PB0752 - Platelet peptidylarginine deiminase 4 is not essential for platelet activation or development of cardiac fibrosis in mice

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Katrien Bomhals (she/her/hers)

PhD student
Center of Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Diest, Vlaams-Brabant, Belgium

Co-Author(s)

SK

Sirima Kraisin

Post-doctoral researcher
Center for Molecular and Vascular Biology - KU Leuven
Leuven, Vlaams-Brabant, Belgium
VS

Valérie Spalart

PhD Student
Center for Molecular and Vascular Biology - KU Leuven
Leuven, Vlaams-Brabant, Belgium
SM

Severien Meyers

Researcher
Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
MS

Mathias Stroobants

Lab Technician
Center of Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium
LF

Liesbeth Frederix

Lab technician
Center for Molecular and Vascular Biology
Leuven, Vlaams-Brabant, Belgium
Kimberly Martinod, PhD

Assistant Professor
Center of Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium
Leuven, Vlaams-Brabant, Belgium

Background: Excessive release of pro-inflammatory and cytotoxic neutrophil extracellular traps (NETs) in the heart can promote an environment for detrimental fibrotic remodeling. Activated platelets play a key role in this process. Systemic deficiency of peptidylarginine deiminase 4 (PAD4), the enzyme which catalyzes the initial step of NET formation, protects mice against age-related cardiac fibrosis and functional decline. Although expression of the Padi4 gene has been identified in human platelets, the consequence of PAD4 on platelet function is unknown.

Aims: To investigate the impact of platelet PAD4 on platelet activation and development of age-related cardiac fibrosis.

Methods: We bred platelet/megakaryocyte-selective PAD4 knockout mice (PAD4fl/flPF4Cre+) and isolated their platelets for ex vivo functional testing. Light transmission aggregometry was performed after stimulation with various agonists (i.e. ADP, U46619, collagen, thrombin). Additionally, platelet activation was quantitatively assessed by flow cytometry, with markers selected to represent hallmarks of platelet activation: P-selectin, and TGF-β for alpha-granule release, CD63 for dense-granule release, and JON/A for the open conformation of αIIbβIII. Spontaneous cardiac fibrosis was assessed in mice aged to at least 18 months, with collagen content quantified by Sirius Red/Fast Green staining.

Results: PAD4-deficiency did not alter ex vivo platelet activation and aggregation. No significant difference in hypertrophy or fibrotic response was observed when comparing aged platelet-PAD4 knockouts to wild-type littermate controls. Accordingly, collagen content was increased in old compared to young (5-15 weeks) PAD4fl/flPF4Cre+ animals, and was similar to the PAD4fl/fl littermates. Platelet-PAD4 deficiency thus did not prevent cardiac fibrosis development with age.

Conclusion(s): These data suggest that deletion of platelet/megakaryocyte PAD4 is insufficient to phenocopy the cardioprotective phenotype seen in systemic PAD4-deficient mice. With no major side effects observed on platelet function, PAD4 inhibition to minimize NET formation in disease, is unlikely to affect platelets negatively and remains a viable option.