PB0853 - The potential of pre-activated platelets to contribute to thrombus formation

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Ilaria de simone, MA

PhD
Department of Biochemistry, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands
Maastricht, Limburg, Netherlands

Co-Author(s)

Constance Baaten, PhD

postdoctoral researcher
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
Maastricht, Limburg, Netherlands
JG

Jonathan M. Gibbins, PhD

Professor
Institute for Cardiovascular and Metabolic Research, School of Biological sciences, University of Reading, Reading, UK
Reading, England, United Kingdom
Hugo ten Cate, MD, PhD, FAHA

Professor, MD, PhD
Thrombosis Expertise Center, Heart & Vascular Center, Maastricht University Medical Center, Maastricht, the Netherlands
Maastricht, Limburg, Netherlands
JW

Johan W. M. Heemskerk

Emeritus Professor
Synapse research insitute
MAASTRICHT, Limburg, Netherlands
CJ

Chris Jones

Associate Professor
Reading School of Biological Sciences, University of Reading, Whiteknights, Reading, United Kingdom
Reading, England, United Kingdom
Paola van der Meijden, PhD

Assistant Professor
Department of Biochemistry, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands
Maastricht, Limburg, Netherlands

Background: Platelet activation results in a conformational change of integrin αIIbβ3, which regulates the adhesiveness of platelets to fibrinogen and VWF and is necessary for thrombus formation. Prior integrin activation can be reversed. We hypothesize that platelet activation with different agonists results in diverse integrin (in)activating effects over time, thereby affecting the participation of pre-activated platelets in thrombus formation.

Aims: To study short-and long-term platelet responses upon GPVI and PAR stimulation and to investigate whether pre-activated platelets contribute to thrombus formation.

Methods: Short-and long-term effects of GPVI and PAR stimulation were evaluated via flow cytometry (PAC-1 and fibrinogen binding) and plate-based aggregation. Pre-activated washed platelets were added to washed red blood cells and fibrinogen and were perfused over collagen type I or fibrinogen surfaces, to assess the role of pre-activated platelets in thrombus formation.

Results: After 30 minutes of PAR stimulation, with thrombin or TRAP6, platelets showed 30-50% less PAC-1 (p < 0.01) and fibrinogen binding (p < 0.05) than after 10 minutes. The ability of platelets to aggregate also decreased after 30 minutes of PAR stimulation compared to immediately after agonist addition (p < 0.05). Contrarily, neither PAC-1 and fibrinogen binding nor aggregation were diminished after 30 minutes of GPVI stimulation with CRP-XL. Compared to unstimulated platelets, short- or long-term pre-activation of platelets (0 or 30 minutes, respectively) with CRP-XL resulted in the formation of larger heterogeneous thrombi on collagen and fibrinogen surfaces. Large heterogeneous thrombi were also formed when platelets were pre-activated short-term with TRAP6. However, after longer incubation with TRAP6, the formed homogeneous thrombi on collagen and fibrinogen resembled those of control, unstimulated platelets.

Conclusion(s): The long-term integrin αIIbβ3 activation by prior platelet triggering with GPVI agonists is accompanied by a more prolonged response and enhanced thrombus formation in comparison to PAR triggering. When integrin αIIbβ3 reverses, platelets retain their normal aggregation pattern under flow.