Procoagulant Platelet Subpopulation Formation in Whole Blood: Correlation with Whole Blood Clotting Time and 5-Year Mean Bleeding Score in Individuals with Factor VIII Deficiency.

Abstract

Abstract 4022Poster Board III-958Different clinical phenotypes are often observed among individuals with hemorrhagic or thrombotic disorders despite the presence of the same mutation and/or factor levels suggesting that additional genetic and/or environmental factors influence clinical presentation. As thrombin plays a central role in hemostasis, factors that affect an individual's ability to generate thrombin may lead to an increased risk of hemorrhage or thrombosis. The goal of this study was to assess procoagulant platelet formation in individuals with hemophilia A using a rapid, whole blood flow cytometric assay of prothrombinase complex assembly on platelets described previously. In this assay, Ca2+-dependent factor Xa binding to activated platelets is used as a marker of thrombin generating potential as it has been shown previously to correlate with platelet prothrombinase activity in a washed platelet system. Procoagulant platelet subpopulation formation in 15 men with varying degrees of factor VIII-deficiency was evaluated and compared to two independent measures of hemostatic competence: a 5-year mean bleeding score and whole blood clot time. In these individuals, the % activated platelets binding factor Xa in whole blood varied from 2.35 – 9.0% (n = 30), which is consistent with what is observed in unaffected individuals (1.5 – 41.5%, n = 136). Bleeding was scored independently by two experienced hemophilia nurses and one hematologist at Centre Hospitalier Universitaire Sainte Justine based on each individual's bleeding history, including hemarthroses, soft tissue hematoma, and annual factor VIII usage, and averaged. The 5-year mean bleeding scores in these individuals ranged from 0 – 20.4. Linear regression analyses indicated that in this population the % activated platelets binding factor Xa correlated indirectly with the 5-year mean bleeding score, where individuals with lower bleeding scores (i.e. less bleeding episodes and/or factor VIII usage) generated larger procoagulant platelet subpopulations. The time to clot formation in a tissue factor-dependent, contact pathway-suppressed whole blood clotting assay described previously, was also determined. The clot time, which was determined visually, and ranged from ∼3 – 7 min, also correlated indirectly, though less well, with the % activated platelets binding factor Xa in whole blood (r = -0.23). Thus, consistent with what was observed with the bleeding score, those individuals who clotted more quickly (i.e. exhibited a greater degree of hemostatic competence) generated more proacoagulant platelets. Platelet procoagulant subpopulation formation was also compared to other hematological measures. An inverse correlation was observed between the % activated platelets binding factor Xa and mean platelet volume (r = -0.347), suggesting that in this population, individuals with smaller platelets (i.e. platelets with fewer prothrombinase binding sites) may generate a greater number of platelets capable of assembling prothrombinase. In contrast, no correlation was observed between whole blood platelet number and % activated platelets binding factor Xa. Interestingly, factor Xa binding was also negatively correlated with plasma levels of factor IX (r = -0.56) and factor V (r = -0.62). Platelet subpopulation formation was also weakly and negatively associated with the aPTT (r = - 0.28). In this small population of individuals with hemophilia A, whole blood platelet factor Xa binding correlates with bleeding phenotype. These observations support the notion that this measurement can be used to predict an individuals propensity to bleed or clot. Disclosures:No relevant conflicts of interest to declare.