1-stage Assay Research Articles

Rapid diagnosis of coagulopathies from vitamin K deficiency in a consecutive case cohort evaluated by comparative assessment of factor II by 1-stage assays with prothrombin time vs Ecarin reagents

BackgroundVitamin K (VK) deficiency (VKD) impairs γ-carboxylation of VK-dependent factors (VKDFs), resulting in higher factor (F)II levels measured by Ecarin (FIIE) reagents (that convert des-γ-carboxylated FII to meizothrombin) than by prothrombin time (FII) reagents. ObjectivesTo evaluate FII/FIIE abnormalities among patients assessed for coagulopathies and identify findings predictive of coagulopathy improvement after VK. MethodsWe retrospectively assessed consecutive cases from 2002 to 2021 with FII/FIIE tests and the sensitivity and specificity of FII/FIIE ratios and FIIE-FII differences for VKD defined as international normalized ratio correction/improvement of ≥0.5 after VK. ResultsTwo hundred ninety-two patients (males, 58.2%; adults, 85.6%; median age, 73 years) were evaluated (84.2% hospitalized, 48.3% in intensive care, 71.6% with active liver disease, and 28% deceased at discharge) and 25% to 38% had FII/FIIE findings suggestive of VKD. Among 170 patients assessed for response to VK, FII/FIIE ratios of ≤0.84 to 0.91 and FIIE-FII differences of >0.04 U/mL had similar modest sensitivity (47.7%-69.3%) and modest to good specificity (67.1%-91.5%) for VKD. FII/FIIE ratios of <0.86, suggestive of VKD (sensitivity, 47.7%; specificity, 90.2%), were more common in patients deficient in only VKDF (P = .0001), but were detected in 16% with non-VKDF deficiencies. Low FIIE was commonly associated with active liver disease (P = .0002). Patients with and without probable VKD (based on FII/FIIE ratios of <0.86) had similar mortality, bleeding, and rates of prothrombin complex concentrate and red cell transfusions (P ≥ .78), but fewer with probable VKD received plasma and fibrinogen replacement (P ≤ .024). ConclusionFII/FIIE comparison aids the diagnosis of VKD and predicts clinical responses to VK treatment among patients with coagulopathies.

Cell Salvage Using the Autotransfusion Device CATSmart®: A Randomized Controlled Bicentric Trial Evaluating the Quality of Two New Flex Wash Programs

Background: The use of cell salvage and autologous blood transfusion is an important and widespread method of blood conservation during surgeries with expected high blood loss. The continuous autotransfusion device CATSmart^® (Fresenius Kabi, Germany) contains two new washing programs on the device called Flex wash 3 and Flex wash 5. To the best of our knowledge, there are no published clinical data regarding the performance of the two new washing programs. Methods: In total, 69 patients undergoing cardiac or orthopedic surgery were included in this randomized, controlled, bicentric trial to validate the red cell separation process and washout quality of Flex wash 3 compared to Flex wash 5. After washing, the primary quality target was to determine hematocrit value, recovery rate, albumin, and total protein elimination rate in the packed red cells (PRCs). The secondary objective was to assess the elimination of heparin by measuring the factor anti-Xa activity by a 1- and 2-stage assay in PRC after washing. Results: In the whole cohort of patients, hematocrit was 16.00% [9.15%; 21.30%] (median [Q1; Q3]) in the wound blood and 69.90% [51.10%; 80.90%] in the PRC resulting in a recovery rate of 63.92% [47.06%; 88.13%]. The albumin elimination rate was 98.77% [97.94%; 99.27%], and the total protein elimination rate was 98.85% [97.76%; 99.42%]. The heparin elimination rate was 99.95% [99.90%; 99.97%] in the 1-stage assay and 99.70% [99.41%; 99.87%] in the 2-stage assay. There was no difference between Flex wash 3 and Flex wash 5 washing procedure regarding the recovery rate 63.75% [46.64%; 78.65%] versus 67.89% [47.20%; 92.69%] (p = 0.85), albumin elimination rate 98.74% [97.67%; 99.27%] versus 98.78% [98.10%; 99.28%] (p = 0.97), protein elimination rate 98.79% [97.94%; 99.47%] versus 98.92% [97.58%; 99.42%] (p = 0.88), and anti-Xa elimination rate in the 1-stage assay 99.94% [99.79%; 99.97%] versus 99.95% [99.92%; 99.97%] (p = 0.24) and in 2-stage assay 99.66% [99.20%; 99.86%] versus 99.77% [99.47%; 99.90%] (p = 0.23). Conclusions: The two new washing procedures, Flex wash 3 and Flex wash 5, enable sufficient and comparable red cell separation and washout quality of albumin, total protein, as well as heparin.

Four-Year Follow-up of the Alta Study, a Phase 1/2 Study of Giroctocogene Fitelparvovec (PF-07055480/SB-525) Gene Therapy in Adults with Severe Hemophilia Α

Introduction: Hemophilia A is an X-linked ( F8 gene) recessive disorder of hemostasis that results in insufficient factor VIII (FVIII) activity. Adeno-associated virus (AAV)-based gene therapy enables delivery of a modified F8 cDNA, allowing synthesis of functional endogenous FVIII, which prevents bleeding events. We present updated results with nearly 4 years of follow-up on an ongoing gene therapy study in participants with severe hemophilia A (FVIII activity &amp;lt;1%). Methods:The phase 1/2 Alta study (NCT03061201) is a dose-ranging study of giroctocogene fitelparvovec (PF-07055480, previously called SB-525), a recombinant AAV serotype 6 vector encoding a modified B-domain-deleted F8 coding sequence. Four ascending doses of giroctocogene fitelparvovec (9e11, 2e12, 1e13, and 3e13 vg/kg) were infused into adults aged ≥18 years with severe hemophilia A across 4 cohorts (n=2 each). The high-dose (3e13 vg/kg) cohort was expanded to 5 participants. Key endpoints included safety, circulating FVIII activity, use of FVIII replacement therapy, and frequency of bleeding events. Results: Eleven male participants were enrolled in the study (mean [SD] age, 30.3 [7.8] years; White, 81.8%). As of the cutoff date (May 19, 2023), participants had been followed for 153 to 290 weeks. Two participants left the study after Week 156. Of the remaining, 1 participant had not yet completed 4 years (208 weeks). The most common treatment-related adverse events (AEs) reported in the high-dose cohort (n=5) were elevated liver enzymes and infusion-related reactions: increased alanine aminotransferase (ALT; n=3 [60.0%]), increased aspartate aminotransferase (AST; n=2 [40.0%]), pyrexia (n=3 [60.0%]), and tachycardia (n=2 [40.0%]). Treatment-related serious AEs were reported in 1 participant in the high-dose cohort who experienced hypotension and fever, with onset ≈6 h after infusion; the events fully resolved with treatment. AEs (all causality) of ALT increases requiring ≥7 days of corticosteroids were observed in 4 of 5 participants in the high-dose cohort. ALT elevations were managed with tapering courses of corticosteroids (median duration: 56 days; range: 7-135 days), with maintenance of efficacious levels of FVIII activity. Participants in the high-dose cohort have not required steroids since Week 65, have had ALT values in the normal range (follow-up: 156-208 weeks) and normal findings via liver MRI (follow-up: 104-208 weeks). No participant developed a confirmed inhibitor to FVIII. No thrombotic events or liver masses have been detected. Of the 5 participants in the high-dose cohort, 2 had data available through Week 208 and FVIII activity was maintained in the mild to normal range ( Table), consistent with Week 156 results. Of those without Week 208 data, 2 had data through Week 182. One participant maintained FVIII activity in the mild range (14.1% and 24.1% of normal, measured with a chromogenic and 1-stage assay, respectively); the other had FVIII activity of 3.1% and 7.2%. The remaining participant left the study after Week 156, with FVIII activity maintained in the mild range (11.8% and 22.9%). In the high-dose cohort, the mean annualized total bleeding rate [(number of all bleeding episodes starting 3 weeks after study drug infusion) / (observation period in years)] was 0 for the first year post infusion and 1.2 (SD 2.58) throughout the total duration of follow-up. In this cohort, the participant with the lowest FVIII activity level experienced a total of 22 bleeds, with 21 necessitating treatment (8 traumatic; 7 spontaneous; 6 unknown). The other 4 participants had no or very minimal bleeds, including 1 who experienced a bleed in a target joint. No participants in the high-dose cohort have resumed prophylaxis. Conclusion:A single infusion of giroctocogene fitelparvovec gene therapy in participants with severe hemophilia A remains generally well tolerated over a period of nearly 4 years post infusion, with associated increases in FVIII levels in the moderate to normal range, without sustained AEs and with no AEs associated with increased liver function tests since Week 59. The ongoing phase 3 study (NCT04370054) in a larger cohort will provide more long-term data on the safety and durability of giroctocogene fitelparvovec in participants with moderately severe to severe hemophilia A.

Effect of emicizumab-neutralizing antibodies on activated partial thromboplastin time–based clotting time test results in patients treated with emicizumab

BackgroundEmicizumab is a bispecific humanized monoclonal antibody that shortens the activated partial thromboplastin time (aPTT), making aPTT-based tests unreliable. ObjectivesTo evaluate the efficacy of a mixture of 2 anti-idiotype monoclonal antibodies (anti-emi) in neutralizing emicizumab in samples from persons with hemophilia A treated with emicizumab. MethodsFifty samples from persons with hemophilia A treated with emicizumab were analyzed for aPTT and factor VIII procoagulant activity; FVIII inhibitor titer was measured using Nijmegen-Bethesda assay in 50 plasma samples of additional patients (positive for FVIII inhibitor) treated with emicizumab. FVIII procoagulant activity and inhibitor titer were measured using 1-stage (Actin FS, Siemens) and chromogenic assays with bovine regents (Factor VIII Chromogenic Assay, Siemens). Emicizumab concentration was measured by modified a 1-stage assay calibrated with a drug-specific calibrator (r2 Diagnostics Inc). All the tests were performed on Sysmex CS-2400 (Sysmex) before and after the addition of anti-emi (Chugai Pharmaceutical). ResultsEmicizumab concentrations measured after neutralization were <1.6 μg/mL in all samples. FVIII levels were >480 IU/dL with an aPTT of <30.8 seconds in all samples before neutralization and were <1 IU/dL with an aPTT of >70 seconds after adding anti-emi. FVIII inhibitor resulted in a false negative result in 44 of 50 samples measured with the 1-stage assay before neutralization. A good correlation (r = 0.98) was found between inhibitor titer measured using the chromogenic (insensitive to emicizumab) and 1-stage assays after neutralization. ConclusionThe anti-emi antibodies were shown to completely neutralize emicizumab, making samples pretreated with anti-emi analyzable with the 1-stage assay.

Updated Results of the Alta Study, a Phase 1/2 Study of Giroctocogene Fitelparvovec (PF-07055480/SB-525) Gene Therapy in Adults with Severe Hemophilia Α

Introduction: Hemophilia A, a sex-linked (F8 gene) disorder of hemostasis, results in insufficient factor VIII (FVIII) activity. Adeno-associated virus (AAV)-mediated gene transfer enables the delivery of a modified functional F8 coding sequence to hepatocytes. This subsequently synthesizes FVIII at levels preventing unprovoked bleeding events in the absence of exogenous FVIII. We present updated results with nearly 3-year follow-up on all participants from Alta, an ongoing gene therapy study in patients with severe hemophilia A (FVIII activity <1%). Methods: The phase 1/2 Alta study (NCT03061201) is a dose-ranging study of giroctocogene fitelparvovec (PF-07055480, previously called SB-525), which is a recombinant AAV serotype 6 vector encoding a modified B-domain-deleted F8 coding sequence. Giroctocogene fitelparvovec was infused into adults aged ≥18 years with severe hemophilia A in 4 cohorts (n=2 each) across 4 ascending doses: 9e11, 2e12, 1e13, and 3e13 vg/kg. The 3e13-vg/kg dose cohort was expanded with 3 additional participants. Key endpoints included safety, circulating FVIII activity, use of FVIII replacement therapy, and frequency of bleeding events. We present data with nearly 3 years of follow-up (data cutoff date: May 20, 2022). Results: Eleven male patients participated in the study (mean [SD] age, 30.3 [7.8] years; White, 81.8%). As of the cutoff date, participants had been followed for 147 to 247 weeks and 2 had not completed 3 years (156 weeks) . The most common treatment-related adverse events (AEs) reported in the highest dose cohort (3e13-vg/kg) included elevated liver enzymes and infusion-related reactions (n/N [%]): increased alanine aminotransferase (ALT; 3/5 [60.0%]), increased aspartate aminotransferase (AST; 2/5 [40.0%]), pyrexia (3/5 [60.0%]), and tachycardia (2/5 [40.0%]). Treatment-related serious AEs were reported in 1 participant (in the 3e13-vg/kg cohort) who experienced hypotension and fever with onset ≈6 h after giroctocogene fitelparvovec infusion; the events fully resolved with treatment and did not delay post-infusion discharge the next day. AEs (all causality) of ALT increases requiring ≥7 days of corticosteroids were observed in 4 of the 5 participants in the 3e13-vg/kg cohort: elevations in ALT were managed with a tapering course of corticosteroids (median duration: 56 days; range: 7-135 days), with maintenance of efficacious levels of FVIII activity, as evidenced by a lack of bleeding events around the time of corticosteroid treatment and minimal bleeding events afterwards. No participant in the 3e13-vg/kg cohort developed a confirmed inhibitor to FVIII, and there have been no thrombotic events or hepatoma detected. The three of the 5 participants in the 3e13-vg/kg cohort with available data through Week 156 had mean FVIII activity maintained in the mild to normal range (Table). Of the data available for the remaining 2 participants, 1 maintained FVIII activity levels in the mild range through Week 130 and 1 participant had FVIII activity levels below lower level of quantification (<3%) as measured with a chromogenic assay and 5.4% measured with a 1-stage assay at Week 130. In this cohort, the mean annualized total bleeding rate [(number of all bleeding episodes starting 3 weeks after study drug infusion) / (observation period in years)] was 0 for the first year postinfusion and 0.9 throughout the total duration of follow-up. In the 3e13-vg/kg cohort, 2 participants experienced an overall total of 12 bleeding events necessitating treatment with exogenous FVIII (participant 1 (n=1 event): circumstances of bleed unknown, event occurred in a target joint; participant 2 (n=11): 6 traumatic; 4 spontaneous; 1 unknown, no events occurred in a target joint). No participants in the 3e13-vg/kg cohort have resumed prophylaxis. Conclusions: A single infusion of giroctocogene fitelparvovec gene therapy in participants with severe hemophilia A remains generally well tolerated over a period of nearly 3 years postinfusion, with associated increases in FVIII levels in the moderate to normal range, without sustained AEs and with minimal overall bleeding in the highest-dose cohort (3e13 vg/kg). The ongoing phase 3 study (NCT04370054) in a larger cohort will provide more long-term data to further characterize the safety and durability of giroctocogene fitelparvovec in patients with moderately severe to severe hemophilia A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dosing for Personalized Prophylaxis in Hemophilia A Highly Varies on the Underlying Population Pharmacokinetic Models.

Model-informed personalized prophylaxis with factor VIII (FVIII) replacement therapy aimed at higher trough levels is becoming indispensable for patients with severe hemophilia A. This study aimed to identify the most suitable population pharmacokinetic (PK) models for personalized prophylaxis using various FVIII products and 2 clinical assays and to implement the most suitable one in open-access software. Twelve published population PK models were systematically compared to predict the time above target (TaT) for a reference dosing occasion. External validation was performed using a 5-point PK data from 39 adult patients with hemophilia A with FVIII measured by chromogenic substrate (CSA) and 1-stage assays (OSAs) using NONMEM under 3 different conditions: a priori (with all FVIII samples blinded), a posteriori (with 1 trough sample), and general model fit (with all FVIII samples including the reference dosing occasion provided). On average, the baseline covariate models overpredicted TaT (a priori; bias -3.8 hours to 49.6 hours). When additionally including 1 previous trough FVIII sample before the reference dosing occasion (a posteriori), only 50% of the models improved in bias (-1.0 hours to 36.5 hours) and imprecision (22.4 hours and 60.7 hours). Using all the time points (general model fit), the models accurately predicted (individual TaT less than ±12 hours compared with the reference) 62%-90% and 33%-74% of the patients using CSA and OSA data, respectively. Across all scenarios, predictions using CSA data were more accurate than those using the OSA data. One model performed best across the population (bias: -3.8 hours a priori, -1.0 hours a posteriori , and 0.6 hours general model fit ) and acceptably predicted 44% (a priori) to 90% ( general model fit ) of the patients. To allow the community-based evaluation of patient-individual FVIII dosing, this model was implemented in the open-access model-informed precision dosing software "TDMx."

Guidelines on the investigation and management of antiphospholipid syndrome

This guidance updates and replaces the previous guideline on the investigation and management of antiphospholipid syndrome (APS) published in 2000 (Greaves et al, 2000), though where there have not been changes we refer back to them when appropriate. The guidance is updated with reference to relevant publications since 2000. Publications known to the writing group were supplemented with additional papers identified by searching PubMed for publications in the last 11 years using the key words: lupus anticoagulant, anticardiolipin, antiphospholipid, b2–glycoprotein I, antiprothrombin and limits (clinical trial, randomized control trial, meta-analysis, humans, core clinical journals, English language). The writing group produced the draft guideline, which was subsequently revised by consensus by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 50 UK haematologists, the Royal College of Obstetricians and Gynaecologists (RCOG), and the British Committee for Standards in Haematology (BCSH) Committee and comments incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found at http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMEN DATION/43_GRADE.html. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of patients with antiphospholipid syndrome though individual patient circumstances may dictate an alternative approach.

Development of a microplate coagulation assay for Factor V in human plasma

BackgroundFactor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available.MethodsThe FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma.ResultsThe FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP.ConclusionsThe FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and clinical laboratories to provide measurement of the functional coagulation activity of FV in human plasma.

Identification of von Willebrand disease type 2N (Normandy) in Australia: a cross-laboratory investigation using different methods.

We report on a cross-laboratory study of type 2N von Willebrand disease (vWD). We tested 101 selected plasma samples for factor VIII and factor VIII binding activity of von Willebrand factor (vWF). Of these plasma samples, 31 were cotested by 2 specialist centers using different detection procedures for vWF-factor VIII binding: there was good agreement between results obtained by chromogenic assay and enzyme-linked immunosorbent assay. In total, 8 patients with type 2N vWD were identified. The 2-stage factor VIII assay detected a deficiency of factor VIII relative to vWF antigen in all 8 patients; the 1-stage factor VIII assay detected a relative deficiency in only 3 patients. Four patients were homozygous for the most common type 2N mutation (R854Q), 3 patients were presumed to be compound heterozygotes, and in 1 patient no type 2N mutations were identified. In this study of patients from 5 specialist centers in Australia, type 2N vWD was found in 5 families. The 2-stage factor VIII assay was more useful as a screening test than the 1-stage assay, and both vWF-factor VIII binding assays were equally effective.

Activated Factor VIII inTherapeutic Preparations:Analysis by Ultracentrifugation

Activation of factor VIII is signaled by an increase in the 1-stage factor VIII activity and release from von Willebrand factor. Ultracentrifugation in 10-40% sucrose gradients was used to identify such activation in therapeutic concentrates. Plasma-derived factor VIII lots were examined and factor VIII sedimenting independently of von Willebrand factor was identified in some of the preparations. In addition there was slower sedimentation of factor VIII by the 1-stage assay than by the chromogenic assay. These results are consistent with a factor VIII cleaved at residue 1689, a site important for von Willebrand binding. This activated form leads to some of the assay discrepancies between the 1-stage assay and the chromogenic or the 2-stage assay. There is more rapid sedimentation of the factor VIII measured by the chromogenic assay than the von Willebrand factor in some manufacturers' samples indicating that the method of fractionation may select a low molecular weight von Willebrand factor which does not bind factor VIII. Routine comparison between the 1-stage and chromogenic assays during fractionation may be able to identify such activated preparations. Other assay discrepancies may be due to structural differences between the standards and the tested product.

In Vivo Studies of Activated Porcine Factor VIII

The haemostatic effectiveness of activated FVIII was compared to that of non-activated FVIII in a cross-over study in a canine model of haemophilia. Activation of FVIII in porcine concentrate was achieved by the addition of 3 x 10(-5) IU thrombin per ml of concentrate, which gave consistent increases in 1-stage FVIII activity of 13- to 14-fold and slow decay. The haemostatic effect was monitored by measurements of the cuticle bleeding time 10 and 45 min after infusion and there were no consistent differences between the activated and non-activated concentrates. One-stage factor VIII assays on plasmas 5 min after infusion showed identical mean values for activated and non-activated concentrates, indicating that most of the higher activity observed in vitro had disappeared rapidly from the circulation. These results suggest that controlled activation of FVIII by thrombin, which increases its activity in 1-stage assays, is unlikely to be of therapeutic benefit. For therapeutic concentrates which may contain small amounts of activated FVIII, the 1-stage assay may be an unreliable guide to their therapeutic effect.

Single or multicellular origin of human T lymphocyte colonies in vitro: modification by 12-o-tetradecanoylphorbol 13-acetate (TPA).

Abstract The assumption that human T lymphocyte colonies have a unicellular origin has been directly tested with peripheral blood mononuclear cells from 2 women heterozygous for the common X-linked glucose-6-phosphate dehydrogenase (G-6-PD) gene (GdB) and the variant GdA. T cells were cultured in semisolid medium in the presence of phytohemagglutinin (PHA) and T lymphocyte growth factor with or without preincubation in suspension culture with PHA (2-stage and 1-stage assays, respectively). The enzyme type of individual T cell colonies was then determined electrophoretically at the lowest colony density with adequate growth (usually less than 100 colonies/dish). In the 2-stage system, 90 of 97 tested colonies had equal amounts of A and B enzyme activities suggesting multicellular origin of the colonies. Similarly, in the single-stage system, 21 of 31 colonies had both A and B enzymes. Increasing the density of the soft agar did not influence the frequency of A/B colonies. However, when 12-O-tetradecanoylphorbol 13-acetate (TPA), a promoter of T cell colony growth shown in other systems to inhibit metabolic cooperation, was added, a striking decrease in frequency of colonies with both G-6-PD types was found. In the 2-stage culture, 0 of 9 colonies had a double-enzyme type and in the single-stage system, the frequency of A/B colonies declined to 9 of 34 (p less than 0.025). The data suggest that despite the apparent multicellular origin of T cell colonies in cultures with TPA, most colonies do originate from single cells when cultured with TPA at low colony densities. Stimulation of cell growth or inhibition of metabolic cooperation between cells by TPA are possible explanations for these differences.

Parallel destruction of factor VIII procoagulant activity and an 85,000 dalton protein in highly purified factor VIII/VWF

Concentrated, highly purified human factor VIII/von Willebrand factor (FVIII/vWF) (produced by two different methods) in O.1M ammonium bicarbonate, pH 7.4 was digested with human plasmin (0.004 cu/ml) or human or bovine thrombin (0.1 U/m), and the digests were assayed for procoagulant (VIIIC) activity. Digestion samples were lyophilized and then subjected to two different new electrophoretic methods to examine trace proteins, which would not be detectable by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The results show that both thrombin and plasmin proteolyse a single-chain protein with an apparent molecular weight of 85,000 daltons, but at different sites. The initial thrombin-induced proteolysis of this protein, which comprised about 2% of highly purified FVIII/vWF, was largely complete by the time VIIIC was maximally activated as judged by the 1-stage assay. The loss of VIIIC induced by both thrombin and plasmin, as measured by the 2-stage assay (which shows no activation) paralleled the rate of proteolysis of the protein by both enzymes. Since no other proteolytic events were observed which even remotely paralleled the loss of VIIIC, we conclude that the biological activity defective in haemophilia may be expressed by the 85,000 dalton protein reported in this study.

A Quantitative Two-Stage Assay for Factor IX (Christmas Factor) Using Plasma from Dogs with Christmas Disease

SummaryA modified 2-stage assay for factor IX which utilizes factor IX deficient plasma plasma from a colony of dogs with Christmas factor deficiency, has been described. The assay is technically less difficult and less time consuming than the established 2-stage assay for factor IX. The assay is more sensitive than other methods currently in use and uses only 4 μl of deficient plasma for a test. Results are comparable to those reported for 1-stage assays, both in vitro and in vivo, with an apparent increase in activity during the conversion of plasma to serum, and with apparently low in vivo recovery of factor IX when infused into a deficient patient.