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Molecular basis of antithrombin deficiency

Antithrombin (AT) is the most important physiological inhibitor of coagulation proteases. It is activated by glycosaminoglycans such as heparin. Hereditary antithrombin deficiency is a rare disease that is mainly associated with venous thromboembolism. So far, more than 200 different mutations in the antithrombin gene (SERPINC1) have been described. The aim of our study was to characterise the molecular background in a large cohort of patients with AT deficiency. Mutation analysis was performed by direct sequencing of SERPINC1 in 272 AT-deficient patients. Large deletions were identified by multiplex PCR coupled with liquid chromatography or multiplex ligation-dependent probe amplification (MLPA) analysis. To predict the effect of SERPINC1 sequence variations on the pathogenesis of AT deficiency, in silico assessments, multiple sequence alignment, and molecular graphic imaging were performed. The mutation profile consisted of 59% missense, 10% nonsense, 8% splice site mutations, 15% small deletions/insertions/duplications, and 8% large deletions. Altogether 87 different mutations, including 42 novel mutations (22 missense and 20 null mutations), were identified. Of the novel missense mutations, nine are suspected to impair the conformational changes that are needed for AT activation, two to affect the central reactive loop or the heparin binding site, and six to impair the structural integrity of the molecule. Despite the heterogeneous background of AT deficiency, 10 AT variants occurred in multiple index patients. Characterisation of the SERPINC1 mutation profile in large cohorts of patients may help to further elucidate the pathogenesis of AT deficiency and to establish genotype-phenotype associations.

Influence of blood collection techniques on platelet function

For investigations of platelet function it is recommended that venipuncture should be performed using ordinary needle systems instead of butterfly cannulae systems. Platelets might be activated in the long plastic tubes of butterfly systems. The aim of this study was to investigate the dependency of platelet function results on blood sampling using different collection systems. Therefore, blood of 25 healthy volunteers was collected from both arms using at the same time on one side a 21-gauge needle and on the other side a 21-gauge butterfly cannula system. Both samples of each volunteer were analyzed on the PFA-100®. Platelet aggregation was performed on the Behring Coagulation Timer (BCT) and the optical aggregometer PAP-4 using ADP, collagen and arachidonic acid to induce platelet aggregation in platelet-rich plasma. No significant prolongation of the closure times on the PFA-100 with the COL/EPI cartridge and the COL/ADP cartridge was observed when using butterfly cannulae. The results of optical aggregometry were not significantly different. The maximum aggregation response did not differ significantly for both collection systems. Aggregometry and the PFA-100 system are not affected by different blood collection systems. Therefore butterfly cannulae can be used for sample collection to investigate platelet function.

Response to aspirin and clopidogrel monitored with different platelet function methods

Laboratory non-response to aspirin or clopidogrel is defined as an inability to cause in vitro detectable platelet function inhibition. It would be beneficial to monitor response to aspirin or clopidogrel with widely available and routinely used platelet function methods, like the platelet function analyzer (PFA-100®) or the fully automated coagulation analyzer BCT®. The aim of this study was to assess the potential of the coagulation analyzer BCT® and the platelet function analyzer PFA-100® in monitoring the response of aspirin and clopidogrel. A group of 125 consecutive patients with arterial occlusive disease treated either with aspirin 100 mg/day (82 patients) or clopidogrel 75 mg/day (43 patients) as only antiplatelet drug were investigated. For the first time platelet-enriched plasma (PRP), not adjusted to a fixed predetermined concentration of platelets, was used for aggregation studies and the effect of clopidogrel alone without combination of aspirin treatment on platelet function was investigated. Response to aspirin was observed in 85% (70/82) of patients using PFA-100®, while performing the arachidonic acid-induced aggregation on the BCT showed an inhibitory effect to aspirin in 91% (75/82) of patients. Non-response to aspirin was assessed with both platelet function methods in 7% (6/82) of patients. Clopidogrel response was observed in 58% (25/43) of patients when performing ADP-induced aggregation on the BCT. On the PFA-100® the antiplatelet effect of clopidogrel could not be detected. In conclusion, measurement of platelet aggregation on the BCT using native platelet-enriched plasma allows the quantification of individual inhibitory effects to aspirin as well as to clopidogrel, while the PFA-100® seems only suitable to investigate the degree of platelet inhibition induced by aspirin but not by clopidogrel.

Cardiovascular risk factors in idiopathic compared to risk-associated venous thromboembolism: a focus on fibrinogen, factor VIII, and high-sensitivity C-reactive protein (hs-CRP).

There have recently been reports of an increased incidence of arterial cardiovascular events in patients with idiopathic venous thromboembolism (VTE) compared to patients with risk-associated VTE. The aim of our study was to evaluate whether elevated clotting factors, which have been linked to chronic sub-clinical inflammation and arterial thromboembolic disease, have a higher prevalence in idiopathic VTE compared to secondary VTE. Plasma fibrinogen, factor VIII, and high-sensitivity C-reactive protein (hs-CRP) levels were determined in a cohort of sex- and age-matched patients with unprovoked VTE (n=101), patients with secondary VTE (n=101), and controls (n=202). Fibrinogen and hs-CRP levels were higher in patients with idiopathic VTE (fibrinogen: median/range: 331/214-524 mg/dl; hs-CRP: median/interquartile range: 1.8/0.8-3.7 mg/l) than in those with risk-associated VTE (299/162-458 mg/dl, p=0.004; 1.5/0.8-2.2 mg/l, p=0.05) and controls (302/185-644 mg/dl, p=0.001; 1.2/0.5-2.2 mg/l, p=0.02). Fibrinogen levels in the upper tertile of the controls were seen in 53% of patients with unprovoked VTE, compared to 35% of patients with secondary VTE. According to their hs-CRP levels (>3 mg/l), 26% of patients with idiopathic VTE were categorised as being at high risk for cardiovascular disease, as opposed to just 9% of those with risk-associated VTE. Factor VIII activity was significantly higher in patients with both idiopathic and secondary VTE than in controls, with the highest median value in patients with idiopathic VTE. Our data show that markers of inflammation, such as hs-CRP, fibrinogen, and factor VIII, are at higher levels in patients with idiopathic compared to secondary VTE, supporting the hypothesis that idiopathic VTE and arterial thromboembolism share common risk factors.

Development and clinical evaluation of two chromogenic substrate methods for monitoring fondaparinux sodium

This study was designed to develop methods for monitoring of the selective factor Xa inhibitor fondaparinux sodium (ARIXTRA®) based on standard laboratory methods for the chromogenic determination of the anti-factor Xa activity of low molecular weight heparin. To examine the biologic activity of fondaparinux in comparison to its plasma concentration, 2 methods were investigated: 1 working with the addition of antithrombin (AT), the other without exogenous AT. Both methods showed a linear relationship of fondaparinux concentration and OD/min on a log-lin scale in the range from 0.1 to 2 μg/mL. Inter- and intra-assay variability was <6% in all cases. The results of spiked samples from patients on vitamin K antagonists (VKA) were in good agreement with both methods, and the determination of the fondaparinux concentration was not influenced by reduced levels of factor X in plasma caused by VKA-intake. Ex vivo samples from orthopedic patients (n=18) on prophylactic treatment with fondaparinux showed concentrations between 0.2 to 0.7 μg/mL 3 hours after s.c. injection. No significant differences were detected between both methods with these samples. The presented methods are suitable tools for monitoring of fondaparinux. The linear calibration curve in the range 0.1 to 2 μg/mL is suitable for determination of prophylactic and therapeutic application of fondaparinux. Both methods, with and without addition of AT, can be performed fully automated in clinical routine on an automated coagulation analyzer (STA coagulation analyzer®). No significant differences were detected between both methods with these samples.

Prediction of phenprocoumon maintenance dose and phenprocoumon plasma concentration by genetic and non-genetic parameters

PurposeThe anticoagulation response to vitamin K antagonists is characterised by high inter-individual variability. The impact of single nucleotide polymorphisms (SNPs) in several genes of enzymes involved in the vitamin K cycle on phenprocoumon dose variability and phenprocoumon plasma concentrations is still under investigation.MethodsWe assessed the influence of VKORC1 c.-1639G>A, CYP2C9*2, CYP2C9*3, CYP4F2 c.1297G>A, CALU c.*4A>G, EPHX1 c.337T>C, GGCX c.214+597G>A, F7 c.-402G>A, F7 c.-401G>T, PROC c.-228C>T and PROC c.-215G>A along with clinical and demographic parameters on steady-state phenprocoumon therapy in 75 patients. A prediction model was developed for total phenprocoumon plasma concentrations and daily phenprocoumon doses required for therapeutic anticoagulation.ResultsThe VKORC1 c.-1639 genotype was the main predictor of the phenprocoumon daily dose (adjusted R2 = 37.6%) and the total phenprocoumon concentration (adjusted R2 = 38.3%). CYP2C9 affected the phenprocoumon concentration, but not the dose requirements. SNPs in the other genes of the vitamin K cycle, concomitant medication, nicotine use and alcohol consumption did not predict phenprocoumon concentrations and phenprocoumon dose requirements in a multiple linear regression model. Phenprocoumon concentrations were predicted by VKORC1 c.-1639, CYP2C9 genotype, age and BMI. The final prediction model for the daily phenprocoumon dose requirements comprised VKORC1 c.-1639 genotype, age and height accounting for 48.6% of the inter-individual variability.ConclusionsA rough prediction of phenprocoumon maintenance doses can be achieved by a limited set of parameters (VKORC1, age, height). The investigated SNPs in CYP4F2, CALU, EPHX1, GGCX, F7, and PROC did not improve the predictive value of a pharmacogenetic-based dosing equation for phenprocoumon.

Impact of the type of SERPINC1 mutation and subtype of antithrombin deficiency on the thrombotic phenotype in hereditary antithrombin deficiency

Mutations in the antithrombin (AT) gene can impair the capacity of AT to bind heparin (AT deficiency type IIHBS), its target proteases such as thrombin (type IIRS), or both (type IIPE). Type II AT deficiencies are almost exclusively caused by missense mutations, whereas type I AT deficiency can originate from missense or null mutations. In a retrospective cohort study, we investigated the impact of the type of mutation and type of AT deficiency on the manifestation of thromboembolic events in 377 patients with hereditary AT deficiencies (133 from our own cohort, 244 reported in the literature). Carriers of missense mutations showed a lower risk of venous thromboembolism (VTE) than those of null mutations (adjusted hazard ratio [HR] 0.39, 95% confidence interval [CI] 0.27-0.58, p<0.001), and the risk of VTE was significantly decreased among patients with type IIHBS AT deficiency compared to patients with other types of AT deficiency (HR 0.23, 95%CI 0.13-0.41, p<0.001). The risk of pulmonary embolism complicating deep-vein thrombosis was lower in all type II AT deficiencies compared to type I AT deficiency (relative risk 0.69, 95%CI 0.56-0.84). By contrast, the risk of arterial thromboembolism tended to be higher in carriers of missense mutations than in those with null mutations (HR 6.08-fold, 95%CI 0.74-49.81, p=0.093) and was 5.9-fold increased (95%CI 1.22-28.62, p=0.028) in type IIHBS versus other types of AT deficiency. Our data indicate that the type of inherited AT defect modulates not only the risk of thromboembolism but also the localisation and encourage further studies to unravel this phenomenon.

Impact of pharmacokinetic (CYP2C9) and pharmacodynamic (VKORC1 , F7 , GGCX , CALU , EPHX1) gene variants on the initiation and maintenance phases of phenprocoumon therapy

Compared to warfarin, little is known about the effect of pharmacogenomics on the inter-individual variability of phenprocoumon therapy. In a retrospective cohort study, we investigated the impact of VKORC1 c.-1639G>A; CYP2C9*2 , CYP2C9*3 ; GGCX c.214+597G>A; CALU c.*4A>G; EPHX1 c.337T>C; F7 c.-402G>A, and F7 c.-401G>T on the initiation (n=54) and maintenance phases (n=91) of phenprocoumon therapy. We assessed the following outcome parameters: time to stable international normalised ratio (INR), time to first supra-therapeutic INR, time out of INR range, probability of over-anticoagulation, number of anticoagulation clinic visits. During the initiation phase, homozygotes for the VKORC1 c.-1639 A and G alleles achieved stable INRs later (p<0.001), spent more time at supra-therapeutic INRs (p<0.001), had increased risks of over-anticoagulation (odds ratio 19.83, p=0.003 and 4.45, p=0.045, respectively), and had higher frequencies of anticoagulation clinic visits (p<0.001) compared to GA carriers. CYP2C9*2, *3 carriers reached stable INRs faster (p=0.024) with fewer anticoagulation clinic visits (p=0.001) than wild-type carriers. EPHX1 c.337 C carrier spent significantly more time above range in the initiation phase (p=0.023). GGCX , CALU , and F7 gene variants did not affect outcome parameters of the initiation phase and none of the genotypes had an impact on maintenance phase parameters. Compared to the VKORC1 genotype, early INR values were less informative in the prediction of outcome parameters such as time to stable INR and time above the INR range. Our study is limited by the retrospective study design with no standardised protocol in a usual care setting. Therefore, our findings should be validated in a larger, controlled prospective study.

Intrinsic clotting factors in dependency of age, sex, body mass index, and oral contraceptives: definition and risk of elevated clotting factor levels.

Elevated clotting factors have been demonstrated to be a risk factor for venous thromboembolism (VTE). The aim of our study was to investigate the impact of age, sex, body mass index, and oral contraceptives on the clotting factor activities of factors VIII, IX, XI, and XII and their impact on the cutoff definition and risk of VTE associated with elevated clotting factors. Factor VIII, IX, XI, and XII activities were measured in 499 blood donors and 286 patients with VTE. Age and body mass index predicted significantly and independently the clotting factor activities of factors VIII, IX, and XI, whereas use of oral contraceptives predicted factor IX, XI, and XII levels. Percentiles of clotting factor activities, which are often used for the cutoff definition of elevated clotting factors, varied due to the effect of age, body mass index, and oral contraceptives. The adjusted odds ratios for VTE were 10.3 [95% confidence interval (CI) 5.1–20.7], 6.1 (95% CI 3.1–12.0), and 3.3 (95% CI 1.9–5.8) for elevated factors VIII, IX, and XI, respectively. Furthermore, our study demonstrates for the first time that elevated factor XII is associated with an increased risk of VTE (adjusted odds ratio 2.9, 95% CI 1.6–5.3).

Spleen Size Is Significantly Influenced by Body Height and Sex: Establishment of Normal Values for Spleen Size at US with a Cohort of 1200 Healthy Individuals

PURPOSE To define height- and sex-corrected normal values for spleen length and volume determined with ultrasonography (US). MATERIALS AND METHODS The authors performed a retrospective data review of stem cell donors who had provided written informed consent for stem cell donation and use of anonymized data and biologic materials for scientific and quality control purposes. Spleen length, spleen volume, and anthrophometric data were correlated in 1230 healthy volunteers to identify variables that affect spleen size. Multiple linear regression analysis was performed to weight effects of various variables on spleen size. Linear regression through the 95th percentile for men and women of different height cohorts generated the formula for the upper limit of normal for spleen length and volume. For validation, the upper limit of normal was calculated for each volunteer and compared with the observed value. Formulae to calculate the additional percentiles were similarly generated and validated. A cohort of 75 volunteers was analyzed twice to assess the stability of spleen length and volume over time. RESULTS Spleen length and volume were significantly and independently associated with sex (length: P < .001; volume: P = .012), body height (P < .001 for both), and weight (P < .001 for both), with men and taller and heavier individuals having longer and larger spleens. The spleen length of 20 of 324 women (6%) and 234 of 906 men (26%) exceeded the previously reported upper limit of normal of 12 cm. Repeat measurements indicated that spleen length (median difference, 0.10 cm; range, -1.8 to 1.7 cm) and volume (median difference, 3 cm(3); range, -106 to 142 cm(3)) were quite stable. A mobile application that performs these calculations is available for download. CONCLUSION The authors define height- and sex-corrected normal values for spleen length and volume for women with a body height of 155-179 cm and men with a body height of 165-199 cm and propose validated algorithms to gauge the percentile of an individuals spleen size.