William H. Velander
Date of this Version
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