The complement system is a long and complicated event of reactions where activation leads to cleavage of different factors and ends with either inflammation or cell lysis.     Recent studies have shown that the complement system and coagulation have some elements in common. Therefore in this study it was relevant to look at the inhibition of the complement system in two different whole blood analyses of coagulation activation, thromboelastography and impedance aggregometry. Thromboelastography, or TEG®, measures the clot forming properties of whole blood and the impedance aggregometry, or Multiplate®, measures platelets’ ability to adhere and aggregate to an electrode. Four different inhibitors where used: Eculizumab, C1 inhibitor, Compstatin and OMS721, which all inhibits different parts of the complement system.     The curves from Multiplate® was presented in standard deviation and the number of reduction, while the results from TEG® was presented in before and after added inhibitor in graphs.     In conclusion, impedance aggregometry show a more specific and secure results of the inhibitors effect, which was seen by that both C1 inihibitor and Compstatin had a major influence on the area under the curve (AUC). In TEG® there were no detectable difference, which could mean TEG® is not specific enough for platelets efficiency, which is affected by the complement inhibition.

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While some rare and severe forms of platelet function disorders are now well characterized, many common types of platelet function disorders are not yet characterized. My hypothesis was that uncharacterized platelet function disorders that impair platelet function in aggregation and/or dense granule ATP release assays are associated with increased bleeding risk. The main goal of the thesis was to study the phenotype and bleeding risks for uncharacterized platelet function disorders, through analysis of the results from clinical laboratory tests of platelet function and for a detailed analysis of their reported bleeding symptoms. First, I assessed if lumi-aggregometry provides useful diagnostic information on platelet function and can be used to help decide if an individual has a bleeding disorder. Two cohorts of individuals were studied that had dense granule ATP release assessed in response to multiple agonists as part of a work-up for a bleeding disorder. Cohort I was comprised of individuals tested between January 2007 and June 2013 and cohort II was comprised of subjects tested at least twice by this assay prior to September 2015. Among subjects tested more than once for dense granule release defects as part of the work up for a bleeding disorder (cohort I; n=133; cohort II; n=17), normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), but impaired release with multiple agonists was not often confirmed (cohort I: 34%; cohort II: 54%) and even if it was present, the finding was not predictive of a bleeding disorder. Consequentially, it was recommended that lumi-aggregometry should not be used to diagnose platelet function disorders. Next, I studied the bleeding risks associated with uncharacterized platelet function disorders, by evaluating subjects who had abnormal findings by validated assays, namely subjects who had defective aggregation responses to two or more agonists and/or dense granule deficiency. Bleeding history was evaluated using the International Society for Thrombosis and Haemostasis bleeding assessment tool (ISTH BAT) and the likelihood for bleeding symptoms/ problems, was estimated using odds ratios (OR) collected using the clinical history assessment tool - platelet (CHAT-P) for all affected subjects, a subgroup family with a mutation RUNX1, unaffected family v members and general population controls. Individuals with platelet function disorders (n=29) and the affected members of the family with the RUNX1 mutation (n=6) had elevated ISTH BAT scores (median: 9; range:0-18 and median: 8.5, range 4-15, respectively) and an increased risk of abnormal bruising (OR 15-65 and 11-67), nosebleeds (OR 23-40 and 19-121), menorrhagia (OR 6.5-29) and excessive bleeding after trauma or dental/surgical procedures (OR 9.5-44 and 15-77 ) and wound healing problems (OR 13 and 38) compared to general population control (n=60) and unaffected (n=12) family members. Overall, the platelet function disorders in the study present with a significantly increased risk of mild, rather than severe bleeding problems. These findings are important for individuals and healthcare providers to promote evidence-based care of common uncharacterized inherited platelet function disorders for individuals with RUNX1 mutations, dense granule deficiency and/or impaired aggregation responses. / Thesis / Master of Science (MSc) / Platelets are small blood cells that help stop bleeding. People who have platelets that do not work properly are more likely to bleed. Determining who has platelet problems can be challenging as there are limitations to diagnostic tests for these conditions. Additionally, the risks for bleeding in individuals with platelet problems are unknown. We looked at individuals with bleeding problems and found that a recommended test to assess platelet dense granule release, called lumi-aggregometry, wasn’t able to reliably identify persons with bleeding problems. Based on this, we recommend that lumi-aggregometry should not be used to diagnose platelet function disorders. We also found that individuals with uncharacterized platelet function disorders have increased risks for wound healing problems and experiencing bruising, nosebleeds, menorrhagia, and excessive bleeding after dental or surgical procedures. These risks are common among other mild bleeding disorders and will be important to differentiate bleeding risk from other platelet disorders.

Introduction : L’effet biologique variable de l’aspirine a été attribué à un état de résistance pharmacologique. L’incidence de cette « résistance » varie selon la population ou la technologie étudiée. Méthodes : Nous avons déterminé la performance de 5 techniques évaluant l’effet de l’aspirine chez des sujets sains, non fumeurs et ne prenant aucune médication pouvant interférer avec la fonction plaquettaire. Des spécimens de sang et d’urine ont été obtenus avant et après 8-10 jours de prise de 80 mg d’aspirine. Résultats: Chez 45 sujets de 19-59 ans, la sensibilité (SE), la spécificité (SP), et la valeur optimale de coupure (CO) pour détecter l’effet de l’aspirine sont : agrégométrie par transmission optique induite avec 1,6 mM d’acide arachidonique (ATO-AA) - SE 100%, SP 95,9%, CO 20%; ATO-ADP 10 μM - SE 84,4%, SP 77,7%, CO 70%; VerifyNow® Aspirin - SE 100%, SP 95,6%, CO 550 ARU; agrégation en tube - SE 82,2%, SP 86,7%, CO 55%; TEG® - SE 82,9%, SP 75,8%, CO 90%; et le dosage de 11-dehydrothromboxane B2 urinaire - SE 62,2%, SP 82,2%, CO 60 pg/ml. Conclusions: La résistance à l’aspirine chez les sujets sains définie par ATO-AA et VerifyNow® Aspirin est rare. Puisque les autres techniques étudiées discriminent de façon sous optimale l’effet de l’aspirine, leur utilité dans la définition de la résistance pharmacologique à l’aspirine semble marginale. Ces résultats suggèrent qu’une proportion de la variabilité de l’incidence rapportée de “résistance à l’aspirine” est artefactuelle et reliée aux limitations technologiques de certaines analyses. / Background: Variable biological effect of aspirin is suggested to be related to pharmacological resistance. The incidence of this so-called “resistant” state varies with the study population and the assay used. Methods: We determined performance features of five assays used to assess aspirin effects in non smoking healthy volunteers not taking any drug known to interfere with platelet function. Blood and urine samples were obtained immediately before and after 8-10 days of aspirin 80 mg intake. Results: Forty-five participants 19-59 years old were enrolled. The sensitivity (SE), specificity (SP), and optimal cut-off (CO) value to detect the effect of aspirin were: light transmission aggregometry (LTA) with 1.6 mM arachidonic acid - SE 100%, SP 95.9%, CO 20%; LTA with ADP 10 μM - SE 84.4%, SP 77.7%, CO 70%; VerifyNow® Aspirin - SE 100%, SP 95.6%, CO 550 ARU; platelet count drop - SE 82.2%, SP 86.7%, CO 55%; TEG® - SE 82.9%, SP 75.8%, CO 90%; and urinary 11-dehydrothromboxane B2 levels - SE 62.2%, SP 82.2%, CO 60 pg/ml. Conclusions: Aspirin resistance in normal individuals as defined by arachidonic acid-induced LTA and the VerifyNow® assay is rare. Because the other assays discriminate suboptimally aspirin effect, they should not be used to define pharmacological “aspirin resistance”. These results suggest that a proportion of the variability in the reported incidence of aspirin resistance is artefactual and related to technical limitations of some assays.

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