Concentrations Of FVIII Research Articles

An Electrochemical Biosensor for the Detection of Pulmonary Embolism and Myocardial Infarction.

Due to the clinical similarities between pulmonary embolism (PE) and myocardial infarction (MI), physicians often encounter challenges in promptly distinguishing between them, potentially missing the critical window for the correct emergency response. This paper presents a biosensor, termed the PEMI biosensor, which is designed for the identification and quantitative detection of pulmonary embolism or myocardial infarction. The surface of the working electrode of the PEMI biosensor was modified with graphene oxide and silk fibroin to immobilize the mixture of antibodies. Linear sweep voltammetry was employed to measure the current-to-potential mapping of analytes, with the calculated curvature serving as a judgment index. Experimental results showed that the curvature exhibited a linear correlation with the concentration of antigen FVIII, and a linear inverse correlation with the concentration of antigen cTnI. Given that FVIII and cTnI coexist in humans, the upper and lower limits were determined from the curvatures of a set of normal concentrations of FVIII and cTnI. An analyte with a curvature exceeding the upper limit can be identified as pulmonary embolism, while a curvature falling below the lower limit indicates myocardial infarction. Additionally, the further the curvature deviates from the upper or lower limits, the more severe the condition. The PEMI biosensor can serve as an effective detection platform for physicians.

High factor VIII concentrations interfere with glycoprotein VI-mediated platelet activation in vitro

BackgroundThe recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound αIIbβ3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. ObjectivesTo characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function. MethodsFVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay. ResultsFVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. ConclusionWe showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined.

Increasing the time-to-freezing for clinical apheresis plasma meets quality specifications.

In Australia, the vast distances between blood collection centres and processing facilities make it challenging to align supply with demand. Increasing the time to freezing for clinical plasma beyond 6 h would alleviate supply issues. This study aimed to determine the quality of clinical apheresis plasma frozen within 12 h of collection. Apheresis plasma (n = 20) collected at donor centres was immediately transported to a blood processing facility, stored at 26°C and sampled aseptically at 6, 8 and 12 h post collection. Frozen samples were thawed, and coagulation factors (F) II, V, VII, VIII and XIII, von Willebrand factor (vWF) and fibrinogen were measured using a coagulation analyser. FVIII concentrations declined in plasma frozen at 6, 8 and 12 h post collection (1.22 ± 0.27, 1.21 ± 0.25 and 1.16 ± 0.24 IU/mL, respectively) but not significantly (p = 0.3338). Importantly, all components met the FVIII specification (>0.7 IU/mL) for clinical plasma. Fibrinogen concentrations were stable from 6 to 12 h (p = 0.3100), as were vWF concentrations (p = 0.1281). Coagulation factors II, V, VII and XIII were not significantly different (p > 0.05 for all factors). Clinical apheresis plasma can be frozen within 12 h of collection, allowing collections from donor centres further from processing centres and increasing supply.

Optimization of Thrombin Generation in Hemophilia a

Background: Hemophilia A (HA) is a bleeding disorder characterized by decreased or absent FVIII. Clinical analysis of coagulation potential in this patient population is classically based on APTT based FVIII assays. Although both the one-stage FVIII assay and the chromogenic FVIII assay can measure FVIII concentrations reliably these types of assays only give insight on the initiation of coagulation. Global coagulation assays, like thrombin generation (TG), can be used to measure the full coagulation spectrum of initiation, amplification and propagation. However the frequently used commercially available TG kits lack sensitivity for measurements of hemophilia plasma within the lower FVIII ranges which are essential in explaining differences in bleeding phenotype.Aim: We aim to optimize the sensitivity of the TG-assay for measurements in hemophilia A patients, especially in the lower FVIII ranges.Methods: In order to minimize patient specific sensitivity a hemophilia A pool plasma (HAPP) was created. Analysis of the influence of pre-analytical variables, like contact activation inhibitors, on the assay was performed. Initiation of coagulation by different reagents was compared for sensitivity towards factor FVIII titrations in patient plasma. Other assay variables like phospholipids and temperature were adjusted to increase sensitivity even further.Results: Commonly used tissue factor (TF) initiated TG at varying concentrations was unable to significantly differentiate in FVIII levels below 20%. In contrast, TG activation with low concentrations of TF in presence of FXIa appeared to be highly sensitive for FVIII changes both in high and low ranges. Additionally, a representative baseline TG-curve in severe HA plasma could only be produced using this dual TF/FXIa-activation. There was a value in the addition of contact activation inhibitors in the assay. Higher phospholipid concentrations seem to benefit this assay setup compared to a TF only setup.Conclusion: TF/FXIa dual activation thrombin generation increased assay sensitivity in severe hemophilia plasma, allows for dose-dependent measurements in low FVIII ranges and provides a solid baseline curve that can be used for further clinical evaluation of coagulation potential and possibly therapeutic monitoring in hemophilia A. [Display omitted] DisclosuresHackeng: ACS Biomarker BV: Current Employment, Current equity holder in publicly-traded company; Coagulation Profile BV: Current Employment, Current equity holder in publicly-traded company.

The Impact of Specific Factor VIII Domains on Uptake By Dendritic Cells

Introduction: The role of dendritic cells in the presentation of foreign antigens to induce an adaptive immune response is critical for protection from potentially harmful pathogens. However in the treatment of hemophilia A, the recognition, processing, and presentation of factor VIII (fVIII) by dendritic cells that results in anti-fVIII inhibitors is a devastating complication of modern hemophilia A care. FVIII structure consists of A1, A2, B, A3, C1, and C2 domains. While prior studies have described a role for the C1 and C2 domains in mediating fVIII uptake by dendritic cells, the contribution of the remaining fVIII domains has not been fully explored. In this study we analyzed the full spectrum of fVIII domains on uptake by dendritic cells as a means to better understand the pathogenic innate immune response to fVIII. Methods: Bone marrow cells were harvested from the femurs and tibias of 8-10 week old E16 hemophilia A knockout mice, stimulated with GM-CSF, and cultured at 37°C for 6-7 days to induce bone marrow derived dendritic cells (BMDCs). BMDCs were incubated with dylight650-conjugated recombinant full length fVIII (DyL650-rfVIII) in combination with a panel of 17 previously characterized anti-fVIII monoclonal antibodies (MAbs) from our repertoire of anti-human murine derived MAbs representing B cell epitopes from all fVIII domains. Internalization of 10 nM fVIII by CD11c+ BMDCs in the presence of molar excess of individual anti-fVIII MAbs (80 nM) following a 30 minute incubation at 37°C was compared to uptake of fVIII alone. FVIII uptake by BMDCs at 4°C was used as a negative control. FVIII internalization was analyzed by flow cytometry and imaged using an Imagestream X Mark II flow cytometer. A supraphysiologic dose of fVIII was utilized in these experiments for optimal visualization and measurement of fVIII internalization by flow cytometry, though similar trends in uptake pattern at more physiologic concentrations of fVIII have also been observed in previous studies performed. Results: The panel of 17 anti-fVIII MAbs tested included 1 anti-A1, 2 anti-A2, 2 anti-B, 2 anti-A3, 3 anti-C1, and 7 anti-C2 domain MAbs. In this model, the presence of anti-C1 domain and anti-C2 domain MAbs reduced fVIII uptake by BMDCs as shown in prior studies. Anti-C1 domain MAb B136, a group B MAb that binds a critical structural epitope within the 2077-2084 region of the C1 domain, reduced fVIII uptake by 56%. Additionally classical group A anti-C2 domain MAb 3E6 reduced fVIII uptake by 35%. Both MAbs B136 and 3E6 bind to epitopes that are important for fVIII binding to von Willebrand factor and phospholipid surfaces suggesting that these epitopes may play a role in fVIII recognition by dendritic cells, particularly in instances when fVIII is unbound by either ligand in plasma. Interestingly anti-A1 domain MAb 2-116 and anti-A3 domain MAb I92 that binds residues 1818-1916 located at the C terminal end of the A3 domain significantly increased fVIII uptake by BMDCs two-fold (113% and 95% respectively) compared to fVIII alone. These findings indicate that epitopes within the A1 and A3 domains may play a role in limiting fVIII recognition by dendritic cells. All of the anti-A2 and anti-B domain MAbs, 2 of the 3 anti-C1 MAbs, and 5 of the 7 anti-C2 MAbs tested did not alter fVIII uptake. The figure below depicts a bar graph summarizing fVIII internalization by BMDCs with or without the anti-fVIII MAbs tested (figure 1A) and a representative Imagestream image of a dendritic cell (Ch01), CD11c+ surface staining of the dendritic cell (Ch07), DyL650-rfVIII alone without an anti-fVIII MAb (Ch11), and a merged image of CD11c+ dendritic cell and DyL650-rfVIII (Ch07/Ch11) (figure 1B). Conclusions: The C1 and C2 domains of fVIII primarily mediate fVIII uptake by dendritic cells. However the A1 and A3 domains may play a protective role in antigen uptake and ultimately antigen presentation by dendritic cells. Further investigation into the innate immune response to fVIII in patients with hemophilia A and differences between patients who do and do not develop inhibitors would provide insight into key aspects of the immune response that regulates fVIII tolerance. Download : Download high-res image (136KB) Download : Download full-size image Figure 1 . Disclosures Batsuli: Genentech, Octapharma: Membership on an entity's Board of Directors or advisory committees.

The Evaluation of APTT Reagents in Reference Plasma, Recombinant FVIII Products; Kovaltry® and Jivi® Using CWA, Including sTF/7FIX Assay.

The FVIII activity in patients treated with several extended half-life FVIII (EHL-FVIII) agents different when various activated partial thromboplastin time (APTT) reagents were used. The present study examined the difference in clot waveform analysis (CWA) findings and FVIII activity when various APTT reagents and CWA were used. The CWA including FVIII activity was measured using 12 APTT reagents, and the FIX activation based on a small amount of tissue factor assay (sTF/FIX) were examined in reference plasma (RP), EHL-FVIII (Jivi®) and Kovaltry®. The 3 APTT reagents were associated with high variation in the peak time and height in the CWA when analyzing low concentrations of FVIII. The peak time and height could not be measured with one APTT reagent, and there were marked differences in the CWA findings between Jivi® and Kovaltry® among APTT reagents. Several APTT reagents showed a markedly lower FVIII activity with Jivi® than with Kovaltry®. In the FVIII assay, the peak time measured with sTF/FIX did not differ markedly between Jivi® and Kovaltry®; however, the FVIII activity in Jivi® (as measured by the peak height) tended to be higher than in Kovaltry®. The CWA findings for monitoring Jivi® varied for monitoring Jivi® depending on the APTT reagents used, and sTF/FIX assay may be able to measure the EHL-FVIII.

Emicizumab improves the stability and structure of fibrin clot derived from factor VIII-deficient plasma, similar to the addition of factor VIII.

Emicizumab is an antifactor (F)IXa/FX bispecific antibody, mimicking FVIIIa cofactor function. Emi prophylaxis effectively reduces bleeding events in patients with haemophilia A. The physical properties of emicizumab-induced fibrin clots remain to be investigated, however. We have investigated the stability and structure of emicizumab-induced fibrin clots. Coagulation was initiated by activated partial thromboplastin time (aPTT) trigger and prothrombin time (PT)/aPTT-mixed trigger in FVIII-deficient plasma with various concentrations of emicizumab or recombinant FVIII. The turbidity and stability of fibrin clots were assessed by clot waveform and clot-fibrinolysis waveform analyses, respectively. The resulting fibrin was analysed by scanning electron microscopy (SEM). Using an aPTT trigger, the turbidity was decreased and the fibrinolysis times were prolonged in the presence of emicizumab dose-dependently. Scanning electron microscopy imaging demonstrated that emicizumab improved the structure of fibrin network with thinner fibres than in its absence. Although emicizumab shortened the aPTT dramatically, the nature of emicizumab-induced fibrin clots did not reflect the hypercoagulable state. Similarly, using a PT/aPTT-mixed trigger that could evaluate potential emicizumab activity, emicizumab improved the stability and structure of fibrin clot in a series of experiments. In this circumstance, fibrin clot properties with emicizumab at 50 and 100µg/mL appeared to be comparable to those with FVIII at ~12 and ~24-32IU/dL, respectively. Emicizumab effectively improved fibrin clot stability and structure in FVIII-deficient plasma, and the physical properties of emicizumab-induced fibrin clots were similar to those with FVIII.

The association between leptin concentration and blood coagulation: Results from the NEO study

BackgroundThe adipocyte-derived hormone leptin has been associated with altered blood coagulation in in vitro studies. However, it is unclear whether this association is relevant in vivo and to what extent this association is influenced by total body fat. Therefore, we aimed to examine the association between serum leptin and blood coagulation while taking total body fat into account in a population-based cohort study. MethodsWe performed a cross-sectional analysis with baseline measurements of 5797 participants of the Netherlands Epidemiology of Obesity (NEO) study, a population-based cohort of middle-aged men and women. We examined associations between serum leptin concentration and coagulation factor concentrations and parameters of platelet activation in linear regression analyses. All analyses were adjusted for multiple covariates, including total body fat. ResultsIn multivariable adjusted analyses a 1 μg/L higher serum leptin concentration was associated with a 0.22 IU/dL (95% CI: 0.11, 0.32) higher FVIII concentration and a 0.20 IU/dL (95% CI: 0.14, 0.27) higher FIX concentration (3.5 IU/dL FVIII and 3.2 IU/dL FIX per SD leptin). Serum leptin concentration was not associated with FXI, fibrinogen, platelet count, mean platelet volume and platelet distribution width in multivariable adjusted analyses. DiscussionThis study showed that serum leptin concentration was associated with higher concentrations of FVIII and FIX in an observational study, which could be clinically relevant.

Measurement of plasma and platelet tissue factor pathway inhibitor, factor V and Protein S in people with haemophilia.

Tissue factor pathway inhibitor (TFPI) is a naturally occurring anticoagulant found in plasma, where it circulates bound to lipoproteins, factor V (FV) or Protein S (PS), and in platelets. Therapeutic agents targeting TFPI are under development for the treatment of haemophilia A and haemophilia B. To begin to understand how TFPI, FV and PS interact to modulate haemophilia bleeding. Plasma and platelet antigen concentrations of these factors were determined in 73 people with haemophilia A and 18 with haemophilia B. Using multiple regression models, these were compared to the same analytes measured in 224 male blood donors. There were no differences in plasma or platelet TFPI, FV or PS concentrations between haemophilia types or severities. However, compared to blood donors, people with haemophilia had approximately one-third lower plasma PS, 9% lower plasma TFPIα, 50% higher platelet FV and 26% lower platelet Protein S. Together, the presented data suggest that individuals with haemophilia may have a compensatory procoagulant response of both plasma and platelet proteins to the decreased concentrations of FVIII or FIX.

Elevated fibrinogen, von Willebrand factor, and Factor VIII confer resistance to dilutional coagulopathy and activated protein C in normal pregnant women

BackgroundGestational changes in coagulation factor concentrations include elevations in fibrinogen, Factor VIII, and von Willebrand factor (vWF). We hypothesised that blood samples from term pregnant (TP) subjects are less prone to coagulation disturbances from haemodilution compared with those from non-pregnant (NP) females. MethodsBlood samples were collected from 15 NP and 15 TP subjects. In vitro haemodilution with normal saline was assessed by modified Clauss fibrinogen assay, factor activity, flow-chamber assay, and thromboelastometry. The impact of human fibrinogen concentrate (hFC), cryoprecipitate, and vWF/Factor VIII (FVIII) concentrate replacement in diluted TP and NP blood was compared. Thrombin generation and activated protein C sensitivity were assessed. ResultsTP blood contained twice the concentrations of fibrinogen, FVIII, and vWF relative to NP blood (P<0.0001). Platelet thrombus formation (PTF) under flow was reduced by 99.2% and 69.2% in diluted NP and TP blood, respectively. Platelet thrombus formation was partially restored by adding vWF/FVIII, but not hFC or cryoprecipitate. Fibrin clot firmness approached the threshold of 10 mm in diluted NP blood, and clot firmness was effectively restored by hFC, but not by vWF/FVIII. In the presence of thrombomodulin, peak thrombin generation was decreased by 86.7% in NP plasma, but by 31.8% in TP plasma (P<0.0001 vs NP plasma), indicating reduced activated protein C sensitivity in TP plasma. Both elevated FVIII and haemodilution contributed to activated protein C insensitivity. ConclusionsOur in vitro model showed relative resistance of TP blood to dilutional coagulation changes with respect to platelet adhesion, fibrin polymerisation, and thrombin generation. Careful therapeutic monitoring for different pro-haemostatic agents in pregnant women is warranted.

Standardization and evaluation of the performance of the thrombin generation test under hypo- and hypercoagulability conditions

BackgroundIn order to standardize a thrombin generation() protocol, we analyzed the analytical variables and sensitivity of this test to hypo/hypercoagulability states. MethodsThe effect of the tissue factor concentration and the intra- and interassay precision were analyzed. To evaluate the hypercoagulability status, the plasma of women under an oral contraceptive was tested, while plasma from hemophilia A patients at 1, 3 and 7 days after recombinant FVIII infusion, and lyophilized plasma deficient in FVII or FVIII were used for the evaluation of hypocoagulability. ResultsThe intra-assay coefficient of variation was <10% with 1 and 5pM of low and high TF. The oral contraceptive users showed increased thrombin generation in comparison to non-users, which was more pronounced with low TF (endogenous thrombin potential ETP) p=0.0009; peak p=0.0009; lagtime p=0.0008). In relation to the FVIII-deficient plasma, a higher TG was observed as FVIII levels were increased and a better discrimination was obtained for different concentrations of FVIII with low TF (ETP p<0.0001; peak p<0.0001; lagtime p=0.0004). Using low TF, plasma from hemophilia A patients showed higher TG values after 1 day of recombinant FVIII infusion vs after 3 days (ETP p<0.0001; peak p<0.0001; lagtime p=0.0407), while the lowest values were observed after 7 days. With FVII-deficient plasma, thrombin generation was lower than normal plasma and a more pronounced difference was observed with high TF compared to low TF (ETP p<0.0001; peak p<0.0001; lagtime p<0.0001). ConclusionUnder our conditions the thrombin generation test seems to be sensitive to evaluation of hyper/hypocoagulability states. Standardization of the thrombin generation test may have an application in the evaluation of bleeding and thrombotic disorders.

Prevalence and Burden of Major Bleeding Events in Patients with Von Willebrand Disease Based on Claims Data from the USA

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder (clinically symptomatic prevalence rate ~1:10,000). Patients with VWD have impaired hemostasis due to a quantitative or qualitative deficit in von Willebrand Factor (VWF) that alters platelet adhesion and collagen binding and/or decreases FVIII concentrations. Most patients with VWD present with mild-to-moderate mucosal bleeding (epistaxis, menorrhagia), although life-threatening bleeding may also occur, especially in patients with VWD type 3 and some forms of VWD type 2. While bleeds associated with VWD have been well described in the literature, information on the burden associated with major bleeding events (MBE) is limited. Real-world data can be used to help understand the clinical and economic impact of these events.Aims: To estimate the prevalence, healthcare resource utilization (HCRU), and costs associated with MBE among patients with VWD.Methods: Patients with VWD with ≥1 year of continuous enrollment since the eligibility start date were identified from Truven databases (01/2008-12/2016). Patients with MBEs were identified using a medical claim associated with an ICD-9/10 CM diagnosis code for intracranial, GI, or eye bleed; or menorrhagia, epistaxis, and joint bleeds that required red blood cell transfusion in an inpatient (IP) setting or within 7 days of diagnosis in an outpatient (OP) setting. Prevalence was calculated as the proportion of eligible patients with ≥1 MBE during the observation period (from start to the end of continuous eligibility). To evaluate economic burden, patients with ≥1 MBE on or after the first diagnosis of VWD were compared with patients with no MBEs. HCRU and cost in the 12-month continuous enrollment period following the first MBE were compared with those from a similar 12-month period for patients without MBEs. Regression models were used, controlling for demographics, health plan, index year, Charlson Comorbidity Index (CCI), comorbidities, thrombotic events, and HCRU during the 12-month continuously enrolled baseline period. For patients with MBEs, the proportion of patients with comorbidities was compared between the 12-month baseline and study periods using McNemar test.Results: 19,785 VWD patients were identified (mean age 34 years, 75% female) During a median observation of 4 years, 15.1% of patients experienced ≥1 MBE (mean rate: 0.11 ± 0.64 MBE/year). GI bleeding was the most prevalent MBE, occurring in 13.4% of all patients. Although not common, the prevalence of intracranial bleeds (1.1%) was slightly higher in males than females (1.7% vs 0.9%). In the sample to evaluate economic burden, 773 patients with ≥1 MBE (age 44.5 ± 20.1 years) and 4285 patients without MBEs (age 34.2 ± 19.5 years) were selected. Patients with MBEs were significantly (p<0.01) more likely to have an IP admission (OR, 4.1; 95% CI, 3.4-5.0), ER visit (1.8; 1.5-2.1), or OP visit (4.9; 1.8-13.4); they also had significantly longer IP stays (IRR, 3.9; 95% CI, 3.1-4.9) and more frequent IP admissions (3.2; 2.8-3.8), ER visits (2.0; 1.8-2.3), and OP visits (1.3; 1.2-1.3), compared to those without MBEs. Patients with MBEs incurred significantly (p<0.01) higher total healthcare costs (adjusted mean difference, $20,890; 95% CI, $15,524-$29,254) than those without MBEs. Among the 773 patients with MBEs, approximately 1 in 4 patients had a MBE (26.8%) diagnosed in the IP setting. The overall annual mean (± SD) IP length of stay (LOS) was 7.4 ± 19.4 days, with intracranial bleeds associated with the longest mean IP LOS (14.3 ± 19.4 days). The readmission rate was 3.1% for any MBE, and 2.5% for the same type of MBE as the initial bleed. The proportions of patients with anemia (24.2% vs 15.9%; p<0.01) and anxiety (18.6% vs 14.2%; p<0.01) were significantly higher after the MBE than before.Conclusions: In this large retrospective analysis of data from a US commercial healthcare plan, ~15% of patients with VWD experienced MBEs, mostly GI bleeds. While our estimation of some MBEs may be conservative, this is the first study to use a large dataset with sound statistical methods to evaluate the burden associated with MBEs in this population. MBEs were associated with additional comorbidities and high HCRU and costs (driven by inpatient costs), so optimal therapy is essential to prevent MBEs in patients with VWD. DisclosuresLu:Shire: Employment, Equity Ownership. Oladapo:Shire: Employment, Equity Ownership. Wu:Shire: Employment. Farahbakhshian:Shire: Employment, Equity Ownership. Ewenstein:Shire: Employment, Equity Ownership.

Evaluating the thrombin generation profiles of four different rFVIII products in FVIII-deficient plasma using FIXa and FXIa activation.

The thrombin generation assay (TGA) can be used to monitor factor replacement therapy in patients with haemophilia. The TGA assay is typically performed using tissue factor as the reaction activator; however, activating with FIXa or FXIa can enhance assay sensitivity when FVIII<1%. To evaluate the sensitivity of the TGA when FIXa (5nmol/L) and FXIa (0.22nmol/L) are used to activate the assay in platelet-poor plasma and to compare these data to the one-stage and chromogenic assays. Plasma from 10 severe FVIII-deficient subjects was supplemented with FVIII (0%, 0.1%, 0.4%, 1.2%, 4%, 11% and 33%), using either Novo Eight® , Advate® , Eloctate® , turoctocog alfa pegol or a control standard. The one-stage and chromogenic assays quantified the FVIII levels. The TGA assay was activated using either FIXa or FXIa. Both FIXa- and FXIa-activated TGA were sensitive across FVIII concentrations, with intra-assay coefficient of variation (CV)<10%. The FXIa-activated assay had 25% CV at the lowest level of FVIII compared to 10% CV with FIXa activation. There were strong correlations between the FIXa- and FXIa-activated TGA tests (R2 =0.9912) and between the one-stage and chromogenic assays (R2 =0.9469). However, there were poor relationships between the TGA tests and one-stage and chromogenic assays. Both FIXa- and FXIa activation results in similar TGA profiles across a FVIII range of 0.1%-33%; however, FIXa activation was more robust at the lowest levels of FVIII compared with FXIa activation.

Modified clot waveform analysis to measure plasma coagulation potential in the presence of the anti‐factor IXa/factor X bispecific antibody emicizumab

Background Emicizumab is an anti-activated factorIX/FX bispecific antibody that mimics activated FVIII cofactor function. Emicizumab does not require activation by thrombin, and its effect on shortening the activated partial thromboplastin time (APTT) is much greater than that of FVIII. Therefore, the APTT has limited utility in hemophiliaA (HA) patients treated with emicizumab. Aim To evaluate the global coagulation potential of emicizumab. Methods Clot waveform analysis (CWA) with prothrombin time (PT)/APTT mixed reagents was used to define hemostatic monitoring protocols in HA patients. A modified parameter, adjusted-|min1| (Ad|min1|), was developed. Maximum and minimum percentage transmittance were defined as 100% and 0% in the precoagulation and postcoagulation phases, respectively. Ad|min1| was calculated as an index of the maximum velocity of the coagulation process. Results Ad|min1| obtained with mixed-trigger reagent (PT/APTT/buffer, 1:15:135) in the presence of emicizumab optimally corresponded to the conversion rate estimated in animals; 0.2-0.4IUdL-1 equivalent FVIII per 1μgmL-1 emicizumab). Exvivo addition of emicizumab to HA plasma with or without inhibitors resulted in concentration-dependent increases in Ad|min1|, with some individual variations. The addition of various concentrations of FVIII to HA plasma mixed with emicizumab resulted in dose-dependent increases in Ad|min1|. Similarly, mixtures of activated prothrombin complex concentrate and emicizumab added to HA plasma resulted in dose-dependent increases in Ad|min1|. In contrast, enhanced coagulation potential appeared to be better defined by the clot time than by Ad|min1| in experiments using recombinant activated FVII. Conclusion The PT/APTT reagent-triggered adjusted CWA could provide a useful means of assessing global coagulation potential in emicizumab-treated HA patients, with enhanced activity neither masking nor being masked by FVIII or bypassing agents.

In silico thrombin and factor Xa generation profiles in adult patients after Fontan operation.

: Single-ventricle defects are associated with increased risk of thromboembolic events. To analyze the prothrombotic potential in a long-term follow-up on Fontan patients via plasma contribution to thrombin and factor (F)Xa generation profiles. Thrombin and FXa generation was simulated from plasma concentrations of FII, FV, FVII, FVIII, FIX, FX, antithrombin and tissue factor (TF) pathway inhibitor from Fontan patients (n = 48) and healthy controls (n = 34). TF and thrombin-antithrombin complex (TAT) were measured by ELISA. Fontan patients had significantly reduced procoagulant protein concentrations and increased anticoagulant protein concentrations over controls, resulting in a lowered procoagulant potential. However, Fontan patients showed increased hemostatic activation as evidenced by increased TF and TAT. Modeling this increased TF showed a more prothrombotic profile. Observed changes in procoagulant and anticoagulant proteins may be a compensatory mechanism aimed at mitigating the underlying disease effects characterized by elevated TF and TAT.

A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.

Discrepancies have been previously reported for one-stage clotting and chromogenic assays for FVIII activity analysis. Inter-laboratory variations in instruments, method of clot detection, assay set-up, reference standard calibration, reagent source and reagent composition all contribute to assay variability. To characterise multilaboratory assay variability in measuring ADYNOVATE, OBIZUR and ADVATE FVIII activity in human plasma and survey multinational FVIII activity assay preferences. As samples from patients treated with either of the FVIII products are not available in the quantities required for a systematic collaborative study, haemophilia A plasma was spiked in vitro with either ADYNOVATE (PEGylated rFVIII), OBIZUR [Porcine Sequence Antihaemophilic Factor (Recombinant)] or ADVATE at high (0.80 IU or U mL-1 ), medium (0.20 IU or U mL-1 ) and low (0.05 IU or U mL-1 ) FVIII concentrations, based on labelled potencies. Clinical laboratories used their routine FVIII activity assay to determine FVIII activity of each product. Thirty-five data sets using one-stage clotting assay and 11 sets using chromogenic assay were obtained. A vast majority of laboratories (98%) prefer and rely on the one-stage clotting assay. Mean recoveries across all concentrations were 113%, 120% and 127% for ADYNOVATE, OBIZUR and ADVATE respectively. Assay variation was comparable between ADVATE, ADYNOVATE and OBIZUR with inter-laboratory percent coefficients of variation (%CV) ranging from 11 to 22%. Mean chromogenic assay results were 116%, 51% and 113% for ADYNOVATE, OBIZUR and ADVATE respectively. Inter-laboratory CV's were similar for ADYNOVATE, OBIZUR and ADVATE. One-stage clotting assays can and will be used with sufficient accuracy and precision for the measurement of ADYNOVATE, OBIZUR and ADVATE in plasma samples from subjects with haemophilia A. Chromogenic assay underestimates OBIZUR potency, particularly at lower concentrations.

Recombinant human prothrombin (MEDI8111) prevents bleeding in haemophilia A and B mice.

Haemophilia A and B are treated with FVIII and FIX replacement therapy. Treatment may be complicated by inhibitory antibodies that require bypass therapy such as FEIBA(®) in which prothrombin (FII) is suggested to be the main active component. To evaluate the effect of FII on haemophilia recombinant human (rh) FII (MEDI8111) or plasma-derived human FII (pdhFII) was given as single doses to anaesthetized haemophilia A and B mice 3 min before tail transection and rhFVIII or rhFIX was used for comparison. After tail transection, automatic bleeding registration was used to continuously measure blood loss (BL) and bleeding time (BT). Thrombin generation and plasma concentrations of human FVIII, FIX, FII and thrombin-antithrombin complex (TAT) were measured. Blood loss and BT were dose-dependently decreased by rhFVIII or rhFIX. The concentrations that decreased BL and BT for rhFVIII by 50% (EC50) were 0.06 and 0.01 IU mL(-1) and for rhFIX 0.07 and 0.07 IU mL(-1) , respectively. Administration of rhFVIII and rhFIX dose-dependently increased thrombin generation potential but did not affect TAT. MEDI8111 and pdhFII dose-dependently decreased BL and BT in haemophilia A mice, EC50 37 and 87 and 100 and 155 mg L(-1) respectively. In haemophilia B mice given MEDI8111 EC50 was for BL 56 mg L(-1) and for BT 67 mg L(-1) . TAT and thrombin generation increased dose-dependently for MEDI8111 and pdhFII. MEDI8111 dose-dependently decreased bleeding and increased procoagulant activity in haemophilia A and B mice and suggest that MEDI8111 may be useful for preventing bleeding in patients with haemophilia A and B.

Influencing Factors Control of Cryoprecipitate Preparation by Centrifugation

Objective To reduce the loss of coagulation factors by influencing factors control of cryoprecipitate preparation by centrifugation.Methods From July 2013 to June 2014,a total of 60 samples of cryoprecipitate prepared by centrifugation with strict control of influencing factors at Jingmen Red Cross Blood Center were selected into this study via simple random sampling method,as study group (n =60).From July 2012 to June 2013,a total of 54 samples of cryoprecipitate prepared by general centrifugation approach were selected into control group via simple random sampling method (n=54).The concentrations of factor Ⅷ (F Ⅷ) and fibronectin (Fib) in cryoprecipitate were analyzed.Comparison of average concentrations of FⅧ and Fib in cryoprecipitate between study group and control group were retrospectively analyzed.Results Average concentration of FⅧ and Fib in cryoprecipitate of study group and control group were both qualified.Average concentration of FⅧ in study group was (102.8±12.3) IU/bag,and average concentration of FⅧ in control group was (90.4 ± 4.2) IU/bag.Average concentration of F Ⅷ in study group was significantly higher than that in control group (t =8.04,P＜0.001).Average concentration of Fib in study group was (179.9 ± 6.7) mg/bag,and average concentration of Fib in control group was (170.2± 8.3) mg/bag.Average concentration of Fib in study group was also significantly higher than that in control group (t =7.72,P ＜0.01).Conclusion Strict control of influencing factors during cryoprecipitate preparation by centrifugation can reduce the loss of concentrations of FⅧ and Fib. Key words: Centrifugation; Cryoprecipitate coagulum; Factor Ⅷ; Fibronectin; Quality control

Changes in haemostatic parameters during the menstrual cycle and subsequent use of drospirenone-containing oral contraceptives

IntroductionOral contraceptives (OC) increase the risk of venous thromboembolism that depends on the OC formulation and could at least partially be explained by impaired function of the protein C-system (APC resistance) and the tissue factor pathway inhibitor (TFPI)-system. There is limited information available on the effects of OC, containing a newer progestogen- drospirenone (DRSP-OC) on these two major anticoagulant pathways, thrombin generation, reflecting the overall state of coagulation, and other coagulation parameters. MethodsIn a study population consisting of 14 healthy women (age 21–33years) we investigated the effect of the menstrual cycle and subsequent use of DRSP-OC on APC resistance, the function of the TFPI-system, thrombin generation and on their major determinants, i.e. prothrombin, antithrombin, FV, FX, FVIII, protein C, protein S(total and free) and TFPI(full-length and free). ResultsAll studied parameters remained unchanged during the menstrual cycle. During DRSP-OC use we observed a significant increase in APC resistance (~2.4-fold), thrombin generation measured at low (~2.2-fold) and high tissue factor concentrations (~1.4-fold), plasma concentrations of prothrombin (19%), FX (31%), FVIII (17%) and protein C (43%). DRSP-OC use impaired the function of the TFPI-system and decreased plasma levels of antithrombin (−6%), FV (−22%), protein Stotal (−21%), protein Sfree (−20%), TFPIfull-length (−36%) and TFPIfree (−46%). ConclusionsDRSP-OC caused procoagulant changes in all studied haemostatic parameters. The impairment of the protein C- and TFPI-systems was more pronounced than the impairment of the coagulation pathways and can at least partially account for the increased risk of venous thromboembolism in users of DRSP-OC.

High levels of procoagulant factors mediate the association between free thyroxine and the risk of venous thrombosis: the MEGA study

Thyroid hormone affects the coagulation system, but its effect on clinical disease is not clear. We determined the associations of levels of free thyroxine (FT4), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase antibodies (antiTPO) with levels of coagulation factors and the risk of venous thrombosis. In a large population based case-control study (Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis study) on the etiology of venous thrombosis, we determined the levels of FT4, TSH, antiTPO, factor FII, FVII, FVIII, FIX, FX, von Willebrand factor (VWF), antithrombin, protein C, protein S and fibrinogen in 2177 cases and 2826 controls. High levels of FT4 were associated with increased concentrations of procoagulant factors, and not with levels of anticoagulant factors. High levels of FT4 were also associated with the risk of venous thrombosis, up to an odds ratio (OR) of 2.2 (95% confidence interval [CI] 1.0-4.6) for levels above 24.4 pm relative to FT4 levels between 15.5 and 18.9 pm. In 11 cases and one control, clinical hyperthyroidism had been diagnosed within a year of the thrombotic event, leading to an OR of 17.0 (95% CI 2.2-133.0) for thrombosis. The ORs approached unity after adjustment for FVIII and VWF, which suggests that the effect was mediated by these factors. Low TSH levels were also, but less evidently, associated with thrombosis, whereas there was no association between antiTPO and venous thrombosis risk. High levels of FT4 increase the concentrations of the procoagulant proteins FVIII, FIX, fibrinogen, and VWF, and by this mechanism increase the risk of venous thrombosis.