Activated Protein C Resistance Research Articles

Thrombophilia Screening: Not So Straightforward.

Although inherited thrombophilias are lifelong risk factors for a first thrombotic episode, progression to thrombosis is multifactorial and not all individuals with inherited thrombophilia develop thrombosis in their lifetimes. Consequently, indiscriminate screening in patients with idiopathic thrombosis is not recommended, since presence of a thrombophilia does not necessarily predict recurrence or influence management, and testing should be selective. It follows that a decision to undertake laboratory detection of thrombophilia should be aligned with a concerted effort to identify any significant abnormalities, because it will inform patient management. Deficiencies of antithrombin and protein C are rare and usually determined using phenotypic assays assessing biological activities, whereas protein S deficiency (also rare) is commonly detected with antigenic assays for the free form of protein S since available activity assays are considered to lack specificity. In each case, no single phenotypic assay is capable of detecting every deficiency, because the various mutations express different molecular characteristics, rendering thrombophilia screening repertoires employing one assay per potential deficiency, of limited effectiveness. Activated protein C resistance (APCR) is more common than discrete deficiencies of antithrombin, protein C, and protein S and also often detected initially with phenotypic assays; however, some centres perform only genetic analysis for factor V Leiden, as this is responsible for most cases of hereditary APCR, accepting that acquired APCR and rare F5 mutations conferring APCR will go undetected if only factor V Leiden is evaluated. All phenotypic assays have interferences and limitations, which must be factored into decisions about if, and when, to test, and be given consideration in the laboratory during assay performance and interpretation. This review looks in detail at performance and limitations of routine phenotypic thrombophilia assays.

Thrombin Generation Assay in Antiphospholipid Antibodies Positive Subjects as a Personalized Thrombotic Risk Assessment: State of the Art and Perspectives.

Thrombotic risk assessment in antiphospholipid positive (aPL +) subjects is a major challenge, and the study of in vitro thrombin generation (thrombin generation assays (TGA)) could provide useful information. Activated protein C (APC) sensitivity is involved in thrombotic events in antiphospholipid syndrome patients. We summarized methods used to assess APC sensitivity with TGA and evaluated the prognostic role of APC resistance through literature search. APC resistance induced by aPL is a complex pathway. Several cross-sectional studies assessed APC sensitivity to understand thrombotic event mechanisms in aPL + subjects. Only one prospective cohort had investigated the prognostic impact of APC resistance in aPL + subjects, with a positive and significant correlation between APC sensitivity and the risk of thrombosis during the follow up (hazard ratio, 6.07 [95% CI, 1.69-21.87]). APC resistance assessed with TGA could be associated with thrombotic events in aPL + subjects.

A Multicentric Verification of Hereditary and Acquired Thrombophilia Coagulation Assays

Aims: With an ever-growing number of patients being admitted to our University Hospital Sveti Duh (Zagreb, Croatia), there was a need to positively respond to clinicians' demands about hereditary and acquired thrombophilia testing.
 Study Design: An extensive and multicentric verification of coagulation assays included in thrombophilia testing was performed on the BCS XP coagulation analyzer (Siemens Healthineers, Marburg, Germany): antithrombin activity (AT) (Innovance Antithrombin), protein C activity (PC) (Berichrom Protein C), protein S activity (PS) (Protein S Ac), free protein S antigen (free PS:Ag) (Innovance Free PS Ag), activated protein C resistance (APCR) (ProC Ac R and ProC Global + Coagulation Factor V Deficient Plasma), lupus anticoagulant (LA) screening (LA1 and activated partial thromboplastin time by using Dade Actin FSL as reagent) and confirmation test (LA2), factor VIII activity (FVIII) (Dade Actin FS and coagulation FVIII Deficient Plasma).
 Place and Duration of Study: Department of Medical Laboratory Diagnostics, University Hospital Sveti Duh, Zagreb, Croatia, between January 2020 and December 2021.
 Methodology: Verification protocol included: the precision study (CLSI EP15-A3 protocol), trueness estimation by comparison of seventy remnant plasma samples with two large Croatian hospital laboratories with established thrombophilia testing, and verification of reference intervals and cut-off values (CLSI EP28-A3C and CLSI HA-60 guidelines).
 Results: All of the obtained imprecision CVs were within the manufacturer's claims (<5/10/15%). While the observed bias for PC (+1.9%) was within the EFLM performance specifications (6.7%), the average bias for AT was higher than acceptance criteria (+10.8% vs 3.2% allowed). P&B regression revealed a significant positive proportional difference (slope=1.14). Comparison of PS activity yielded a high negative bias (-33.4%) that exceeded the acceptance criteria of 8.1%. Regarding LA testing, the diagnostic accuracy was 99% when compared to Sestre milosrdnice UHC (N=70) and 95% when compared to University Hospital Centre Zagreb (N=19). All manufacturer's reference intervals and cut-offs were verified.
 Conclusion: The verification study confirmed all manufacturer's claims, except for PS which has been replaced with free PS:Ag determinations.

Influence of Activated Protein C Resistance on Short-Term Hospital Outcomes Following Admission for Acute Ischemic Stroke in the United States

Introduction: Activated protein C (APC) plays a crucial role in breaking down factor Va and VIIIa, preventing excessive blood clotting. APC resistance occurs when protein C is unable to degrade these factors, leading to a higher risk of thrombosis. This condition can be either hereditary or acquired, with the most common cause being the factor V Leiden mutation. While previous studies have indicated that APC resistance increases the likelihood of acute ischemic stroke (AIS), their specific outcomes have not been extensively studied. Our research aims to bridge this gap via a national study. Methods: We used hospital records via the National Inpatient Sample (NIS) for our study. It consists of de-identified in-patient data, released on an annual basis by HCUP, AHRQ, and collaborating hospitals. Patients admitted with a primary diagnosis of AIS were recruited. We excluded all hospitalizations of ages <18 years. The impact of APCR status on outcomes and in-hospital complications such as acute kidney injury (AKI), myocardial infarction (MI), thrombolysis, mechanical thrombectomy (MT), vasopressor use, seizures, unfavorable discharge (discharge to another center such as short-term hospital, skilled nursing facility, intermediate care facility, and another type of facility) was studied using multivariable regression analysis, while adjusting for the impact of several potential patient and hospital characteristics. Results: Our selection criteria identified 2631890 admissions for AIS between 2016-2020, including 5910 (0.2%) patients also reporting a diagnosis of APCR (Table 1). In addition, APCR patients were younger (mean age 60.1 vs. 70.1 years, p<0.01) with a lower mean Charlson Comorbidity Index (CCI) score (3.50 vs. 3.87, p<0.01), and showed higher odds of a history of prior stroke (19.3% vs. 15.4%, aOR 1.482, 95% CI 1.387-1.584, p<0.01). While both groups were primarily covered by Medicare (65.0% in non-APCR group and 47.3% in APCR patients), a higher proportion of patients with APCR were also covered by private insurance (33.0% vs 19%, p<0.01). The APCR group also showed a higher prevalence of Whites (85.9% vs. 68.2%, p<0.01). Most cases in both cohorts were treated in Urban teaching centers. While higher prevalences of smoking(43.2% vs. 39.8%, p<0.01), obesity (20.3% vs. 14.5%, p<0.01), and drug abuse (4.4% vs. 2.8%, p<0.01) were seen in APCR patients, they also expressed fewer cases of cirrhosis (0.3% vs. 0.7%, p<0.01), diabetes (30.6% vs 39.2%, p<0.01), hypertension (51.9% vs 58.2%, p<0.01), chronic kidney disease (13.7% vs 18.3%, p<0.01), atrial fibrillation (18.8% vs 24.9%, p<0.01), and lipid disorder (53.8% vs 60.3%, p<0.01). After careful adjustments of confounders, APCR patients showed a higher use of mechanical thrombectomy (aOR 1.523, 95% CI 1.374-1.689, p<0.01) and vasopressor use (aOR 1.877, 95% CI 1.435-2.455, p<0.01) and also reported higher odds of seizures (aOR 1.413, 95% CI 1.265-1.578, p<0.01) and unfavorable discharge (aOR 1.065, 95% CI 1.006-1.128, p=0.03). However, lower odds of acute kidney injury (aOR 0.87, 95% CI 0.794-0.952, p<0.01) and death(aOR 0.708, 95% CI 0.597-0.838, p<0.01) were noted. No differences in events of MI (aOR 1.161, 95% CI 0.994-1.358, p=0.06) and thrombolysis (aOR 0.94, 95% CI 0.863-1.024, p=0.159) use were seen between the two groups. Conclusion: The study reported higher short-term outcomes, including increased use of mechanical thrombectomy and vasopressors and higher odds of seizures and unfavorable discharge in APCR patients with acute stroke. However, no similar increased odds of in-patient death, acute kidney injury, thrombolysis, and myocardial infarction were noted. While the impact of APCR on stroke recurrence has been highly debated in prior studies, our analysis provides a novel update showing an increased risk of subsequent stroke in patients with a history of stroke. Encouraging more extensive studies in a clinical setting, with larger groups of people and more extended observation periods, can provide a clearer understanding of the situation and thus improve their long-term outcomes.

Survival Advantage Associated with Heterozygous Factor V Leiden Mutation in Sars-Cov-2-Infected K18-hACE2 Mice

The pathogenesis of coronavirus disease 2019 (COVID-19) complications in critically ill patients involves virus-mediated cellular damage in infected tissue and secondary organ damage associated with a hyperinflammatory response. The proinflammatory milieu can result in endothelial dysfunction, predisposing patients to thrombosis leading to a life-threatening condition known as COVID-19-associated coagulopathy (CAC) which is driven by cellular and molecular mechanisms that are still unclear. We developed a mouse model based on the K18-hACE2 transgenic mouse recapitulating many of the CAC-associated pathological findings observed in human patients offering a reliable animal model for the study of SARS-CoV-2 pathogenesis. Plasma analysis revealed virus-induced upregulation of endothelial activation markers and alterations of all the measured coagulation factors' activities, resulting first in increased vascular leakage in the lung on day 5 post-infection, followed by the appearance of focal hemorrhagic lesions potentially associated with local thrombotic events on day 7. The infection-associated procoagulant phenotype appeared as early as day 3 post-infection (p.i.). Prothrombin time showed a 1/3 reduction in the normal clotting time in males. The overall procoagulant state was also reflected in the alteration of Factor(F) VIII and FX activity increasing to ~150% compared to uninfected controls. FIX showed the most significant alteration and was ~200% of the normal activity on day 3 p.i. and gradually increased over time, reaching ~350%. We also observed an increase in the activity of FV starting 3 days p.i. at ~210% of the normal level and persisting unchanged up to day 7 p.i. As reported during human infections, FV activity increased without significant changes in protein concentration or FVa levels. Natural anticoagulant and fibrinolytic mechanisms were dysregulated during virus infection resulting in a profound imbalance in thrombus formation and resolution. Infected K18-hACE2 mice showed downregulation of plasma protein C concentration which was not associated with any alteration of liver function, while plasma protein S concentration remained unaltered. Given the procoagulant environment generated after SARS-CoV-2 infection, reducing activated protein C (APC) anticoagulant and cytoprotective activities by blocking APC's active site with the SPC-54 monoclonal antibody worsened disease outcome and brought the mortality rate from 50% to 90%. In this observed procoagulant pathological context, where we observe increased activity of FV and where a deficiency of APC functions resulted in increased mortality, it wouldn't be surprising to observe an even more severe phenotype with the addition of APC resistance. To test this idea, we generated K18-hACE2 mice harboring the homologous human FV Leiden (FV L) mutation (R504Q). The Leiden mutation in FV eliminates one of several sites in FVa that are substrates for proteolysis by APC, resulting in diminished inactivation of FVa by APC and preventing the formation of the anticoagulant form of FV that serves as a cofactor for protein S-dependent inactivation of FVIIIa by APC. Upon SARS-CoV-2 infection, the presence of the homozygous FV L mutation in K18-hACE2 mice didn't affect the severity of the disease as measured by body weight loss during the course of the infection, and homozygous mutated mice had the same mortality rate as FV WT mice. Surprisingly, K18-hACE2 male mice carrying a heterozygous FV L mutation were extremely protected from SARS-CoV-2 infection, as their mortality dropped from 50% to only 15%. Histological analysis reveals how heterozygous FV L mice displayed a great reduction in immune infiltrate-associated fibrin deposition, suggesting an unexpected protective role of this prothrombotic mutation in the development of CAC. The protective effect of FV L heterozygosity may be attributable to enhanced thrombin formation which boosts inflammation and enhanced APC generation controlling proinflammatory and prothrombotic signaling, thus showing a delicate balance achieved by FV L heterozygosity that was lost for FV L homozygosity. As seen also in mouse models of bacterial sepsis, such favorable effects associated with FV L support the somewhat counterintuitive speculation that a partial increase in thrombin production in the early phase of the infection may support the resolution of COVID-19 complications.

NXT007-mediated hemostatic potential is suppressed by activated protein C-catalyzed inactivation of activated factor V

BackgroundActivated protein C (APC) inactivates activated factor (F) V (FVa) and FVIIIa. NXT007, an emicizumab-based engineered therapeutic bispecific antibody, enhances the coagulation potential of FVIII-deficient plasma (FVIIIdef-plasma) to near normal levels. However, little is known about the effect of APC-induced inactivation in NXT007-mediated hemostatic function. ObjectivesTo investigate the contribution of APC-mediated reactions to NXT007-driven hemostasis. MethodsIn pooled normal plasma (PNP) or FVIIIdef-plasma spiked with NXT007 (10 μg/mL), effects of APC (0-16 nM) were measured using a thrombin generation assay (TGA). The direct effects of APC on cofactor activity of NXT007 or FVIIIa in a FXa generation assay were also measured. The FVdef-plasma and FV Leiden plasma (FVLeiden plasma) were preincubated with 2 anti-FVIII monoclonal antibodies (termed FVIII-depleted), and the APC effect in the presence of NXT007 in FVIII-depleted FVdef-plasma with the addition of exogenous FV (7.5-30 nM) or FVIII-depleted FVLeiden plasma was investigated. ResultsThe APC dose-dependent suppression effect in TGA of FVIIIdef-plasma spiked with NXT007 was similar to that of PNP. FXa generation with NXT007 was not impaired by the addition of APC, suggesting that the APC-induced reaction in TGA with NXT007 was attributed to the direct inactivation of FVa. The addition of APC to FVIII-depleted FVdef-plasma, along with NXT007 and various FV concentrations, showed a similar attenuation to PNP. The NXT007-driven thrombin generation in FVIII-depleted FVLeiden plasma was suppressed by APC, similar to the reaction in native FVLeiden plasma. ConclusionNXT007 did not impair APC-mediated downregulation of FVa in FVIIIdef-plasmas, regardless of the presence of FV mutation with APC resistance.

OC 35.3 Correlation between ETP-Based APC Resistance and the Relative Risk of Venous Thromboembolism in Women Using Combined Oral Contraceptives

OC 35.3 Correlation between ETP-Based APC Resistance and the Relative Risk of Venous Thromboembolism in Women Using Combined Oral Contraceptives

Impact of Increased Activated Protein-C Resistance, Decreased Antithrombin III Activity and Hypocomplementemia on the Gestational Outcomes of Pregnancies with MTHFR Polymorphisms.

To evaluate the impact of increased Activated Protein C (APC) resistance, decreased antithrombin III activity and hypocomplementemia on the pregnancy outcomes of the patients with methylentetrahydrofolate reductase (MTHFR) polymorphisms. This study was composed of 83 pregnancies with MTHFR polymorphisms. Increased APC resistance, decreased antithrombin III activity and hypocomplementemia were accepted as risk factors for poor gestational outcome. Having at least one risk factor resulted in significantly higher rates of "APGAR score of<7" at the first ten minutes (p=0.009). Composite adverse outcome rate was also higher in patients with at least one of the defined risk factors despite lack of statistical significance (p=0.241). Rate of newborn with an "APGAR score of<7" at first ten minutes was significantly higher at patients with hypocomplementemia (p=0.03). Hypocomplementemia is a risk factor for poor gestational outcome in pregnancies with MTHFR polymorphisms.

Laboratory Diagnosis of Activated Protein C Resistance and Factor V Leiden.

The factor V Leiden (FVL) polymorphism is known as the most common inherited risk factor for venous thrombosis. In turn, FVL is the leading cause of an activated protein C resistance (APCR) phenotype, in which the addition of exogenous activated protein C to plasma does not result in the expected anticoagulant effect. In the routine laboratory approach to the formal diagnosis of FVL, an initial positive screening plasma-based method for APCR is often performed, and only if needed, this is followed by a confirmatory DNA-based assay for FVL. Multiple methods with accepted sensitivity and specificity for determining an APCR/FVL phenotype are commonly categorized into two separate groups: (1) screening plasma-based assays, including qualitative functional clot-based assays, for APCR, and (2) confirmatory DNA-based molecular assays, entailing several tests and platforms, including polymerase chain reaction-based and non-PCR-based techniques, for FVL. This review will describe the methodological aspects of each laboratory test and prepare suggestions on the indication of APCR and FVL testing and method selection.

Anti–β2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies interfere with cleavage of factor V(a) by activated protein C

BackgroundThe acquired thrombotic risk factor known as lupus anticoagulant (LA) interferes with laboratory clotting assays and can be caused by autoantibodies against β2-glycoprotein I (β2GPI) and prothrombin. LA is associated with activated protein C (APC) resistance, which might contribute to thrombotic risk in patients with antiphospholipid syndrome. How antibodies against β2GPI and prothrombin cause APC resistance is currently unclear. ObjectivesTo investigate how anti-β2GPI and antiphosphatidylserine/prothrombin (PS/PT) antibodies induce APC resistance. MethodsThe effects of anti-β2GPI and anti-PS/PT antibodies on APC resistance were studied in plasma (of patients with antiphospholipid syndrome) and with purified coagulation factors and antibodies. ResultsAPC resistance was observed in LA-positive patients with anti-β2GPI or anti-PS/PT antibodies and in normal plasma spiked with monoclonal anti-β2GPI or anti-PS/PT antibodies with LA activity. Analysis of factor (F)V cleavage patterns after APC incubation indicated that anti-β2GPI antibodies attenuated APC-mediated FV cleavage at R506 and R306. APC-mediated cleavage at R506 is required for FV cofactor activity during inactivation of FVIIIa. Assays with purified coagulation factors confirmed that anti-β2GPI antibodies interfered with the cofactor function of FV during FVIIIa inactivation but not with FVa inactivation. Anti-PS/PT antibodies attenuated APC-mediated FVa and FVIIIa inactivation. Analysis of FV(a) cleavage patterns after APC incubation indicated that anti-PS/PT antibodies interfere with APC-mediated cleavage of FV at positions R506 and R306. ConclusionAnti-β2GPI antibodies with LA activity contribute to a procoagulant state by causing APC resistance via interference with the cofactor function of FV during FVIIIa inactivation. LA-causing anti-PS/PT antibodies interfere with the anticoagulant function of APC by preventing FV(a) cleavage.

Role of tissue factor pathway inhibitor in hormone-induced venous thromboembolism.

Exposure to higher levels of steroid hormones, like that in pregnancy or during combined hormonal contraception, increases the risk of venous thromboembolism. Development of resistance to activated protein C (APC) thought to be the underlying pathomechanism of this prothrombotic state. This coagulation phenomena is largely to be explained by the hormone-induced impairment of the protein S/ tissue factor pathway inhibitor (TFPI) leading to a less efficient inactivation of factor Va and factor VIIIa by APC. APC resistance and decreased protein S/TFPI function were associated with the risk of first as well as recurrent venous thromboembolism. Preexisting disturbances in these pathways are likely to predispose to thrombosis during hormone exposure and can persist over years after the thrombosis event.Further studies are necessary to investigate the predictive value of forgoing APC resistance and decreased protein S/TFPI function or an excessive alteration in these parameters during hormone intake on the development of hormone-induced venous thromboembolism.

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.

Elucidation of an altered anticoagulant function due to Factor V abnormality and development of a simple screening assay for thrombophilia

Coagulation factor V (FV) is both procoagulant and anticoagulant functions. Congenital FV abnormality, which are caused by mutations in the FV gene, are characterized by a tendency to bleed. However, FV-R506Q (FVLeiden) is the most common FV abnormality that eliminates an activated protein C (APC) cleavage site, resulting in the occurrence of deep venous thrombosis (DVT). In Japan, the thrombotic predisposition caused by FVLeiden and FV molecular abnormalities was believed to be nonexistent. We did, however, report the first case in Japan of a young patient with FV abnormality-related thrombosis. The recurrent DVT in this case was caused by a novel mutation of FV-W1920R (FVNara), located in the C1 domain and far from the APC cleavage sites. We considered the possibility that there were cases of FV-related thrombotic predisposition that had gone undetected in Japan. We thoroughly examined FV-related anticoagulant function to understand the pathogenesis of thrombosis caused by FV abnormality. Furthermore, using recombinant thrombomodulin, we successfully developed a novel assay with clot waveform analysis for the rapid detection of FV deficiency with APC resistance. Other FV abnormality-related thrombosis has been reported in Japan in recent years, and we hope to further clarify the FV-related thrombotic predisposition in the future.

Factor V Leiden-independent activated protein C resistance: Communication from the plasma coagulation inhibitors subcommittee of the International Society on Thrombosis and Haemostasis Scientific and Standardisation Committee

Activated protein C resistance (APC-R) due to the single-nucleotide polymorphism factor V Leiden (FVL) is the most common cause of hereditary thrombophilia. It is found predominantly in Caucasians and is uncommon or absent in other populations. Although FVL is responsible for >90% of cases of hereditary APC-R, a number of other F5 variants that also confer various degrees of APC-R and thrombotic risk have been described. Acquired APC-R due to increased levels of coagulation factors, reduced levels of inhibitors, or the presence of autoantibodies occurs in a variety of conditions and is an independent risk factor for thrombosis. It is common for thrombophilia screening protocols to restrict assessment for APC-R to demonstrating the presence or absence of FVL. The aim of this Scientific and Standardisation Committee communication is to detail the causes of FVL-independent APC-R to widen the diagnostic net, particularly in situations in which in vitro APC-R is encountered in the absence of FVL. Predilution clotting assays are not FVL specific and are used to detect clinically significant F5 variants conferring APC-R, whereas different forms of acquired APC-R are preferentially detected using the classical activated partial thromboplastin time-based APC-R assay without predilution and/or endogenous thrombin potential APC-R assays. Resource-specific recommendations are given to guide the detection of FVL-independent APC-R.

Activated protein C resistance impact on Syrian candidates for in vitro fertilisation and the benefit of anticoagulation therapy: a retrospective cohort study

Activated protein C resistance (APCR) is a common thrombophilia, caused mainly by a mutation. The impact of APCR on the efficacy of In Vitro Fertilization (IVF) are still unclear, and no solid recommendations for its management were published. To investigate the effect of APCR on IVF outcomes and assess the efficacy of our management protocol, we retrospectively scanned the medical records of women who were tested with APCR assay in 2019 at our fertility centre. The 66 women (12%) positive for APCR had lower odds of reaching clinical pregnancies after IVF 0.18 [95% CI: 0.07–0.47] and fewer live births. The administration of low-molecular-weight heparin and aspirin associated with more implantation in treated compared to untreated APCR-positive women with an odds ratio of 43.2 [7.51–248.6]. In conclusion, APCR negatively affects the number of clinical pregnancies after IVF, but anticoagulation therapy can mitigate this effect and significantly increase clinical pregnancies. Impact Statement What is already known on this subject? The evidence about the impact of APCR on IVF outcomes is still inconclusive. According to the Canadian guideline, routine screening for thrombophilia in patients with recurrent pregnancy loss is not recommended. No clear recommendations regarding the management of APCR in the planning for IVF are yet available. What do the results of this study add? APCR significantly increases implantation failure among infertile women who conduct IVF. Management of APCR using LMWH and aspirin was effective in mitigating this effect and increasing successful implantation. What are the implications of these findings for clinical practice and/or further research? Our findings can support the recommendation to include APCR assay in the routine tests for infertile women conducting IVF, and suggest the combination between LMWH and aspirin as an effective therapy to increase successful implantation in APCR positive candidates. However, more controlled clinical trials are still needed to confirm our results.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Antiphospholipid syndrome: Reversal of antiphosphatidylserine/prothrombin-induced activated protein C resistance

BackgroundAnti-phosphatidylserine/prothrombin (aPS/PT) antibodies are the major contributor to activated Protein C resistance (APC-R) in tetra-positive thrombotic high-risk patients with Antiphospholipid Syndrome (APS). ObjectivesTo evaluate the role of phospholipids (PL) on aPS/PT mediated APC-R. Patients/methodsTotal IgG were purified from plasma of 6 tetra-positive patients and IgG aPS/PT were affinity-purified from 3 of these patients. Purified material was spiked into Normal Pooled Plasma (NPP) and tested for APC-R in thrombin generation assay and in Factor Va inactivation assay using increasing amounts of phospholipids. Results and conclusionsTotal IgG showed APC-R at low PL concentration (1.5 μg/mL) that disappeared at increasing PL concentrations (5.8, 11.6 and 46.6 μg/mL). In the same way, affinity purified aPS/PT showed a robust (59 %, 52 %, 36 %) APC-R in patients #4, #5 and #6, respectively at low PL concentration (1.5 μg/mL) that was completely reversed at higher concentration (11.6 μg/mL). The inactivation of FVa by activated Protein C (aPC) was impaired in the presence of aPS/PT at low aPL concentration and reversed by increasing amounts of PL. These data point out the relevance of PL in reversing APC-R in this ‘in vitro’ system. The mechanism for reversal might be explained by loss of PL availability for aPC. These results may give some insight into the pathogenesis of thrombosis or suggestions for alternative treatments.

Determination of Thrombogenicity Levels of Various Antiphospholipid Antibodies by a Modified Thrombin Generation Assay in Patients with Suspected Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a hypercoagulable state accompanied by the presence of heterogeneous antiphospholipid antibodies (aPL), which nonspecifically affect hemostasis by the presence of lupus anticoagulans (LA), anticardiolipin antibodies (aCL), antibodies against β2-glycoprotein-I (anti-β2GPI), but also non-criteria antibodies such as antibodies against β2-glycoprotein-I domain I (anti-DI), anti-phosphatidylserine/prothrombin (anti-PS/PT), anti-annexin V, and many others. The main target of the antibodies is the activated protein C (APC) system, the elimination of which can manifest itself as a thrombotic complication. The aim of this study was to determine the thrombogenicity of antibodies using a modified protein C-activated thrombin generation assay (TGA) on a group of 175 samples suspected of APS. TGA was measured with/without APC and the ratio of both measurements was evaluated (as for APC resistance), where a cut-off was calculated ≤4.5 (90th percentile) using 21 patients with heterozygous factor V Leiden mutation (FV Leiden heterozygous). Our study demonstrates the well-known fact that multiple positivity of different aPLs is a more severe risk for thrombosis than single positivity. Of the single antibody positivity, LA antibodies are the most serious (p value < 0.01), followed by aCL and their subgroup anti-DI (p value < 0.05). Non-criteria antibodies anti-annexin V and anti-PT/PS has a similar frequency occurrence of thrombogenicity as LA antibodies but without statistical significance or anti-β2GPI1 positivity. The modified TGA test can help us identify patients in all groups who are also at risk for recurrent thrombotic and pregnancy complications; thus, long-term prophylactic treatment is appropriate. For this reason, it is proving increasingly beneficial to include the determination antibodies in combination with modified TGA test.