OC 28.1 - Released, but not platelet-associated FXIII-A, is associated with crosslinking activity exposed on the platelet membrane

Sunday, June 25, 2023

14:45 – 15:00 ET

Room: 518

Session Available As: On-Demand

Presenting Author(s)

Yaqiu Sang, PhD

Postdoctoral Research Associate
UNC at Chapel Hill
Chapel Hill, North Carolina, United States

Co-Author(s)

FA

Fahad S.M. Alshehri

Graduate trainee
Aberdeen Cardiovascular & Diabetes Centre, School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen
Aberdeen, Scotland, United Kingdom
Claire S. Whyte, PhD (she/her/hers)

Advanced Research Fellow
Aberdeen Cardiovascular & Diabetes Centre, School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen
Aberdeen, Scotland, United Kingdom
RL

Robert H. H. Lee, PhD

Research Assistant Professor
University of North Carolina at Chapel Hill
Chapel Hill, North Carolina, United States
AB

Abigail Rene R. Ballard

Graduate Research Assistant
Department of Biochemistry and Biophysics, The University of North Carolina
Chapel Hill, North Carolina, United States
Éva Katona, PhD

Associate professor
Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen
Debrecen, Hajdu-Bihar, Hungary
Wolfgang Bergmeier, PhD

Professor
University of North Carolina at Chapel Hill
Chapel Hill, North Carolina, United States
Nicola J. J. Mutch, PhD (she/her/hers)

Professor
Aberdeen Cardiovascular & Diabetes Centre, School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen
Aberdeen, Scotland, United Kingdom
Alisa S. Wolberg, PhD

Professor
Univerisity of North Carolina at Chapel Hill
Chapel Hill, North Carolina, United States

Background: Factor (F)XIII-A accounts for ~3% of platelet proteins and ~50% of total circulating FXIII. Platelet FXIII-A resides in the cytoplasm and is externalized to the surface of platelets and can be released as platelet-derived microvesicles after strong dual-agonist stimulation (convulxin+thrombin [CVX+IIa]), similar to its cytoplasm neighbor, tissue factor pathway inhibitor (TFPI). Platelets also carry F13a1 mRNA. Mechanisms mediating externalization and destiny of platelet FXIII-A are unclear.

Aims: To characterize mechanisms regulating platelet FXIII-A externalization and function

Methods: Washed platelets from healthy humans were activated by IIa, CVX+IIa or A23187, in the absence/presence of inhibitors for mRNA translation (cycloheximide), microtubules (colchicine), calpain (calpeptin), and Rho GTPase-RhoA (rhosin). Platelets and releasate were separated by centrifugation, and FXIII-A and TFPI in these fractions were detected by immunoblotting. FXIII-A antigen (anti-FXIII-A antibody) and crosslinking activity (peptide from α2-antiplasmin N-terminus, A15) exposed on the platelet membrane were detected by flow cytometry.

Results: CVX+IIa stimulation released essentially all TFPI to the supernatant whereas only ~20% of FXIII-A was released. Cycloheximide did not reduce platelet-associated or released FXIII-A. Compared to IIa, A23187 or CVX+IIa stimulation released ~2-fold more FXIII-A to the supernatant. Whereas platelet-associated FXIII-A remained intact, FXIII-A in the releasate of CVX+IIa-, but not A23187-, stimulated platelets was cleaved to FXIII* and then degraded by exogenous plasmin. Inhibitors of microtubules, calpain, or RhoA did not change the percentage of FXIII-A+ platelets, but reduced both the amount of FXIII-A released to the supernatant and the percentage of A15+ platelets.

Conclusion(s): Platelet cytoskeletal rearrangements lead to the release of FXIII-A that can be proteolytically activated to FXIII*. Crosslinking on the platelet surface is associated with FXIII-A* in the supernatant but not FXIII-A on the platelet surface. The different susceptibility of platelet-associated and released FXIII-A to proteolysis suggests platelet-bound FXIII-A is protected and may have unique function during hemostasis and wound healing.

Learning Objectives:

Upon completion, participants will be able to recognize that only a subset of platelet agonists leads to externalization of platelet FXIII-A2.
Upon completion, participants will be able to describe distinct characteristics of platelet-associated and free FXIII-A2.
Upon completion, participants will be able to predict that platelet-associated and free FXIII-A2 have different roles in vivo.