PB0889 - Endogenous nitroxyl (HNO) production in a normal biological system and its role in platelet function.

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Roberto G. Pozner, PhD

Researcher
Experimental Medicine Institute. National Academy of Medicine. CONICET.
CABA, Ciudad Autonoma de Buenos Aires, Argentina

Co-Author(s)

YD

Yvan Doctorovich

Fellow
Experimental Medicine Institute. National Academy of Medicine. CONICET.
CABA, Ciudad Autonoma de Buenos Aires, Argentina
MM

Maria Chiara Maccaferro

Fellow
Experimental Medicine Institute. National Academy of Medicine. CONICET.
CABA, Ciudad Autonoma de Buenos Aires, Argentina
SS

Sebastian Suarez

Researcher
INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C.A.B.A. Argentina.
CABA, Ciudad Autonoma de Buenos Aires, Argentina
MM

Marcelo Adrian Marti

Researcher
Depto. de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C.A.B.A. Argentina.
CABA, Ciudad Autonoma de Buenos Aires, Argentina
FD

Fabio Doctorovich

Researcher
INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C.A.B.A. Argentina.
CABA, Ciudad Autonoma de Buenos Aires, Argentina
PI

Paola Ivani

Fellow
Experimental Medicine Institute. National Academy of Medicine. CONICET.
CABA, Ciudad Autonoma de Buenos Aires, Argentina
MS

Mirta Ana Schattner

Researcher
Experimental Medicine Institute. National Academy of Medicine. CONICET.
CABA, Ciudad Autonoma de Buenos Aires, Argentina

Background: Platelet inhibition is the focus of numerous investigations. Although several inhibitors are widely used, HNO (a reduced form of NO) is raising a great clinical interest. HNO shares vasodilatory properties with NO, but also showed increased muscle contractility, acceleration of ventricular relaxation and decrease in cardiac load. These data come from the use of HNO-releasing drugs, however, it is unknown if this gasomediator can be synthesized endogenously by platelets.

Aims: In this study, we evaluated the generation of HNO by platelets (PRP) and its impact on functional responses.

Methods: HNO production was quantified in real-time by two experimental strategies: detection of spectral changes of a porphyrin of Mn(III), and current changes using a high sensitivity selective electrochemical sensor -based on a porphyrin of Co(II)-. The concentration of HNO was estimated from a calibration curve made with Angeli´s salt (Fig.1A).
Fibrinogen binding and P-selectin expression were evaluated by cytofluorimetry.

Results: Stimulation of platelets with classical agonist TRAP, ADP, or arachidonic acid (AA) promoted the generation of HNO (Fig.1B, n=5, p < 0.05).
These results were corroborated by spectrophotometry.
In order to modulate HNO levels, and considering that platelets synthesize NO, we use reducing agents such as ascorbic acid (Asc) or 2-mercaptoethanol. The production of HNO by ADP, TRAP, or AA was significantly higher in the presence of both drugs (Fig.1c).
In addition, it was observed that all the platelet activation parameters evaluated (aggregation, fibrinogen binding, and P-selectin externalization) were significantly inhibited by both HNO-elevating agents, in a concentration-dependent manner.
On the contrary, in the presence of L-Cysteine or Glutation, the TRAP- or ADP-induced HNO production were reduced and platelet function improved.

Conclusion(s): These data constitute the first evidence of HNO production by a biological system under normal conditions and suggest a negative feedback loop for this endogenous gasomediator in platelets.