PB0876 - Different agonists stimulate a unique signature of platelet surface markers dependent on aspirin status as determined by platelet mass cytometry

Monday, June 26, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Melissa V. Chan, PhD

Post-Doctoral Fellow
Population Sciences Branch, Division of Intramural Research, National Heart, Lung and Blood Institute, Framingham, MA, USA.
Framingham, Massachusetts, United States

Co-Author(s)

BS

Benjamin Spurgeon

Research Fellow
Center for Platelet Research Studies, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, Massachusetts
Boston, Massachusetts, United States
BN

Bongani B. Nkambule

Associate Professor
University of KwaZulu-Natal
Durban, KwaZulu-Natal, South Africa
AL

Amber Lachapelle

Research Associate
Population Sciences Branch, Division of Intramural Research, National Heart, Lung and Blood Institute, Framingham, MA, USA.
Framingham, Massachusetts, United States
JG

Joseph Grech

Post-Baccalaureate Fellow
Population Sciences Branch, Division of Intramural Research, National Heart, Lung and Blood Institute, Framingham, MA, USA.
Framingham, Massachusetts, United States
AF

Andrew L. Frelinger, III

Assistant Professor
Center for Platelet Research Studies, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, Massachusetts
Boston, Massachusetts, United States
Andrew D. D. Johnson, PhD (he/him/his)

Professor
Population Sciences Branch, Division of Intramural Research, National Heart, Lung and Blood Institute, Framingham, MA, USA.
Framingham, Massachusetts, United States

Background: Flow cytometry is tremendously useful in identifying markers on platelets. However, it is limited in the number of parameters that can be simultaneously analyzed, since adding fluorophores requires complex compensation for analysis. Utilizing mass cytometry (CyTOF), a novel platelet-specific antibody panel of 16 specific surface antigens was developed to create a comprehensive picture of the platelet immunophenotype following agonist stimulation.

Aims: Our aim was to compare platelet surface marker expression by CyTOF with “gold standard” light transmission aggregometry (LTA).

Methods: Blood from age and sex-matched Framingham Heart Study participants (N=46) was collected and platelet-rich plasma (PRP) was isolated (200xg, 10 min). Whole blood was incubated with antibody cocktail and agonist (ADP, PAR-1 activating peptide (AP), PAR-4AP, CRP-XL or arachidonic acid (AA) and a fix/lyse solution was added. Samples were kept at 4°C until washed twice and examined on a CyTOF instrument. Data were analyzed by FlowSOM and FlowJo and output as mean metal intensities. In parallel, LTA was performed on PRP. Aspirin-use was defined as final aggregation to AA in LTA < 40% and Spearman’s rank correlation was performed.

Results: In the non-aspirin group, ADP stimulation in LTA was moderately positively correlated with CyTOF CD154 (ρ=0.418) and PAC-1 (ρ=0.408) expression. In addition, PAR-4AP stimulation in LTA was positively correlated with CD42a (ρ=0.461) and PAR-1AP-LTA with CD107a (ρ=0.507) expression. Following aspirin, AA stimulation in LTA was negatively correlated with CyTOF HLA-A (ρ=-0.559) and GPVI (ρ=-0.441) expression. Furthermore, there was negative correlation in CD63 (ρ=-0.419) following collagen/CRP-XL, CD42a (ρ=-0.444) and CD29 (ρ=-0.444) after ADP and CD31 (ρ=-0.656) following PAR-1AP stimulation. Conversely, PAR-4AP stimulated LTA was positively correlated CD36 (ρ=0.480) and GPVI (ρ=0.565) expression.

Conclusion(s): Each agonist stimulates a unique signature of expression markers which is dependent on aspirin-use, with aspirin generally correlating negatively with the expression of markers. However, this is not strictly correlated with LTA.