Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test – Demonstrated in vitro and ex vivo

Canine models of severe haemophilia resemble their human equivalents both regarding clinical bleeding phenotype and response to treatment. Therefore pre-clinical studies in haemophilia dogs have allowed researchers to make valuable translational predictions regarding the potency and efficacy of new anti-haemophilia drugs (AHDs) in humans. To refine in vivo experiments and reduce number of animals, such translational studies are ideally preceded by in vitro prediction of compound efficacy using a plasma based global coagulation method. One such widely used method is the thrombin generation test (TGT). Unfortunately, commercially available TGTs are incapable of distinguishing between normal and haemophilia canine plasma, and therefore in vitro prediction using TGT has so far not been possible in canine plasma material

Presence of C1-inhibitor polymers in a subset of patients suffering from hereditary angioedema.

Hereditary angioedema (HAE) is a potentially life-threatening disease caused by mutations in the gene encoding the serine protease inhibitor (serpin) C1 inhibitor (C1-inh). The mutations cause decreased functional plasma levels of C1-inh, which triggers unpredictable recurrent edema attacks. Subjects suffering from HAE have been classified in type I patients with decreased functional and antigenic levels of C1-inh, and type II patients with decreased functional but normal antigenic C1-inh levels. However, a few reports have demonstrated that some mutations cause C1-inh polymerization in vitro, and it is speculated that C1-inh polymers may exist in patient plasma, challenging the current classification of HAE patients. To investigate the presence of C1-inh polymers in patient plasma samples, we developed an immunological method, where monoclonal antibodies produced against polymerized C1-inh were applied in native PAGE western blotting. Using this approach we analyzed genuine plasma samples from 31 Danish HAE families, and found that plasma samples from three genotypically distinct HAE type I families (classified upon C1-inh plasma concentrations) contained C1-inh polymers. Identical C1-inh polymerization phenotypes were observed in four affected family members from one of these families. Genotyping of the families revealed that the polymerogenic mutations of two families were located in proximity to the reactive center loop insertion site in C1-inh (p.Ile271Thr and p.Ser258_Pro260del),and one mutation affected helix C (p.Thr167Asn). In conclusion, we demonstrate that C1-inh polymers are present in the plasma of a subgroup of HAE type I patients

Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test – Demonstrated <i>in vitro</i> and <i>ex vivo</i>

<div><p>Introduction</p><p>Canine models of severe haemophilia resemble their human equivalents both regarding clinical bleeding phenotype and response to treatment. Therefore pre-clinical studies in haemophilia dogs have allowed researchers to make valuable translational predictions regarding the potency and efficacy of new anti-haemophilia drugs (AHDs) in humans. To refine <i>in vivo</i> experiments and reduce number of animals, such translational studies are ideally preceded by <i>in vitro</i> prediction of compound efficacy using a plasma based global coagulation method. One such widely used method is the thrombin generation test (TGT). Unfortunately, commercially available TGTs are incapable of distinguishing between normal and haemophilia canine plasma, and therefore <i>in vitro</i> prediction using TGT has so far not been possible in canine plasma material.</p><p>Aim</p><p>Establish a modified TGT capable of: 1) distinguishing between normal and haemophilia canine plasma, 2) monitoring correlation between canine plasma levels of coagulation factor VIII (FVIII) and IX (FIX) and thrombin generation, 3) assessing for agreement between compound activity and thrombin generation in <i>ex vivo</i> samples.</p><p>Methods</p><p>A modified TGT assay was established where coagulation was triggered using a commercially available activated partial thromboplastin time reagent.</p><p>Results</p><p>With the modified TGT a significant difference was observed in thrombin generation between normal and haemophilia canine plasma. A dose dependent thrombin generation was observed when assessing haemophilia A and B plasma spiked with dilution series of FVIII and FIX, respectively. Correlation between FVIII activity and thrombin generation was observed when analyzing samples from haemophilia A dogs dosed with canine FVIII. Limit of detection was 0.1% (v/v) FVIII or FIX.</p><p>Conclusion</p><p>A novel modified TGT suitable for monitoring and prediction of replacement therapy efficacy in plasma from haemophilia A and B dogs was established.</p></div

Correlation between FVIII activity and thrombin generation in <i>ex vivo</i> plasma samples.

<p>Correlation analysis between FVIII activities and peak thrombin generation of samples from six individual haemophilia A dogs dosed with varying levels of rcBDDFVIII and normal canine plasma on several occasions. Each point represents mean of double determinations, and the line represents linear regression analysis of the plots. Y-axes depict peak thrombin generation in nM thrombin, and the log10 transformed x-axes depict FVIII activity given as percentage of FVIII activity in a normal canine plasma pool. IDs represent the identifiers for each dog, r represents Pearson correlation coefficient, and P represents two-tailed P values.</p

Thrombograms from imprecision estimation of the canine optimized TGT.

<p>Twenty replicates of each sample were analyzed per run. Each thrombogram represents a single determination. The following samples were analyzed: A) normal canine plasma pool II, B) normal canine plasma pool II diluted to 0.1% (v/v) in haemophilia A (HA) plasma pool and the HA plasma pool, C) normal canine plasma pool II diluted to 0.1% (v/v) in haemophilia B (HB) plasma pool and the HB plasma pool. Y-axis depicts thrombin generation in nM and x-axis depicts time in minutes.</p

Analysis of normal and haemophilia A canine plasma pools using the CAT method.

<p>Thrombin generation was monitored using the standard CAT method, and was initiated using tissue factor at 5 and 1 pM (PPP and PPPlow reagent, respectively). A) Normal plasma pool II was analyzed in the presence and absence of anti cFVIII PAb. B) Normal plasma pool II and HA plasma pool were analyzed. Thrombograms represent mean of quadruplicates. Y-axis depicts thrombin generation in nM, x-axis depicts time in minutes. All experiments were carried out in one run, and the normal plasma pool II results in A) and B) are identical.</p

Inter-assay imprecision estimates.

<p>Inter-assay imprecision estimates.</p

Analysis of plasma samples from five individual HA dogs using the canine optimized TGT.

<p>Bars represent mean of double determinations, and error bars represent range. Light blue bars represent analysis of plasma samples in the presence of buffer, and dark blue bars represent analysis of plasma samples in the presence of 0.1 mg/mL anti cFVIII PAb. The buffer sample from P16 represents a single determination, as one of the double determinations failed. X-axis represents dog identifier, and y-axis represents peak thrombin generation in nM thrombin.</p