PB0580 - Microvesicles from Stored Platelets Promote High Molecular Weight Kininogen Cleavage Leading to Bradykinin Generation.

Monday, July 11, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Co-Author(s)

DN

DENIS F. NOUBOUOSSIE

Fellow
University of North Carolina Blood Research Center, Chapel Hill, NC, USA.
Chapel Hill, North Carolina, United States
MH

Michael W. Henderson

Postdoctorate Associate
University of North Carolina Blood Research Center, Chapel Hill, NC, USA.
Chapel Hill, North Carolina, United States
Anton Ilich, MD

Research Associate
Division of Hematology and Blood Research Center, University of North Carolina, Chapel Hill
Chapel Hill, North Carolina, United States
PE

Patrick Ellsworth

Assistant Professor
University of North Carolina Blood Research Center, Chapel Hill, NC, USA.
Chapel Hill, North Carolina, United States
MK

Matthew Karafin

Associate Professor
University of North Carolina Blood Research Center, Chapel Hill, NC, USA.
Chapel Hill, North Carolina, United States
RP

Rafal Pawlinski, MD PhD

Professor of Medicine
Division of Hematology and Blood Research Center, University of North Carolina, Chapel Hill
Chapel Hill, North Carolina, United States
MH

Maureane Hoffman

Professor
Veteran Affairs Medical Center, Durham, NC, USA.
Durham, North Carolina, United States
SS

Sidney Strickland

Professor
Patricia and John Rosenwald Laboratory of Neurobiology and Genetics, The Rockefeller University, New York, NY, USA.
New York City, New York, United States
DM

Dougald Monroe

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

Presenting Author(s)

NK

Nigel S. Key

Harold R. Roberts Distinguished Professor
Division of Hematology, University of North Carolina, Chapel Hill, NC, USA
Chapel Hill, North Carolina, United States

Background: The contact system (CS) links coagulation and inflammation. We previously demonstrated that microvesicles isolated from stored platelet apheresis units (sPLT-MVs) activate the CS leading to thrombin generation.

Aims: To investigate if sPLT-MVs also promote high molecular weight kininogen (HK) cleavage and bradykinin generation in a purified system.

Methods: sPLT-MVs were isolated from expired apheresis platelet units by centrifugation, washed twice, and re-suspended in Hepes-buffered saline in the presence of physiologic plasma concentrations of HK and/or prekallikrein (PK). HK cleavage and bradykinin generation were measured after 0.5, 1- or 2-hours incubation at 37⁰C. Cleavage of HK was assessed by a sandwich ELISA measuring residual intact HK (HKi). Bradykinin generation was quantified using a commercial ELISA. In some experiments, lisinopril was added to inhibit angiotensin converting enzyme, while avoralstat was added to block kallikrein (PKa).

Results: Following incubation of sPLT-MVs with HK, the HKi concentration gradually decreased, whereas bradykinin levels increased, suggesting that sPLT-MVs generate bradykinin from HK cleavage, independently of PKa, since these effects were not abolished by avoralstat (Fig 1A&B). However, the addition of PK further increased HK cleavage and bradykinin generation, both of which were inhibited by avoralstat (Fig 1C&D), suggesting the contribution of PKa formed by the activation of PK by sPLT-MVs. Separately, the concentration of exogenously added bradykinin decreased following incubation with sPLT-MVs, which was inhibited by the presence of lisinopril (Fig 1E), indicating the presence of angiotensin-converting enzyme activity associated with sPLT-MVs.

Conclusion(s): sPLT-MVs cleave HK to generate bradykinin, although they also degrade bradykinin at a slower rate. sPLT-MV-mediated bradykinin formation may result from a] direct HK cleavage by sPLT-MVs; and b] an indirect mechanism in which sPLT-MVs activate PK to PKa that subsequently cleaves HK to generate bradykinin. The relevance of these mechanisms in plasma and in vivo following platelet transfusion warrants further studies.