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).