Role of plasma prekallikrein in medical device-induced thrombosis and inflammation

Saturday, October 11, 2025

8:30 AM - 8:35 AM PDT

Location: Room 30B

Presenting Author(s)

Amelia Rodolf

Undergraduate Student
University of California, Los Angeles
Pacific Grove, California, United States

Co-Author(s)

Samantha Moellmer, B.S.

Graduate Student
Oregon Health & Science University
Hillsboro, Oregon, United States
PS

Priyanka Srivastava

Postdoctoral Fellow
Vanderbilt University, United States
AS

Aleksandr Shamanaev

Basic Scientist Assistant 1
Vanderbilt University Medical Center
Nashville, Tennessee, United States
TL

Ting Liu, M.D.

Graduate Student
Oregon Health & Science University, United States
Owen McCarty, PhD

Professor and Chair of Biomedical Engineering
Oregon Health & Science University, Oregon, United States
JS

Joseph J. Shatzel, M.D.

Associate Professor
Oregon Health & Science University, United States
CL

Christina U. Lorentz, Ph.D.

Director of Clinical Operations
Aronora Inc., United States
Karina Nakayama

Associate Professor
Oregon Health & Science University
Portland, Oregon, United States
ET

Erik Tucker, PhD

President & CEO
Aronora Inc., United States
CP

Cristina Puy, Ph.D.

Research Assistant Professor
Oregon Health & Science University, United States
DG

David Gailani, MD

Professor
Vanderbilt University
Nashville, Tennessee, United States

Last Author(s)

Owen McCarty, PhD

Professor and Chair of Biomedical Engineering
Oregon Health & Science University, Oregon, United States

Introduction: : Blood-contacting medical devices are associated with thrombosis, inflammation, and endothelial dysfunction. Plasma prekallikrein (PK)—a zymogen in the contact system—provides a mechanistic link between these pathologic outcomes. In the setting of negatively charged surfaces, PK and coagulation factor (F) XII undergo reciprocal conversion to kallikrein (PKa) and activated FXII (FXIIa), respectively. Activation of FXII triggers the intrinsic pathway of coagulation, a series of reactions between zymogens and activated serine proteases that lead to the formation of a fibrin clot. Concurrently, PKa cleaves high molecular weight kininogen (HK) to generate the proinflammatory peptide bradykinin. This process is termed contact activation. In the absence of an activating surface, prolylcarboxypeptidase (PRCP) on the surface of endothelial cells can activate PK to generate PKa, which can also lead to generation of FXIIa and bradykinin. These surface-dependent and -independent reactions of PK activation and downstream activity suggest PK may be a primary driver of thromboinflammation associated with vascular devices. However, the contributions of the PK apple domains (A1-A4) in these reactions are unclear. We developed a panel of anti-human and anti-mouse PK monoclonal antibodies to investigate the role of the PK heavy chain in the mechanisms of contact activation and bradykinin release. We used these antibodies to study interactions between PK, HK, and endothelial cell PRCP and identify potential therapeutic targets to mitigate the risks of vascular devices.

Materials and

Methods: : PK-deficient mice were immunized with recombinant mouse or human PK. Antibody producing plasma cells were isolated from the spleen and fused with immortalized myeloma cells to generate hybridomas. IgG antibodies were collected from the hybridomas and evaluated for strength of PK binding by ELISA. We screened antibodies with high affinity for anticoagulant properties by activated partial thromboplastin time assay (aPTT) in PK-depleted plasma supplemented with mouse or human PK. We used recombinant PKs with individual apple domains replaced by corresponding domains from the homologous protein FXI to map antibody binding sites.

Using chromogenic substrate assays, we evaluated the effects of each antibody on PK activation, PKa activity, and contact activation initiated by negatively charged ellagic acid in pooled human plasma. To investigate the effects of the antibodies on PK activation by endothelial PRCP, we measured the activity of PK pre-treated with antibodies and incubated with human endothelial cells. We assessed the effect of each antibody on HK cleavage by PKa through SDS-PAGE to identify interactions involved in bradykinin generation.

Results, Conclusions, and Discussions:: We identified and screened 576 anti-human and 192 anti-mouse PK antibodies using binding and clotting time assays. This screening approach yielded 10 anti-human and 14 anti-mouse PK antibodies that exhibited binding and prolonged clotting times in plasma. We further characterized these antibodies using recombinant PKs with individual apple domains replaced by corresponding FXI domains to map each antibody to one of the four non-catalytic apple domains (A1-A4) in PK.

We used a chromogenic bioassay to determine that four antibodies binding the A1, A2, and A3 domains inhibited activation of the contact system in human plasma. One antibody—binding the A3 domain—dramatically decreased PK activation by FXIIa in both human and mouse enzyme systems, suggesting that the PK A3 domain interacts with FXIIa to facilitate PK activation. We investigated the relationship between the contact system and the endothelium through PRCP, a known PK activator expressed by endothelial cells. All of the antibodies selected for screening significantly decreased PK activation by endothelial cells.

We evaluated the effect of each antibody on the kallikrein-kinin system, which involves enzymes of both coagulation and inflammation pathways. Notably, antibodies binding the A1 and A2 domains decreased HK cleavage and bradykinin generation. These results demonstrate that the PK A1 and A2 domains mediate HK cleavage and suggest anti-inflammatory effects of PK inhibition. In addition to releasing bradykinin, HK forms a complex with PK to enhance PK activation by endothelial cell PRCP. Anti-PK antibodies can be used to investigate the mechanism of this interaction, which contributes to inflammation at the endothelial surface.

Our results characterize the role of PK in both coagulation and inflammation and validate anti-PK antibodies as novel tools for investigating pathophysiological mechanisms. The central role of PK in contact activation and HK cleavage presents this enzyme as a promising therapeutic target to prevent thromboinflammatory responses to vascular devices.

Acknowledgements and/or References (Optional): :