The location of the carboxy-terminal region of gamma chains in fibrinogen and fibrin D domains

Abstract

Elongated fibrinogen molecules are comprised of two outer "D" domains, each connected through a "coiled-coil" region to the central "E" domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of gamma chains in each D domain (the gammaXL site) by incorporating intermolecular epsilon-(gamma-glutamyl)lysine bonds between amine donor gamma406 lysine of one gamma chain and a glutamine acceptor at gamma398 or gamma399 of another. Several lines of evidence show that crosslinked gamma chains extend "transversely" between the strands of each fibril, but other data suggest instead that crosslinked gamma chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the gammaXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled gamma398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal gamma chains in fibrinogen or fibrin are oriented toward the central domain and indicate that gammaXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.

Figure 1

Schematic diagram of the 45 nm long fibrin(ogen) molecule includes information that helps to frame the issue of the location of the C-terminal region of the γ chain. End-to-middle assembly of fibrin molecules to form half-staggered double-stranded fibrils is shown, illustrating the polymerization site interaction (Da:E_A) that governs the assembly process. The crosslinking arrangement of γ_XL sites of γ chains in transverse or end-to-end orientations is shown, as is the C-terminal sequence of the γ chain from γ398 to the C-terminus at γ411. The terminal portion of the γ chain emerges 3.5 nm from the distal end of each molecule (27). The positioning of end-to-end-crosslinked γ chains has been adjusted from the originally proposed location at the extreme ends of the D domain (DD-long; ref. 20) to take into account recent data indicating that their location at the D:D site is not a realistic possibility (1, 21, 25, 27).

Figure 2

A molecular ball-and-stick scale model of the Au₁₁ cadaverine compound linked to the γ chain at Gln-399 through an ɛ-(γ-glutamyl)lysine bond. The organic structure has a contour length of 2.7 nm, and its constituent groups are listed. In the conformation shown, the 0.8 nm diameter Au₁₁ cluster is ≈1.5 nm from the γ Gln side chain and is an extended version that was obtained by using an energy minimization procedure (Chem 3D). A schematic drawing showing the general orientation of the cluster compound along the γ chain is given in the lower portion of the diagram.

Figure 3

A gallery of Au₁₁ cluster-labeled fibrinogen molecules. Occasional monoamino Au₁₁ cluster structures contain two Au₁₁ clusters (e.g., molecules 4 and 12). Arrows indicate Au₁₁ cluster locations. Arrowheads (molecules 1 and 2) indicate a filamentous connection between the Au₁₁ cluster and the D domain. A caricature of each molecule is provided for clarity. (Bar = 20 nm.)

Figure 4

A histogram showing the distribution of Au₁₁ clusters on fibrinogen molecules (A) and an aligned diagram of a fibrinogen half-molecule illustrating the orientation of the C-terminal γ chain in the fibrinogen D domain (B). Gold clusters were distributed in four locations on the molecule: 1, distal D domain; 2, middle D domain; 3, proximal D domain; and 4, E domain. The gold clusters (•) that label the γ chains at γ398Q or γ399Q are designated simply as “398.”

Figure 5

Au₁₁ cluster labeling in fibrin fibrils. Several examples of double-stranded fibrin fibrils labeled with Au₁₁ clusters are shown. A diagram showing the molecular arrangement in each fibril is provided for clarity. (Bar = 20 nm.)