OC 70.3 - Effect of High-Molecular-Weight Kininogen Binding to Factor XI and Prekallikrein on Thrombosis

Wednesday, June 28, 2023

10:45 – 11:00 ET

Room: 516

Session Available As: Live-Stream and On-Demand

Presenting Author(s)

Bassem M. Mohammed, BPharm, PhD (he/him/his)

Staff Scientist
Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine
St. Louis, Missouri, United States

Co-Author(s)

MS

Mao-fu Sun

Staff Scientist
Department of Pathology, Immunology and Microbiology, Vanderbilt University Medical Center
Nashville, Tennessee, United States
QC

Qiufang Chen

Staff Scientist
Department of Pathology, Immunology and Microbiology, Vanderbilt University Medical Center
Nashville, Tennessee, United States
Priyanka Srivastava, PhD

Postdoctoral fellow
Department of Pathology, Immunology and Microbiology, Vanderbilt University Medical Center
Nashville, Tennessee, United States
SK

Sunil Kumar

Staff Scientist
Department of Pathology, Immunology and Microbiology, Vanderbilt University Medical Center
Nashville, Tennessee, United States
Owen J.T McCarty, PhD, FAHA

Professor and Chair of Biomedical Engineering
Oregon Health & Science University
Portland, Oregon, United States
David Gailani, MD

Professor
Vanderbilt University Medical Center
Nashville, Tennessee, United States

Background: High-molecular-weight kininogen (HK) is a plasma protein that circulates in plasma in non-covalent complexes with the protease zymogens prekallikrein (PK) and factor XI (FXI). HK serves as a cofactor during contact activation, facilitating PK and FXI binding to surfaces. PK or FXI binding to HK is attributed to sites on the HK D6 domain. KNG1 knockout mice lack HK, and are resistant to FeCl3-induced thrombosis.

Aims: Identify residues in the HK D6 domain responsible for binding of HK to PK and FXI, and demonstrate the importance of the HK/PK or HK/FXI interaction in thrombosis.

Methods: Using alanine scanning mutagenesis, a library of 24 HK proteins was expressed in HEK293 cells. Each protein has 3 consecutive amino acids replaced by alanines in the D6 domain covering amino acids 542-613. Purified HKs were tested for binding to PK or FXI (ELISA) and to reconstitute HK-deficient plasma in an aPTT assay. KNG1 knockout mice were reconstituted with human or mouse HK by intravenous infusion or hydrodynamic tail injection and tested in a FeCl3 carotid artery thrombosis model.

Results: PK binding was disrupted by alanine replacement for residues 560-562, 566-574, and 587-589. The binding site for FXI overlapped that of PK, but was more extensive, involving residues 555-577, 587-589 and 597-599. A defect in FXI binding prolonged the aPTT to a greater extent than a defect in PK binding. Murine HK reconstituted carotid artery thrombosis in mice in KNG1 knockout mice in response to FeCl3 injury, while human HK failed. Binding studies demonstrated that human HK has a specific defect in binding to mouse FXI.

Conclusion(s): HK binding to FXI appears to be more important than HK binding to PK for clotting in aPTT assays. The HK-FXI is required for thrombus formation in a mouse carotid artery model.

Learning Objectives:

Identify residues in High molecular weight kininogen involved in its interaction with FXI and prekallikrein.
Evaluate the role of the interaction of high molecular weight kininogen with FXI and prekallikrein for surface initiated clot formation.
Recognize the effects on thrombosis associated with rapid repletion of high molecular weight kininogen (HK) in HK knockout mice.