Hemostasis (Blood Coagulation / Blood Clotting) is a complex series of cell-cell, cell-protein, and protein-protein interactions designed to stem blood loss. Fibrinogen is a tridomainal disulfide linked plasma protein comprised of two symmetrical halves. Each half is composed of three polypeptide chains termed Aα (red), Bβ (blue), and γ (green). The inner E domain (orange) contains the amino termini of the six polypeptide chains disulfide linked together. Outer D domains are composed of two subdomains, one formed by the Bβ chain and one formed by the γ chain, as well as a region of the Aα chain stabilized by disulfide bonds. The domains are separated / connected by a collagen like coiled-coil region. Aα chains enter the D domain, make a 180° turn, exit the D domains, and the carboxyl terminal regions form a fourth domain that interacts with the E domain in fibrinogen.
In the circulation human fibrinogen exists in two major forms, fibrinogen 1 (‘peak 1 fibrinogen’) and fibrinogen 2 (‘peak 2 fibrinogen’). The two fibrinogens can be separated from each other by ion exchange chromatography. They differ from each other with respect to the composition of their γ chains. Fibrinogen 1 contains two γA chains which are comprised of 411 amino acids. Heterodimeric fibrinogen 2 molecules contain one γA and one γ´ chain. The variant γ´ chain is longer (427 residues), and has a more anionic (acidic), carboxyl terminal sequence than the γA chain beyond position 408. For many years the physiological function of the γ´ chain was unknown. Recently factor XIII (protransglutaminase) has been shown to bind specifically to γ´ chains in fibrinogen 2 and thrombin has been shown to bind to the anionic γ´ extension of fibrin 2.
After a cascading series of enzyme activations the plasma protein fibrinogen is converted to fibrin by the thrombin mediated removal of four peptides. Release of two fibrinopeptide A fragments from the amino terminus of fibrinogen Aα chains exposes a polymerization site, termed ‘A’, in the central E domain, and initiates fibrin fibril assembly via non-covalent pairing of these ‘A’ sites with existing complementary sites, termed ‘a’, in the outer D domains of fibrin. The process of end-to-middle molecular pairing causes staggered overlapping of fibrin molecules, and leads to formation of linear double-stranded fibrin fibrils with intermolecular end-to-end alignment of fibrin D domains.
Release of fibrinopeptide B from the amino terminus of the Bβ chain occurs more slowly that fibrinopeptide A. Fibrinopeptide B release exposes a polymerization site, termed ‘B’, in the central E domain. This site interacts non-covalently with existing complementary sites, termed ‘b’, in the outer D domains of fibrin. The ‘B’ to ‘b’ interaction facilitates lateral association of linear double-stranded fibrin fibrils and the formation of the three dimensional fibrin matrix.
Plasma factor XIII (protransglutaminase) is a non–covalent tetrameric zymogen complex composed of two pairs of polypeptide chains termed A and B respectively. Cleavage at position 37 of the A subunits by thrombin leads to formation of the active enzyme, factor XIIIa (transglutaminase), which requires Ca2+ for dissociation of the activated A subunits (A*2) from B subunits and for full expression of its catalytic activity. Activation of factor XIII and the activity of factor XIIIa is promoted in the presence of fibrinogen and perhaps more specifically, by fibrin. Inhibition of fibrin polymerization eliminates the enhancing effect of fibrin on activation of factor XIII, suggesting that the fibrin effect is mediated through formation of a ternary complex among thrombin, fibrin, and factor XIII. In the presence of factor XIIIa, non-covalent fibrin assembly is accompanied by intermolecular covalent crosslinking, in which ε-amino-(γ-glutamyl) lysine isopeptide bonds are introduced between appropriately positioned donor lysine and acceptor glutamine residues. Crosslinking occurs initially between γ chains to form γ dimers, concomitantly, albeit at a slower rate, among α chains, and to a minor extent between α and γ chains. Continued factor XIIIa activity among the existing g dimer population results in slow progressive evolution of γ chain multimers. Positioning of D domains in the fibrin polymer results in an arrangement of γ chain pairs that facilitates intermolecular covalent crosslinking; resulting in reciprocal ε-amino-(γ-glutamyl) lysine isopeptide bonds between paired donor–acceptor sites in the carboxy-terminal regions of γ chains.
Serendipitously it was discovered that factor XIII cross-links fibrin(ogen) without thrombin activation to factor XIIIa. By mistake a sample of fibrinogen 2 was dialyzed overnight against a buffer that contained PMSF and calcium ions. The following morning it appeared that the sample had clotted suggesting that the PMSF had not inhibited the residual thrombin activity. More careful analysis indicated that the fibrinopeptides were still attached to the fibrinogen, but the γ chains had formed dimers and some of the Aα had formed polymers. Systematic studies subsequently demonstrated that factor XIII, the “inactive” zymogen, could cross-link fibrinogen or fibrin when incubated in the presence of physiological Ca2+ concentrations. A small amount of sulfhydral reagent, dithiolthreitol or mercaptoethanol, increased the rate of the cross-linking reaction.
The interactions between fibrin(ogen) and factor XIII are incompletely understood. To this end I am actively investigating how factor XIII is carried in the plasma by fibrinogen 2, the activation of factor XIII to factor XIIIa by thrombin, the movement of the active enzyme through the fibrin matrix, the factor XIIIa mediated formation of high molecular weight crosslinked fibrin products (γ tetramers & γ trimers), the effects of γ tetramers and γ trimers on the properties of the fibrin matrix, and the intrinsic transglutaminase activity of factor XIII when in the zymogen form. An in vitro fibrinogen and factor XIII expression system is in the process of being established. Using these systems and site directed mutagenesis to introduce specific changes into the proteins the interactions between these molecules will be probed. Studies designed to elucidate the interactions between fibrin(ogen)/fibrin(ogen) and thrombin/fibrin will also use the in vitro fibrinogen expression system. The characterization of clinically relevant abnormal fibrinogens at a molecular level is an active area of investigation. These latter studies are being performed in collaboration with the Fibrinogen Research Laboratory (Dr. M.W. Mosesson) at the Blood Research Institute of The Blood Center of Southeastern Wisconsin.
Siebenlist KR, and Mosesson MW. PROGRESSIVE CROSSLINKING OF FIBRIN γ CHAINS INCREASES RESISTANCE TO FIBRINOLYSIS (1994) J Biol Chem 269, 28414-28419.
Siebenlist KR, Meh DA, Wall JS, Hainfeld JF, and Mosesson MW. ORIENTATION OF THE CARBOXY-TERMINAL REGIONS OF FIBRIN γ CHAIN DIMERS DETERMINED FROM THE CROSSLINKED PRODUCTS FORMED IN MIXTURES OF FIBRIN, FRAGMENT D, AND FACTOR XIIIa. (1995) Thromb Haemostas 74, 1113-1119.
Siebenlist KR, Meh DA, and Mosesson MW. PLASMA FACTOR XIII BINDS SPECIFICALLY TO FIBRINOGEN MOLECULES CONTAINING γ' CHAINS. (1996) Biochemistry 35, 10448-10453.
Meh DA, Siebenlist KR, and Mosesson MW. IDENTIFICATION AND CHARACTERIZATION OF THE THROMBIN BINDING SITES ON FIBRIN. (1996) J. Biol. Chem. 271, 23121-23125.
Siebenlist KR, Meh DA, and Mosesson MW. POSITION OF γ CHAIN CARBOXY-TERMINAL REGIONS IN FIBRINOGEN:FIBRIN CROSSLINKING MIXTURES. (2000) Biochemistry 39, 14171-14175.
Mosesson MW, Siebenlist KR, and Meh DA. THE STRUCTURE AND BIOLOGICAL FEATURES OF FIBRINOGEN AND FIBRIN. (2001) Ann N.Y. Acad Sci 936, 11-30.
Siebenlist KR, Meh DA, and Mosesson MW. PROTRANSGLUTAMINASE (FACTOR XIII) MEDIATED CROSSLINKING OF FIBRINOGEN AND FIBRIN. (2001) Thromb Haemostas 86, 1221-1228.
Meh DA, Mosesson MW, DiOrio JP, Siebenlist KR, Hernandez I, Amrani DL, and Stojanovich L. DISINTEGRATION AND REORGANIZATION OF FIBRIN NETWORKS DURING TPA-INDUCED CLOT LYSIS. (2001) Blood Coag Fibrinol 12, 627-637.
Mosesson MW, Siebenlist KR, Hernandez I, Wall JS, and Hainfeld JF. FIBRIN ASSEMBLY AND CROSSLINKING ON A FIBRIN FRAGMENT E TEMPLATE. (2002) Thromb Haemostas 87, 651-658.
Siebenlist KR, Mosesson MW, Hernandez I, Bush LA, DiCera E, Shainoff JR, DiOrio JP, and Stojanovic L. STUDIES ON THE BASIS FOR THE PROPERTIES OF FIBRIN PRODUCED FROM FIBRINOGEN-CONTAINING GAMMA' CHAINS. (2005) Blood 106, 2730-2736.
Brennan SO, Mosesson MW, Lowen R, Siebenlist KR, Matsunaga A. HYPOFIBRINOGENAEMIA RESULTING FROM NOVEL SINGLE NUCLEOTIDE DELETION AT CODON Bbeta58 (3404del A) ASSOCIATED WITH THROMBOTIC STROKE IN INFANCY. (2006) Thromb Haemost. 95, 738-739.
Mosesson MW, Siebenlist KR, Hernandez I, Lee KN, Christiansen VJ, McKee PA. EVIDENCE THAT α2-ANTIPLASMIN BECOMES COVALENTLY LIGATED TO PLASMA FIBRINOGEN IN THE CIRCULATION: A NEW ROLE FOR PLASMA FACTOR XIII IN FIBRINOLYSIS REGULATION. (2008) Thromb Haemost (Submitted)
Siebenlist KR, Behm RA, Mosesson MW, Ariens R. DIFFERENTIAL CROSS-LINKING ACTIVITY OF THE VAL34 AND LEU34 FACTOR XIII VARIANTS. (In Preparation)
