PB0747 - Platelet reactivity to Dengue and Zika Nonstructural protein 1 (NS1)

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Alan Cano Méndez, PhD (he/him/his)

Facultad de Ciencias Medicas y Biológicas "Dr. Ignacio Chávez". Universidad Michoacana de San Nicolás de Hidalgo
Morelia, Michoacan de Ocampo, Mexico

Co-Author(s)

NG

Nallely I. García-Larragoiti

Posdoc Research
Facultad de Ciencias Medicas y Biológicas "Dr. Ignacio Chávez". Universidad Michoacana de San Nicolás de Hidalgo
Morelia, Michoacan de Ocampo, Mexico
SL

Sandra Edith E. López-Castañeda

Professor
Facultad de Ciencias Medicas y Biológicas "Dr. Ignacio Chávez". Universidad Michoacana de San Nicolás de Hidalgo
Morelia, Michoacan de Ocampo, Mexico
GE

Gabriel Espinosa-Perez

Professor
Facultad de Ciencias Físico Matemáticas. Universidad Michoacana de San Nicolás de Hidalgo.
Morelia, Michoacan de Ocampo, Mexico
YC

Young Chan-Kim

Assistant Professor
The Jenner Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive.
Oxford, England, United Kingdom
AR

Arturo Reyes-Sandoval

Professor
Instituto Politécnico Nacional
CDMX, Distrito Federal, Mexico
MV

Martha Eva Viveros-Sandoval

Professor
Facultad de Ciencias Medicas y Biológicas "Dr. Ignacio Chávez". Universidad Michoacana de San Nicolás de Hidalgo
Morelia, Michoacan de Ocampo, Mexico

Background: Platelets are involved in hemostasis and innate immune processes. Dengue (DENV) and Zika (ZIKV) viruses’ encodes 3 structural and 7 nonstructural proteins. Nonstructural protein 1 (NS1) is mainly involved in the replication of viral genetic material. NS1 can be secreted into the extracellular microenvironment and has been associated with immune response evasion.

Aims: The aim of this work was to evaluate the platelet response to NS1 protein of dengue and Zika viruses.

Methods: Blood samples were obtained from healthy volunteers by venipuncture after signing an informed consent form, using 3.2% sodium citrate as an anticoagulant. Platelet-rich plasma (PRP) was obtained by slow centrifugation (100 g x 10 minutes). PRP was separated and resuspended in Tyrodes buffer. PRP (1X107 cells/ml) was stimulated with DENV and ZIKV NS1 proteins [2.5 ug/ml] for different times, 37°C. Platelet activity was assessed by flow cytometry: CD41-PECy7, CD62-PE, and PAC1-FITC. For Atomic Force Microscopy (AFM) a NT-MDT® NTEGRA AFM and a 0.6 µm SNL-10 BRUKER® cantilever and nitride-based silicone tip were used. For Quartz Crystal Microbalance assays a QCMopen, Novaetech® was used. ADP 20 µM, collagen 20 uM, and epinephrine 100µM were used as platelet activation known agonists.

Results: We observed the highest expression of CD62P and PAC at 60 minutes of stimulation with both proteins. By AFM, platelets´ pseudopod emission and increase in size were observed upon stimulation with the viral proteins. With the QCM assay, a significant increase in the quartz oscillation frequency difference was observed when injecting PRP on quartz coated with the NS1 protein of each of the viruses, in contrast to that observed when injecting PPP and wash buffer.

Conclusion(s): There is platelet reactivity in response to stimulation with DENV and ZIKV NS1 proteins assessed by flow cytometry, atomic force microscopy and QCM. No differences in activation were observed between Zika and Dengue NS1 proteins.