Academic Senate - Meeting Minutes, 4/18/2017

<p>All values are presented with SD. Differences between <i>LDLR−/−</i> and the other two genotypes are significant where indicated, ANOVA: *p<0.05, **p<0.01.</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

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

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

Inter-assay imprecision estimates.

<p>Inter-assay imprecision estimates.</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

Analysis of normal canine plasma pool dilutions in haemophilia B canine plasma pool.

<p>Samples were analyzed using the canine optimized thrombin generation test. A) Dilutions spanning 0–100% normal diluted in HB plasma pool (v/v) were analyzed. 100% represents undiluted normal plasma pool, and 0% represents undiluted HB pool. Thrombograms represent mean of double determinations. Y-axis depicts thrombin generation in nM, x-axis depicts time in minutes. B) Correlation analysis of peak thrombin generation versus dilution of normal canine plasma pool in HB pool from panel A. The log10 transformed x-axis represents plasma dilutions, and the log10 transformed y-axis represents peak thrombin generation in nM. Points represent mean of double determinations, lines represent linear regression analysis.</p

Intra-assay imprecision estimates.

<p>Intra-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

Analysis of normal and HA plasma using the canine optimized TGT.

<p>Plasma samples from five healthy dogs, five HA dogs, a normal canine plasma pool and a HA canine plasma pool were analyzed. A) Thrombograms represent mean of double determinations. Y-axis depicts thrombin generation in nM, x-axis depicts time in minutes. B) Bars represent mean peak thrombin generation in nM, and error bars represent range of double determinations. Y-axis depicts peak thrombin generation in nM, and x-axis depicts samples.</p