Factor XHIa

The Derivative, 5-(biotinamido)pentylamine, contains a 5-carbon cadaverine spacer group attached to the valeric acid side chain of biotin (Thermo Fisher). The compound can be used in a carbodi-imide reaction process to label carboxylate groups in proteins and other molecules, forming amide bond linkages (Chapter 3, Section 1). However, the main use of this biotinylation reagent is in the determination of factor XHIa or transglutaminase enzymes in plasma, cell, or tissue extracts. 

Both intrinsic and extrinsic pathways generate activated factor X. This protease, in turn, catalyses the proteolytic conversion of prothrombin (factor II) into thrombin (Ha). Thrombin, in turn, catalyses the proteolytic conversion of fibrinogen (I) into fibrin (la). Individual fibrin molecules aggregate to form a soft clot. Factor XHIa catalyses the formation of covalent crosslinks between individual fibrin molecules, forming a hard clot (Figures 12.3 and 12.4). 

Fig. 1. Basic scheme of fibrin polymerization and fibrinolysis. The clot is formed on the conversion of fibrinogen to fibrin by cleavage of the fibrinopeptides by thrombin, followed by stabilization of the network with isopeptide bonds by the transglutaminase Factor XHIa. The clot is dissolved through proteolysis by the enzyme plasmin, which is activated on the fibrin surface by plasminogen activators. This process is controlled by several inhibitory reactions (black arrows).

Fibrin polymerization is initiated by the enzymatic cleavage of the fibrinopeptides, converting fibrinogen to fibrin monomer (Fig. 1). Then, several nonenzymatic reactions yield an orderly sequence of macromolec-ular assembly steps. Several other plasma proteins bind specifically to the resulting fibrin network. The clot is stabilized by covalent ligation or crosslinking of specific amino acids by a transglutaminase, Factor XHIa. 

Fig. 8. Formation of isopeptide bond catalyzed by Factor XHIa. The chemical reaction was catalyzed by Factor XHIa, yielding insoluble fibrin crosslinked by Ne-(7 glutamyl) lysine bonds. Factor XIII is activated to Factor XHIa by thrombin in the presence of calcium ions and fibrin.

Fibrin polymers are responsible for the fibrin-dependent enhancement of Factor XIII activation (Greenberg et al, 2003). The mechanism for this effect involves the formation of a tight ternary complex between fibrin, Factor XIII, and thrombin, accompanied by a conformational change of Factor XIII that exposes the active site, after which Factor XHIa remains bound to fibrin. However, the B chains dissociate, which is necessary to expose the active site cysteine of plasma Factor XIII. Platelet Factor XIII without the B chains, is more rapidly activated by thrombin than plasma Factor XIII because of the time that it takes for the B chains to dissociate. 

Many isopeptide bonds can be formed between the side chains of e-lysine (donor) and 7-glutamine (acceptor) residues (Fig. 8). The 7 chains of fibrinogen are crosslinked or ligated first, followed by the C-terminal a chains. In addition, other proteins—notably n2-antiplasmin, plasminogen activator inhibitor-2, and fibronectin—are also covalently ligated to fibrin by Factor XHIa (Greenberg et al, 2003). 

Matsuka, Y. V., Medved, L. V., Migliorini, M. M., and Ingham, K. C. (1996). Factor XHIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen. Biochem. 35, 5810-5816. 

Schense J C, Hubbell J A (1999). Cross-linking exogenous bifunctional peptides into fibrin gels with factor XHIa. Bioconjug. Chem. 10 75-81. 

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