Platelet function.
Platelet function was essentially normal, as suggested by the results obtained with different methodologies. With the use of PFA-100, a tendency to the increase in the obturation time (OT) was observed in patients receiving FVIII, but no significant differences were obtained. There are mixed results in the literature, both with an increase in the baseline OT (13-15) or no differences in patients with HA (16-19). However, we believe our data are the first to show that the administration of FVIII to HA patients does not change OT.
Regarding the Multiplate system, we believe that our study is the first one to use it in HA patients. A significant reduced aggregation was observed between patients after administration of FVIII and the controls in response to TRAP. A data that may be of interest since this decrease was observed clearly in 5 patients, 4 of which were VHC+ and 2 of them HIV+. A reduced platelet function has been also described in the hepatitis C virus infection (20-21), and this possibility clearly merits further study.
The turbidimetric platelet aggregometry remains the gold standard for the diagnosis of platelet function disorders, and our data are also the first to explore it in HA patients. Again, no significant changes were observed after the administration of FVIII in comparison to the baseline or control values.
The expression of P-selectin (CD62P) and CD63, and an increase in the number of platelet-leukocyte conjugates or in the exposure of tissue factor are related to a variety of pathologies with elevated thromboembolic risk. Moreover, P-selectin is considered the gold standard for platelet activation. Our study of flow cytometry, however, could not find a significant difference between groups. There are conflicting results in the literature, even from the same laboratory (22-24), but it seems that our results agree with most of these studies, both in human patients (23, 25) and mice (24), thus suggesting that there is no platelet activation in patients with severe HA.
Calcium study .
Intraplatelet free Ca2+ has been used in the functional study of platelets and in the monitoring of therapies with platelet antagonists (26). In our study, we observed, in all groups, a rapid increase in intracellular calcium and a subsequent decrease in the signal after 30 seconds, although without returning to the previous basal situation, before addition of the agonist. In our study, we used thrombin (more potent physiological platelet activator) and ADP (weaker physiological platelet agonist), which are the most frequently used agonists (27-27), without observing significant differences among the groups studied. However, in the case of ADP, the level of calcium was similar between the control group and the hemophilic patients after infusion of FVIII, although, it was lower in the baseline sample of patients. When TRAP was used as an agonist. it was found that the controls presented a higher response to calcium release than patients (both before and after the administration of factor VIII). Perhaps these results indicate that patients have a lower level of extracellular Ca2+ available to be used when the agonist acts at not very high concentrations, although, in our study, these differences were not statisticallly significant. To the best of our knowledge, this is the first study in which intra-platelet calcium is analyzed in patients with hemophilia. In conclusion, we have not observed significant differences in the response of intraplaquetary Ca2+ to ADP or TRAP stimulation in relationship with the infusion of FVIII compared to a cohort of healthy subjects.