Human samples and reagents.
Blood samples were obtained before (baseline sample, 72h without administering factor VIII) and after infusion of factor VIII (15 minutes after). This time was selected following a previous report of the National Hemophilia Foundation (7). They were always extracted by the same expert nurse, with a 21G needle without the use of compressor or tourniquet, to minimize platelet stimulation. A total of 18 mL were extracted in each subject. The first 2.5 ml were used for measurements other than platelet function, i.e. hemogram, following the recommendations of the International Society on Thrombosis and Haemostasis (8). The rest of blood was distributed as indicated in the supplementary document. Briefly, we determined platelet count, measurement of factor VIII, von Willebrand antigen and cofactor von Willebrand, platelet function by impedance using the Roche Multiplate analyzer system, platelet-related primary hemostasis using the PFA-100 System, platelet aggregation by means of light optical aggregometry, flow cytometry for aggregation, activation and platelet-leucocytes conjugates and platelet cytosolic calcium. Finally, microvesicles were also analyzed in a Gallios flow cytometer in the Laboratory of Flow Cytometry-Coulter Cytometry Center and Related Techniques in Valencia.
The conceptual basis for performing so many different techniques to measure platelet function is that although each one is different, all of them are different from each other, having their advantages and disadvantages, and analyzed differently, with different nuances, sometimes even from very different angles. Therefore, we decided to perform a comprehensive battery of platelet function tests, since it is not known how hemophilia or FVIII interferes with them. Thus, by using a wide spectrum of techniques we will have more possibilities of detecting any change that hemophilia or the administration of FVIII could produce on platelet function, as it is done to assess the efficacy of new antiplatelet agents (9) or to assess platelet function in certain pathologies or circumstances (10) The value, importance and relevance of each of these techniques are very well detailed elsewhere (10-11).