Gln mutation (FV Leiden), which impairs the APC-mediated inactivation of both activated FV (FVa) and activated FVIII (FVIIIa). Objectives Here, we report the identification and characterization of a novel FV mutation (Ala512Val, FVBonn ) in six patients with APC resistance and venous thrombosis or recurrent abortions. Methods FVBonn was expressed in a recombinant system and compared with recombinant wild-type (WT) FV and FV Leiden in several functional assays. Results FVBonn conferred APC resistance to FV-depleted plasma, both in the activated partial thromboplastin time (APTT)-based test (APC sensitivity ratio [APCsr] of 1.98 for FVBonn versus 4.31 for WT FV and 1.59 for FV Leiden) and in the thrombin generation-based test (normalized APCsr of 5.41 for FVBonn versus 1.00 for WT FV and 8.99 for FV Leiden). The APC-mediated inactivation of FVaBonn was slower than that of WT FVa (mainly because of delayed cleavage at Arg506), but was greatly stimulated by protein S. The APC cofactor activity of FVBonn in FVIIIa inactivation was ~ 24% lower than that of WT FV. In line with these findings, an in silico analysis showed that the Ala512Val mutation is located in the same loop as the Arg506 APC cleavage site and might hamper its interaction with APC. Moreover, FVBonn was more procoagulant than WT FV and FV Leiden in the absence of APC, because of an increased activation rate and, possibly, an enhanced interaction with activated FX. Conclusions FVBonn induces hypercoagulability via a combination of increased activation/procoagulant activity, decreased susceptibility to APC-mediated inactivation, and slightly reduced APC cofactor activity.

Hereditary Thrombophilic Factors in Glaucoma.

PURPOSE: To evaluate the hereditary thrombophilic factors in patients with primary open-angle glaucoma, exfoliative glaucoma, and exfoliation syndrome and to compare their results with those of healthy control subjects. MATERIALS AND METHODS: The study included 75 patients [25 patients with primary open-angle glaucoma (group I), 25 patients with exfoliative glaucoma (group II), and 25 patients with exfoliation syndrome (group III)] and 25 healthy control subjects (group IV). Well-known hereditary thrombophilic factors including methylenetetrahydrofolate reductase (MTHFR) gene C677T mutation, prothrombin G20210A mutation, factor V Leiden mutation, activated protein C resistance, protein S, protein C, and antithrombin III activities, and homocysteine levels were measured in venous blood samples of all subjects. RESULTS: Fifty-one males and 49 females were included in the study. The mean age of the patients was 67.8 ± 8.7 years (range, 46 to 87 y). There was no statistically significant difference with regard to the mean age (P=0.057) and distribution of sex (P=0.391) between the study groups. The difference of homocysteine, folate, vitamin B12, antithrombin III activity, protein C activity, free protein S activity, and activated protein C resistance were not statistically significant; and the number of subjects with MTHFR C677T, prothrombin G20210A, and factor V Leiden mutations were similar between the study groups. CONCLUSION: Our results suggest that there is no significant difference between the prothrombotic inherited risk factors of glaucomatous and nonglaucomatous subjects.

Prevalence of the Factor V E666D Mutation and Its Correlation With Activated Protein C Resistance in the Chinese Population.

BACKGROUND: Factor V (FV) Leiden mutation-related activated protein C resistance (APCR) is one of the common inherited risk factors for venous thromboembolism (VTE) in caucasian population. Although APCR could be identified in some of the Chinese healthy people and patients with VTE, it was not related to FV Leiden mutation. In 2008, we have identified a novel FV mutation (FV E666D) in exon 13 in a hereditary APCR family. And we presumed that the novel mutation might be a genetic defect of APCR in the Chinese population. The aim of our study was to evaluate the prevalence of FV E666D mutation and its correlation with APCR in the Chinese population in a larger series. METHODS: From June 2009 to January 2011, 163 consecutive patients who underwent thrombophilia tests in our hospital were recruited. The clinical data were retrospectively reviewed. Thrombophilia tests included APCR, anticoagulant proteins, and antiphospholipid antibodies. Factor V E666D mutation was detected. RESULT: Of the 163 patients, 6 (3.7%) were identified as APCR positive, 2.9% for patients without thrombosis and 5.1% for patients with thrombosis or thrombosis history. Factor V E666D mutation was not detectable in all the 163 patients including 6 APCR-positive patients. CONCLUSIONS: The prevalence of APCR either in the nonthrombotic patients or in the patients with thrombosis was lower than that reported in other Chinese studies. Our study couldn't provide illustration whether FV E666D mutation is correlated with APCR in the Chinese population.

[Resistance to warfarin in a patient with hereditary thrombophilia]. / Warfarinresistens hos en patient med hereditær trombofili.

We present a case report of a young man with spontaneous deep venous thrombosis. He tested positive for heterozygote factor V Leiden mutation and was treated with warfarin. However, he turned out to be resistant to warfarin, and another venous thrombosis occurred during the insufficient treatment. The antithrombotic treatment was then successfully replaced by phenprocoumon. This case report emphasizes the importance of critically evaluating the efficacy of a treatment and substitute if proven insufficient.

Thrombin generation in antiphospholipid syndrome.

Our objective was to study acquired Activated Protein C (APC) resistance in patients with antiphospholipid antibodies (aPL) using a thrombin generation based assay. We compared patients with and without lupus (systemic lupus erythematosus, SLE). A parameter summarizing APC inhibition of thrombin generation with increasing APC concentrations (IC(50)-APC) was increased in all patient groups compared to controls: median values were 15.3 (interquartile range, IQR, 9.7 to 34.0) in patients with primary antiphospholipid syndrome (APS), 27.3 (IQR 23.5 to 43.5) in patients with SLE without APS, 64.1 (IQR 25.9 to 65.0) in patients with SLE/APS compared to 10.4 [IQR 8.5 to 15.8] in controls, respectively p = 0.003, p = 0.0001 and p = 0.0001. In conclusion, patients with SLE and primary APS displayed a hypercoagulable state characterized by APC resistance.

Activated protein C resistance and its correlation with thrombophlebitis in Behçet's disease.

Patients with Behçet's disease (BD) have been recognized to be at an increased risk of thrombosis and thrombotic complications have been reported in 12%-40% of patients. The precise pathogenetic mechanisms underlying the thrombotic tendency of BD are not known. In recent researches, it is reported that procoagulant mutations might play a role in thrombotic process in BD patients. We aimed to evaluate the frequency of activated protein C resistance (APCR) in our BD patients and to investigate the association between thrombophlebitis and APCR. The study included 116 patients with BD who fulfilled the International Study Group criteria and 70 healthy individuals as a control group. APCR levels were measured by the clotting method. APCR levels were 129.63 ± 39.70 and 152.26 ± 22.62 in BD patients and control group, respectively (P<0.01). APCR was found in 47.4% and 8.6% of BD patients and control group, respectively (P<0.01). There was no statistically significant difference regarding APCR levels between patients with thrombophlebitis and without thrombophlebitis (46.4% vs. 48.3%). We found the frequency of APCR to be increased in BD patients with or without thrombophlebitis. The lack of association between thrombophlebitis and APCR in our series of BD patients suggests that some factors like endothelial abnormalities other than thrombophilia play a major role in the pathogenesis of thrombosis in BD.

[Study on activated protein C resistance and disordered coagulation in patients with myeloproliferative neoplasms].

OBJECTIVE: To study the correlation of activated protein C (APC) resistance, coagulation factors and inhibitors abnormality and JAK2V617F mutation burden in patients with myeloproliferative neoplasms (MPN). METHODS: The APC resistance was defined as the ratio of activated partial thromboplastin time (APTT) in the presence and absence of APC, i.e. APC sensitivity ratio (APCsr). Plasma protein C (PC), protein S (PS), prothrombin (FII), factor V (FV), factor VIII levels and CD11b expression on neutrophils were measured. The percentage of mutated JAK2V617F allele (V617F%) was evaluated by real time polymerase chain reaction (qRT-PCR). RESULTS: Expression of CD11b on neutrophils was significantly elevated in MPN patients compared with that of the control group. APCsr, PS and FV levels were reduced in patients with MPN. The APCsr level was decreased mainly in patients with thrombosis and JAK2V617F mutant burden higher than 75%. APCsr was not only positively correlated with PS levels but also inversely correlated with JAK2V617F allele burden in JAK2V617F mutant gene carriers. CONCLUSION: The neutrophil was activated and PS, FV level were reduced in MPN patients. The APCsr level was decreased and the occurrence of relatively acquired APC resistance was found in MPN patients with thrombosis. The APCsr is correlated with the PS level and JAK2V617F mutational furden.

A clinical audit of congenital thrombophilia investigation in tertiary practice.

BACKGROUND: The presumed cause of congenital thrombophilia can now be explained in ~50% of familial thrombosis cases following evaluation of a range of markers, primarily comprising factor V Leiden (FVL), activated protein C resistance (APCR), protein C (PC), protein S (PS) and antithrombin (AT). However, the effectiveness of such evaluations is largely determined by limiting improper investigations, either in inappropriate patients or at unsuitable timepoints. AIM: To evaluate clinical ordering patterns for a range of thrombophilia associated tests at a tertiary level public facility. METHODS: Several independent audits into clinical requests for FVL, APCR, PC, PS, and AT testing were performed at our institution. RESULTS: We identified a wide variety of clinical ordering background, although most requests related to 'thrombosis' or 'obstetric' indications. For FVL, the detection rate of heterozygotes continues to decline and is currently ~10% of investigations. For APCR, review of clinical requests and clinical notes indicated that around 36% of investigations occurred whilst patients were on anticoagulant therapy. For PC, PS and AT investigations, additional testing of samples that yielded low test results for PC, PS and/or AT indicated that an alarming 80% of these cases likely derived from patients on anticoagulant therapy. CONCLUSION: These results continue to reflect on poor patient or timing selection for congenital thrombophilia investigations that compromises the utility of these tests. In total, this would yield a very high rate of false positive identification for disorders that patients do not have, raising the question: are broadly based congenital thrombophilia investigations doing more harm than good?

Hereditary coagulopathies: practical diagnosis and management for the plastic surgeon.

BACKGROUND: Venous thromboembolism is a devastating complication representing one of the major causes of postoperative death in plastic surgery. Within the scope of plastic surgery, body-contouring procedures are often considered to carry a higher risk of venous thromboembolism. Hereditary thrombophilias comprise a group of conditions defined by a genetic predisposition to thrombosis development. Collectively, hereditary thrombophilias are present in at least 15 percent of Western populations and underlie approximately half of thromboembolic events. Although the topic of venous thromboembolism is discussed widely throughout the literature, there is little published on the diagnosis and management of hereditary thrombophilias in the plastic surgery literature. The goals of this study were to present a review of the major inherited thrombophilias, to delineate the risk of these disorders, and to recommend a practical algorithm for patient screening and management before major plastic surgery. METHODS: A MEDLINE search was performed from 1965 to the present to review the literature on inherited thrombophilia disorders. RESULTS: Based on the English language literature and clinical experience, the authors suggest practical guidelines for screening and management of hereditary thrombophilias. A thorough medical history and preoperative evaluation are key to reducing venous thromboembolism complications. CONCLUSIONS: Hereditary thrombophilias are present in a significant number of thromboembolic events. Preoperative vigilance on the part of the plastic surgeon may help to identify patients with undiagnosed hereditary thrombophilias and thereby decrease the incidence of venous thromboembolism.

Usefulness of factor V Leiden mutation testing in clinical practice.

We have investigated the clinical usefulness of the activated protein C resistance (APCR)/factor V Leiden mutation (FVL) test by sending out questionnaires to all Norwegian physicians who ordered these tests from our publicly funded service laboratory during a 3-month period, and of whom 70% (267/383) responded. Indications for testing, patient follow-up, the use of APCR versus FVL tests and differences in practice between hospital doctors and GPs were examined. We found that 46% of the tests were predictive, ordered for risk assessment in healthy individuals with no previous history of venous thromboembolism (VTE). Among these, 42% of the tests were taken on the initiative of the patient and 24% were screening tests before prescription of oral contraceptives. In total, 54% of the tests were classified as diagnostic, among which 42% were ordered owing to a previous history of VTE and 22% to a history of brain stroke or myocardial infarction. The prevalence of FVL heterozygotes was not significantly different between the predictive and diagnostic test groups, that is, 26 and 20%, respectively. Only the predictive tests influenced patient follow-up. Here, the physician's advice to patients depended on the test result. In general, the clinical usefulness of APCR/FVL testing was low. Many tests were performed on unsubstantiated or vague indications. Furthermore, normal test results led to unwarranted refrain from giving advice about antithrombotic measures, leading to potential harm to the patient.

Clinical evaluation of a functional prothrombin time-based assay for identification of factor V Leiden carriers in a group of Italian patients with venous thrombosis.

The efficiency of a new prothrombin-based activated protein C (APC) resistance test to detect factor V Leiden (FVL) was clinically evaluated in 150 Italian patients with deep venous thrombosis. Patient samples are diluted in factor-V-deficient plasma, an APC-containing reagent, and specific factor V activator; after incubation, clotting is initiated by addition of activated-factor-FV-dependent prothrombin activator. Two prothrombin time determinations were performed under identical assay conditions except that no APC was added to one. A ratio over 4.2 for normal individuals and under 2.0 for FVL patients is expected: between 1.3 and 1.9 for FVL heterozygotes, and between 1.0 and 1.1 for FVL homozygotes. Using a predefined cut-off ratio of 2.0, a specificity and a sensitivity of 1.00 for detection of FVL mutation were found. With a cut-off ratio of 1.1, a specificity of 0.98 and a sensitivity of 1.00 were found for discrimination between FVL heterozygous (n = 60) and homozygous (n = 6). No interferences by heparins, oral contraceptives, oral anticoagulant therapy, protein C, protein S, D-dimer, homocysteine, MTHFR mutations and antiphospholipid autoantibodies were detected. In our experience, this new prothrombin time-based APC resistance assay provides improved discrimination between normal individuals and FVL carriers compared with the classical methods. Moreover, this new assay allows good discrimination between homozygous and heterozygous FVL carriers. In the authors' experience this prothrombin time-based method was not influenced by many factors compared with the classical activated partial thromboplastin time-based method.

Coagulation factor V and thrombophilia: background and mechanisms.

Human coagulation factor V (FV) is an essential coagulation protein with functions in both the pro- and anticoagulant pathways. Failure to express and control FV functions can either lead to bleeding, or to thromboembolic disease. Both events may develop into a life-threatening condition. Since the first description of APC resistance, and in particular the description of the so-called factor V(Leiden) mutation, in which a prominent activated protein C cleavage site in FV has been abolished through a mutation in the FV gene, FV has been in the center of attention of thrombosis research. In this review we describe how the functions of FV are expressed and regulated and provide an extensive description of the role that FV plays in the etiology of thromboembolic disease.

Differential impact of conventional and low-dose oral hormone therapy (HT), tibolone and raloxifene on functionality of the activated protein C system.

Recent studies have shown that hormone therapy (HT) is associated with an acquired resistance to activated protein C (APC). The aims of the present study were to evaluate a possible dose-response relationship and differential effects of different HT regimens on functionality of the APC system. Two hundred two healthy women were randomly assigned to receive treatment for 12 weeks with tablets containing either low-dose HT containing 1 mg 17ss-oestradiol + 0.5 mg norethisterone acetate (NETA) (n = 50), conventional-dose HT containing 2 mg 17ss-oestradiol and 1 mg NETA (n = 50), 2.5 mg tibolone (n = 51), or 60 mg raloxifene (n = 51). Normalized APC system sensitivity ratios (nAPCsr) were determined in plasma collected at baseline and after 12 weeks using a thrombin generation-based APC resistance test probed with either recombinant APC (rAPC) or thrombomodulin (rTM). NAPCsr increased in both the conventional- and low-dose HT groups, consistent with reduced sensitivity to APC. The increase was slightly more pronounced in the conventional-dose group, but the difference between the two HT groups was not statistically significant. The sensitivity to APC was only marginally altered in those allocated to tibolone. Consequently, tibolone showed a different phenotype as compared with the low-dose HT group. A small increase in nAPCsr with both rAPC and rTM was seen in the raloxifene-group, but the increase was less than in the low-dose HT group. Our findings indicate that oestrogen-progestin therapy induces an APC resistant phenotype, which may be related to dose, whereas tibolone and raloxifene only marginally alter the sensitivity to APC.

The cytoprotective protein C pathway.

Protein C is best known for its mild deficiency associated with venous thrombosis risk and severe deficiency associated with neonatal purpura fulminans. Activated protein C (APC) anticoagulant activity involves proteolytic inactivation of factors Va and VIIIa, and APC resistance is often caused by factor V Leiden. Less known is the clinical success of APC in reducing mortality in severe sepsis patients (PROWESS trial) that gave impetus to new directions for basic and preclinical research on APC. This review summarizes insights gleaned from recent in vitro and in vivo studies of the direct cytoprotective effects of APC that include beneficial alterations in gene expression profiles, anti-inflammatory actions, antiapoptotic activities, and stabilization of endothelial barriers. APC's cytoprotection requires its receptor, endothelial cell protein C receptor, and protease-activated receptor-1. Because of its pleiotropic activities, APC has potential roles in the treatment of complex disorders, including sepsis, thrombosis, and ischemic stroke. Although much about molecular mechanisms for APC's effects on cells remains unclear, it is clear that APC's structural features mediating anticoagulant actions and related bleeding risks are distinct from those mediating cytoprotective actions, suggesting the possibility of developing APC variants with an improved profile for the ratio of cytoprotective to anticoagulant actions.