Fibrinogen, Factor XIII and Fibronectin: A Biophysical and Kinetic Characterization of Their Interactions

Authors

Frank Fabian, University of Nebraska - LincolnFollow

First Advisor

William H. Velander

Date of this Version

Summer 6-27-2019

Document Type

Article

Comments

A DISSERTATION Presented to The Graduate College at The University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Chemical and Biomolecular Engineering, Under the SUPERVISION of Professor William H. Velander. Lincoln, Nebraska: July, 2019

Copyright 2019 Frank Marco Fabian Sanabria

Abstract

The development of recombinant-based liquid fibrin tissue sealants having enhanced hemostatic and wound healing properties will involve understanding as yet not well characterized interactions between fibrinogen, fibrin (Fbn) factor XIII, thrombin and fibronectin. We study these phenomena in the context of comparing plasma derived fibrinogen to recombinant fibrinogen (rFI) produced in the milk of transgenic cows. An abundance of purified γγ and γγ’ FI subspecies enables detailed study of γγ or γγ’ biomonomer and their respective Fbn biopolymer formation as having different substrate behaviors of activated plasma derived factor XIII (pFXIIIa₂b₂). High pressure size exclusion (HPSEC) and anion chromatography technique were used along with dynamic light scattering (DLS) and an adapted solid phase peptide incorporation assay.

HPSEC analysis of the purified mixtures rFI with pFXIIIa₂b₂ showed greater pFXIIIa₂b₂ avidity for γγ’rFI than γγrFI. This mixture could also be isolatedby weak anion exchange chromatography. The elution profile resolved both γγ and γγ’rFI and transglutaminase activity showed pFXIIIa₂b₂ activity specifically overlapping the γγ’rFI elution peak. DLS analysis of pFXIIIa₂b₂–γγ’rFI mixture showed a high level of polydispersity index suggesting the complexes of different sizes.

Kinetic studies on the effect of the γγ, γγ’rFI and rFbn in the rate of pFXIIIa₂b₂ activation were postulated to exist in three different stages. This mechanism resembles the proposed stages for in-vivo pFXIIIa₂b₂ activation. In stage I, it was showed that γγ’ rFbn accelerates the rate of thrombin proteolytic removal of the pFXIIIa₂b₂ activation peptide. Stage II indicated that γγ’ rFI and γγ’-rFbn induced the faster pFXIIIa₂b₂ B-subunit dissociation. In the final stage, neither rFI nor rFbn showed an effect on the pFXIIIa₂b₂. This was consistent with the previously reported role of B chains in fibrinogen binding.

The goal of this work focused on studying the existence and reversibility of a complex specifically formed between plasma derived γγ’ fibrinogen (γγ’pFI) and plasma fibronectin (pFN) and postulated to exist in plasma circulation. HPSEC was used to identify and isolate the mixture while DLS was used to analyze the hydrodynamic size that ranged between γγ’pFI and pFN. Reconstitution studies using a degraded α-chain γγ’ fibrinogen showed that the γ’ chain of pFI is needed to initiate complex formation.

Advisor: William H. Velander