Frank Vitzthum

Factor VII-activating protease in patients with acute deep venous thrombosis

Factor VII-activating protease (FSAP) is involved in haemostasis and inflammation. FSAP cleaves single chain urokinase-type plasminogen activator (scu-PA). The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene leads to the expression of a FSAP variant with reduced ability to activate scu-PA, without affecting the ability to activate coagulation Factor VII (FVII). Previous studies have investigated the association of the 1601GA genotype with incidence and progression of carotid stenosis and deep venous thrombosis (DVT). The present study is the first to evaluate the potential association between the FSAP phenotype and DVT. We studied the association between the 1601G/A polymorphism, FSAP activity, FSAP antigen, Factor VIIa (FVIIa), prothrombin fragment 1+2 (F1+2), and C-reactive protein (CRP) in plasmas of 170 patients suspected for DVT. FSAP genotypes were equally distributed in patients with (n=64) and without DVT (n=106), (P=0.94). The 1601GA genotype was associated with significant reduction of FSAP activity (P<0.001) and FSAP antigen levels (P=0.04). Patients with DVT showed significantly higher FSAP activity (P=0.008), FSAP antigen (P=0.003), and F1+2 levels (P<0.001) than patients without DVT. The association between the FSAP measures and DVT disappeared when adjusted for CRP levels. F1+2 correlated positively to FSAP antigen (P=0.01), while FVIIa-levels were comparable in patients with and without DVT. We conclude that even though FSAP measures are significantly increased in patients with acute DVT, alterations in the scu-PA activating properties of FSAP are presumably not markedly involved in the development of acute DVT, and that the association between FSAP and DVT disappears after adjustment for CRP.

Tests for the measurement of factor VII-activating protease (FSAP) activity and antigen levels in citrated plasma, their correlation to PCR testing, and utility for the detection of the Marburg I-polymorphism of FSAP

Abstract Background: The single nucleotide Marburg I (MRI) polymorphism of the factor VII-activating protease (FSAP) gene, the prourokinase-activating activity of FSAP, and antigen levels of FSAP in plasma have been associated with incidence and progression of carotid stenosis and venous thromboembolism. However, more information on the extent of these associations, potential further ones, and respective clinical utilities remain to be determined. At present, testing is performed mainly by PCR assays based on probes or SYBR Green I. Some studies include testing for antigen levels of total FSAP and its ability to activate prourokinase. To test large cohorts, it is beneficial to rely on assays that are cost-effective, reliable, easy to use, rapid to perform, and that may eventually be automated. In addition, it appears advantageous to use functional tests or tests that determine antigen levels as they may relate more closely to the phenotype than the genotype does. Methods: Tests for the measurements of antigen levels of FSAP and its prourokinase-activating activity were improved and performance characteristics assessed. To determine the FSAP genotypes, an amplification created restriction site (ACRS) PCR test was developed. Results: Key performance characteristics of the FSAP activity and antigen tests were as follows: measuring range: 350–1400 mPEU/mL and 1.8–120 ng/mL, total coefficients of variation (CV): 5%–20% and 5%–14%, within-run CV: 4%–11% and 2.3%–12%, and run-to-run CV: 2%–17% and 4.3%–8.3%, respectively. The ratio of the activity and antigen level of FSAP correctly identified the FSAP genotypes of 126 samples tested. Conclusions: The ACRS PCR test is useful for laboratories that do not have the equipment to perform probe or SYBR Green I based real-time PCR. Furthermore, the tests developed for the determination of FSAP activity and antigen levels are convenient for determining clinical correlations, even for large population studies. The ratio of activity and antigen level of FSAP appears to be a promising and efficient alternative to molecular diagnostic techniques to detect the MRI polymorphism of FSAP. Clin Chem Lab Med 2008;46:1109–16.

Plasma factor VII-activating protease is increased by oral contraceptives and induces factor VII activation in-vivo.

UNLABELLED Oral contraceptive (OC) use influences the hemostatic system significantly and is a risk factor for development of cardiovascular disease. Factor VII-activating protease (FSAP) has potential effects on hemostasis. The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene expresses a FSAP alloenzyme with reduced pro-fibrinolytic activity. Presently, we address whether OC use and OC formulation affect FSAP measures in human blood. Healthy women (n=588) were allocated to six cycles of OCs with estrogen contents of 20 μg (n=158), 30 μg (n=284), 35 μg (n=79) or 50 μg (n=67) combined with various progestins. FSAP genotypes, FSAP and factor VII (FVII) plasma measures were assessed at baseline and after 6 cycles of OC. The 1601GA genotype was present in 49 (8.3%) of the women and was associated with significantly reduced levels of FSAP (P≤0.001). OC use increased FSAP antigen by 25% and FSAP activity by 59% (P<0.001). The FSAP increase was comparable in the seven different OC treatment groups (P>0.05). The relative increase in FSAP activity was significantly higher in women carrying the 1601GG genotype (63%) than in women carrying 1601GA genotype (50%) (P=0.01) and was associated with an increased activation of FVII. IN CONCLUSION OC use increases the plasma measures of FSAP. The increase in FSAP is comparable in the seven OC-groups studied but is more significant in women carrying the 1601GG genotype than in women with the 1601GA genotype and results in increased activation of FVII suggesting that FSAP-induced activation of FVII takes place in-vivo and not only in-vitro as hitherto described.

Quantification of coagulation factor XIII activity by a thio-NADH based assay using factor XIII immuno-depleted plasma as a diluent for calibration

Abstract Background: Accurate determination of factor XIII (FXIII) activity is crucial for replacement therapy. FXIII activity is typically determined using a coupled enzymatic reaction that measures nicotinamide adenine dinucleotide hydride (NADH) consumption at 340 nm. Methods: Here, we describe the development of a prototype for a novel FXIII activity assay for detection at 405 nm by replacing NADH with thio-NADH, and the application of FXIII immuno-depleted plasma as a diluent for calibration. Results: Performance data show up to two-fold lower susceptibility of the prototype assay to interferences from hemolyzed, icteric, and lipemic samples when compared to a NADH assay format. In addition, the use of FXIII immuno-depleted plasma as diluent for calibration improved recovery almost two-fold in the lower measurement range. The novel prototype assay correlates well with a conventional assay (r=0.98, y=0.99·x+2.17% FXIII, n=173). Conclusions: The described prototype assay has the potential to (a) increase trueness of measurement of low levels of FXIII, (b) improve robustness due to reduction from interferences, and (c) can be used on a broad range of coagulation instruments due to its detection at 405 nm. Clin Chem Lab Med 2010;48:1739–43.

Hormone therapy affects plasma measures of factor VII-activating protease in younger postmenopausal women.

Objectives Current reviews indicate that hormone therapy (HT) has a protective role in coronary heart disease (CHD) in younger postmenopausal women, whereas HT contributes to CHD in older women. Factor VII-activating protease (FSAP) is a serine protease that accumulates in unstable atherosclerotic plaques. FSAP is presumably involved in plaque stability and rupture. Reduced plasma concentration of FSAP may be associated with the development and expression of atherosclerosis and may thus contribute to precipitation of CHD. Here we address the potential influence of various HT regimens on plasma measures of FSAP in postmenopausal women treated for 1 year with different HT formulations or no HT. Methods Six groups of postmenopausal women (n = 139) were allocated to five different HT modalities or no HT. Samples were collected at baseline and after 12 months of treatment. Prototype assays were used for the determination of FSAP antigen and FSAP activity. Results The FSAP measures were comparable at baseline. No significant changes were observed in the control group after 12 months. HT in general induced a significant increase in FSAP antigen (7.7 μg/ml at baseline and 8.0 μg/ml after 12 months, p = 0.05), FSAP activity (1.54 PEU/ml at baseline and 1.68 PEU/ml after 12 months, p < 0.001) and FSAP ratio (202 mPEU/μg at baseline and 210 mPEU/μg after 12 months, p = 0.01). Conclusions HT increases the plasma measures of FSAP. This increase may contribute to the protective effect on CHD induced by HT in younger postmenopausal women.

Factor VII-activating protease: sex-related association with coronary artery calcification

&NA; Factor VII-activating protease (FSAP) may regulate development of cardiovascular disease (CVD). We evaluated sex differences in FSAP measures and examined the association between FSAP and coronary artery calcification (CAC) in a middle-aged population. Participants were randomly selected citizens aged 50 or 60 without CVD, diabetes mellitus, Marburg I polymorphism, or hormone replacement therapy (HRT). FSAP protein concentration (total FSAP), FSAP urokinase-activating capacity (FSAP GP), and FSAP GP/total FSAP (specific FSAP activity) were measured. Cardiac computed tomography (CT) determined the Agatston score, dividing the study population in three groups: (1) Agatston score = 0 U, (2) Agatston score = 1–99 U, or (3) Agatston score more than 99 U. A total of 134 women and 116 men were included. Total FSAP, FSAP GP, and specific FSAP activity were independently higher in women (97.4%, 81.1%, 0.84, respectively) compared with men (87.5%, 68.7%, 0.79, respectively) (P < 0.001). In women, total FSAP was significantly different between (3) Agatston score (111.5%) and (1) Agatston score (95.4%), respectively, (2) Agatston score (96.8%), (P < 0.05). Also, the specific activity of FSAP was significantly different between (3) Agatston score (0.77) and (1) Agatston score (0.85), respectively, (2) Agatston score (0.86) (P < 0.05). No difference in FSAP measures was observed in men. FSAP measures are higher in women compared with age-matched men. The extent of CAC in women is positively associated with total FSAP, but negatively associated with the specific activity of FSAP suggesting that FSAP may play a role in the evolution of CVD in women.

Qualitative detection of the Marburg I alloenzyme of factor VII-activating protease by an immunoassay and its comparison to PCR testing

Abstract Background: The Marburg I (MRI) single nucleotide poly-morphism (SNP) of the factor VII-activating protease (FSAP) gene has been associated with thrombophilia, thromboembolism, atherosclerosis, and the incidence and progression of carotid stenosis. At present, MRI SNP testing is mainly performed using costly nucleic acid analysis. The ratio between FSAP activity and antigen concentrations in citrated plasma has been used to assess the FSAP genotype. Methods: This article describes the development of a prototype ELISA for the detection of the MRI FSAP alloenzyme, and its correlation to FSAP genotypes to assess whether a positive MRI FSAP ELISA result may be used as a surrogate marker for the presence of the MRI SNP. Results: ELISA results were correlated with FSAP genotypes from 523 blood donors measured using PCR. Diagnostic sensitivity and specificity of the assay for determina-tion of the genotype were 100% (95% confidence interval [CI]: 93.36–100) and 99.79% (95% CI: 98.80–99.96), respectively. Maximum run-to-run, within-run, and total coefficients of variation were 7.8%, 7.9%, and 9.9%, respectively. No cross-reactivities with homologues of the MRI FSAP alloenzyme were observed. Test performance was not affected by typical interfering compounds. Conclusions: The data demonstrate that an immunoassay applying antibodies specific to the MRI FSAP alloenzyme can provide sufficiently accurate detection of the MRI SNP. This will significantly simplify MRI FSAP testing, particularly in large cohorts. Clin Chem Lab Med 2010;48:1745–9.

Fully automated immunoassay for quantitative determination of FXIII

UNLABELLED Coagulation factor XIII (FXIII) is essential for clot stabilization. Deficiency of FXIII is associated with a risk of bleeding and impaired wound healing. Substitution therapy with FXIII remedies for patients with low plasma levels of FXIII requires diagnostic quantification of the factor before and during therapy. Here, we describe a prototype of a preliminary research immunoassay for quantification of FXIII antigen on automated coagulation instruments. The prototype assay is based on a monoclonal antibody (mAb) directed against FXIII A chain, whereas the mAbs are coupled to latex particles. FXIII in a plasma specimen causes agglutination of the latex particles, which can be quantified turbidimetrically. Performance data of the assay prototype processed on BCS® XP and Sysmex® CA-1500 instruments demonstrate a good correlation to the Berichrom® factor XIII activity assay1 from Siemens Healthcare Diagnostics (r = 0.94). RESULTS Comparability of instruments was excellent (r = 0.98). Coefficients of variation of total imprecision measurements ranged from 2.2 to 3.4%. Linearity was excellent over the range tested (12-121% FXIII). Analytical sensitivity was 0.51% FXIII on BCS XP and 0.44% FXIII on Sysmex CA-1500, respectively. No interference (>10% bias) was observed with haemoglobin (up to 400 mg/dl), cholesterol (up to 300 mg/dl), bilirubin (up to 60 mg/dl) or triglycerides (up to 3000 mg/dl). CONCLUSION The preliminary research assay prototype has the potential for excellent analytical sensitivity, precision, and dynamic range suitable to measure reliably FXIII antigen levels in human plasma.

Coagulation assays based on the Luminescent Oxygen Channeling Immunoassay technology1)

Abstract Background: The Luminescent Oxygen Channeling Immunoassay (LOCI®) technology is a well-established homogeneous assay format that allows for fast, accurate, and highly sensitive quantitation of analytes. We set out to develop and prove a novel concept to establish a LOCI format that should principally allow for the determination of the activity of coagulation factors and anticoagulants of clinical relevance. Methods: The concept is based on the linkage of LOCI nano-beads by a peptide that can be cleaved by a coagulation factor. To prove the principle, we used a peptide that can be cleaved by thrombin. Results: We were able to show that coagulation activation of plasma or whole blood samples that were combined with the LOCI components degraded the thrombin-sensitive peptide and consequently, led to a reduction of the LOCI signal. Signal reduction was proportional to the amount of active thrombin generated. The research prototype assay allowed for the detection of factor deficiencies in both the extrinsic and intrinsic coagulation pathways, and for the quantification of hirudin, a direct thrombin inhibitor. Conclusions: Taken together, we conclude that the LOCI technology has the potential for extension to functional blood coagulation assays.

Direct chromogenic substrate immuno-capture activity assay for testing of factor VII-activating protease.

Abstract Background: The Marburg I (MRI) single nucleotide polymorphism (SNP) of the factor VII-activating protease (FSAP) gene has been associated with thrombophilia and atherosclerotic disease. PCR is used to detect the SNP. Also, the specific FSAP activity to cleave single-chain urokinase-type plasminogen activator (scu-PA) serves as a surrogate for PCR testing. Development of further assays is indicated in order to increase testing opportunities for future studies. Methods: A direct chromogenic substrate immuno-capture activity assay for FSAP (FSAP dcs activity assay) was established. Performance characteristics of the FSAP dcs activity assay were compared to the FSAP scu-PA activity assay. Results: The FSAP dcs activity assay detects FSAP activity from 25% to 150% of the norm. Total CVs ranged from 6% to 10% for FSAP wild type samples and 9%–18% for MRI samples. Correlation between the FSAP dcs and scu-PA activity assays was low (R=0.7). The FSAP dcs activity determined the presence of the MRI FSAP alloenzyme with a diagnostic sensitivity and specificity of 100% [95% confidence interval (CI): 89.6%–100%] and 96.2% (95% CI: 93.2%–97.4%), respectively, whereas the specific FSAP dcs activity increased specificity to 99.0% (95% CI: 97.2%–99.6%). Conclusions: The specific FSAP dcs activity represents a reliable method for the detection of the FSAP MRI alloenzyme. Due to the limited correlation between the FSAP dcs and scu-PA activity assays, these different measurands may exhibit different utility in research and clinical applications. Thus, the FSAP dcs activity assay can represent a valuable complement or alternative for FSAP testing in future studies.