Platelet Reactivity in Hemophilia a. Flow Cytometry Study to Detect the Hemorragic Phenotype
Authors: Melero-Amor Antonia , García Candel Faustino , García David , Romecin Paola , Iyu David , Cabañas-Perianes Valentin , Pérez Raúl , O´Connor Jose-Enrique , Moraleda Jose M , Marín Noemí , Blanquer Blanquer Miguel 
Published: October 2019

Abstract
INTRODUCTION
Hemophilia A (HA) is a rare inherited genetic disorder linked to the X chromosome, characterized by joint bleeding in patients with severe disease with <1% of factor VIII (FVIII)(White et al. Thromb Haemost 2001). However, there are cases of severe HA with less bleeding than usual and it has been postulated that there must be other contributors to the hemorrhagic phenotype, including a platelet dysfunction (Van Bladel et al. Haematologica 2011).
In this study we analyzed platelet reactivity in a cohort of 16 male patients with severe HA in prophylactic treatment by flow cytometry. The control group was 15 healthy male subjects.
METHODS
A longitudinal prospective observational study cohort was conducted. The study was performed according to the Declaration of Helsinki of 1975 (revised 1983), and was approved by the Ethics Committee of our Institution. Sixteen HA male patients without FVIII inhibitor aged 9-39 years (mean 22,75 years, SD 11,428) and 15 adult male healthy volunteers aged 26-48 years (mean 36,4 years, SD 6,6) were included. All subjects had normal platelet counts (150-450x103/µl). The blood was collected by antecubital venipuncture and avoiding the use of tourniquet to minimize the spontaneous activation of platelets during extraction. For HA patients blood samples were taken immediately before FVIII infusion (basal sample, 72h without FVIII) and 15 minutes after the infusion of FVIII. Levels of factor FVIII in plasma were determined using a chromogenic method. We analyzed platelet responsiveness, using the thrombin receptor activator peptide-6 (TRAP-6) as agonist, measuring the expression of CD62P and CD63 by flow cytometry. For microparticles analysis, percentages and number of events positive for CD62P and CD41 were gated for Annexin-V+ subpopulation. Endothelial cell-derived microparticles were identified as CD144+CD41-. To calculate the absolute count we considered the final volumes of each tube and it was expressed in events/µL for each subpopulation (Annexin-V +, CD41+, CD144+ and CD62P+). For the analysis of cytoplasmic free calcium release, whole blood was labelled with PerCP-Cy5.5-human anti-CD61 and the calcium sensitive dye Fluo-4-AM for 15 min at room temperature. Following adjustment of the basal fluorescence, samples were stimulated with the agonists TRAP-6 or adenine-5«-diphosphate sodium salt (ADP) and analyzed for 5 min by flow cytometer. Ionophore A23187 was used as a positive control for calcium mobilization. Afterwards mean fluorescence intensity values were analysed with the Kaluza flow cytometry analysis software (Beckman Coulter).
RESULTS
Statistical analysis was carried out using SPSS statistical software version 19.0.All values were expressed as mean ± SD for each experimental group.To compare the results in HA patients before and after administration of FVIII we used the t-Student test for means related, to compare HA patients and the control group we used the t-student test for independent samples. Data of calcium were analyzed by one-way analysis of variance followed by Tukey-Kramer for multiple comparisons. The minimum acceptable level of significance wasp<0.05.
Basal levels of FVIII in plasma were4,58 ± 6,4%.
The mean fluorescence intensity for CD62P after stimulation with TRAP was significantly lower in HA patients after infusion of FVIII compared to basal levels (p<0,05)(Table 1). Regarding microparticles (MPs), we detected higher expression of CD41+ MPs in HA patients after infusion of FVIII compared to basal expression, but not for CD41+CD62P+ MPs. Also, percentage of CD144+ MPs were significantly lower in HA patients after FVIII infusion (Table 2). Moreover, we did not find statistically significant differences comparing the release of calcium between the different groups (Table 3).
CONCLUSION
In this study severe HA patients in prophylactic treatment displayed normal platelet activation and microparticles plasmatic levels. However, the platelet activation marker CD62P after TRAP agonist and the endothelial cell origin microparticles expression were lower 15 minutes after the infusion factor VIII.
Acknowledgments
We wish to thank to Pfizer providing economic support to this project.

Disclosures
Melero-Amor: Pfizer: Research Funding. García Candel:Pfizer: Research Funding. García:Pfizer: Research Funding. Romecin:Pfizer: Research Funding. Iyu:Pfizer: Research Funding. Cabañas-Perianes:Pfizer: Research Funding. Pérez:Pfizer: Research Funding. O´Connor:Pfizer: Research Funding. Moraleda:Pfizer: Research Funding. Marín:Pfizer: Research Funding. Blanquer Blanquer:Pfizer: Research Funding.