PB0757 - Divergent functions of canonical integrin activation complex proteins during platelet migration and inflammatory hemostasis

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

MV

Matthew VanderPloeg

Graduate Student
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.
Chapel HIll, North Carolina, United States

Co-Author(s)

MB

Mitchell Butler

Postdoctoral Fellow
UNC Lineberger Comprehensive Cancer Center, UNC-Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599, United States
Chapel Hill, North Carolina, United States
AG

Ansh Gulati

Undergraduate
University of North Carolina at Chapel Hill, Chapel Hill, NC.
Chapel Hill, North Carolina, United States
ED

Elizabeth Davis

Graduate Student
Department of Genetics, University of North Carolina, Chapel Hill, NC 27599, USA.
Chapel Hill, North Carolina, United States
HT

Harrison Truscott

Undergraduate
University of North Carolina at Chapel Hill, Chapel Hill, NC.
Chapel Hill, North Carolina, United States
RL

Robert Lee

Research Assistant Professor
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.
Chapel Hill, North Carolina, United States
JB

James Bear

Professor
UNC Lineberger Comprehensive Cancer Center, UNC-Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599, United States.
Chapel Hill, North Carolina, United States
WB

Wolfgang Bergmeier

Professor
UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.
Chapel Hill, North Carolina, United States

Background: Hemostatic plug formation at sites of mechanical injury requires rapid inside-out activation of platelet integrins by Rap1 and Talin1. In areas of inflammation, single platelets actively migrate to plug holes in the endothelial lining left behind by transmigrating leukocytes. This migration requires sensing of fibrinogen deposition, WAVE-Arp2/3-mediated lamellipodia formation, and outside-in signaling through αIIbβ3 integrins. If and how Rap1 and Talin1 contribute to platelet migration and inflammatory hemostasis is currently unclear.

Aims: Characterize contribution of Rap1 and Talin1 to platelet migration and inflammatory hemostasis.

Methods: Rap1b-mKO (Rap1b deficient in megakaryocyte lineage), Rap1a/b-mKO, Talin1-mKO, and Talin1-R35E,R118E (Talin variant that does not interact with Rap1) mice were used in this study. We developed a TensorFlow convolutional neural network to segment and track ex vivo platelet migration. Super-resolution immunofluorescence microscopy was used to assess cytoskeletal organization, while reverse passive Arthus (rPA) reaction served as inflammatory bleeding model.

Results: Platelet migration was impaired in Talin1-mKO platelets, but contrary to hemostatic plug formation, migration did not require Rap1-Talin1 interaction. Surprisingly, Rap1-deficient platelets migrated up to 30% faster than controls. However, these platelets failed to polarize and retract their trailing edge. Further revealed that Rap1 may facilitate leading edge vs. trailing edge polarity through WAVE2-complex localization. Control platelets reveal greater WAVE2 enrichment at the leading edge vs. trailing edge, while WAVE2 was present around the entire periphery of Rap1-mKO platelets. Consistent with their migratory behavior, an rPA reaction showed impaired vascular intregrity in Talin1-mKO but not Rap1a/b-mKO mice.

Conclusion(s): In summary, our findings indicate that 1) Talin1 operates independently of Rap1 to enable platelet migration, and 2) Rap1 signaling regulates migrating platelet polarity in part by coordinating WAVE2-mediated lamellipodia formation and trailing edge retraction. Ongoing studies seek to clarify whether crosstalk between Rap1 and Rho-family GTPases is critical for polarity and cytoskeletal organization in migrating platelets.