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Cardiovascular Engineering

Cardiovascular Engineering - Poster Session A

Poster H11 - Crosstalk between the contact activation system of coagulation and inflammation: development and use of novel tools to study the role of plasma prekallikrein

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Coagulation factor (F) XII of the contact system is activated by negatively charged surfaces to form FXIIa, promoting thrombogenesis in response to foreign surfaces such as blood-contacting medical devices and bacterial infections. An acute inflammatory response is typically associated with use of vascular devices and infections, suggesting an associative or causative mechanistic link between the coagulation and inflammation cascades. We hypothesize that a member of the contact system, plasma prekallikrein (PK), plays a key role in regulating the crosstalk between inflammation and thrombogenesis. As a zymogen, PK is known to be activated by FXIIa to form kallikrein, which subsequently promotes FXII activation. During inflammation, kallikrein is known to cleave high molecular weight kininogen (HK) to release the vasoactive peptide bradykinin. To test the hypothesis that PK links FXII activation with bradykinin release, we developed and validated a novel panel of anti-human and anti-mouse PK monoclonal antibodies.

Materials and

Methods:  Antibody development and validation: PK-deficient Balb-C 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.

Bioassays for functional studies: Using chromogenic substrate assays, we evaluated the effects of each antibody on kallikrein catalytic activity, PK activation by FXIIa, and FXII activation by kallikrein. We similarly evaluated the effects of the antibodies on contact activation initiated by negatively charged ellagic acid in pooled human plasma. We measured activation of PK pre-treated with antibodies and incubated with human umbilical vein endothelial cells to investigate the effects of the antibodies on PK activation by endothelial prolylcarboxypeptidase (PRCP). Lastly, we assessed the effect of the antibodies on HK cleavage by kallikrein through SDS-PAGE.

Results, Conclusions, and Discussions: We first focused on identifying and screening antibodies generated against prekallikrein. 576 anti-human and 192 anti-mouse PK antibodies were generated. We screened this antibody panel using a binding assay followed by a clotting time assay. With this screening approach, we identified 10 anti-human and 14 anti-mouse PK antibodies that exhibited binding and prolonged clotting times in plasma. We then used a chromogenic bioassay to quantify the effects of these antibodies on kallikrein activity. We found that the antibodies 4H11, 5H1, 3B8, and 6B9 increased kallikrein activity. We then further assessed whether these antibodies inhibited PK activation by FXIIa. We found that one antibody, the anti-mouse PK antibody 4H11, dramatically decreased PK activation by FXIIa in both human and mouse enzyme systems.

We extended our study to determine whether these antibodies prevented activation of the contact system by negatively charged surfaces, which initiate coagulation associated with vascular devices and infections. We modeled these surfaces using ellagic acid in human plasma. In this assay, we found that 6B9, 3B8, 4H11, and 6G10 blocked amplification of the contact system in the presence of a negatively charged surface. 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 further evaluated the effect of each antibody on the kallikrein-kinin system, which involves enzymes of both coagulation and inflammation pathways. Notably, the anti-human antibody 6B9 decreased HK cleavage and bradykinin generation, suggesting anti-inflammatory effects of PK inhibition.

Our results suggest that PK plays a key role in mediating crosstalk between coagulation and inflammation and validate anti-PK antibodies as novel tools for investigating the connections between these systems. The critical role of PK presents this enzyme as a promising target for the treatment of vascular and inflammatory diseases.

Acknowledgements (Optional):