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.