Twofold Symmetry of Human Fibrinogen Proved at the β Chain Distal Domains by Monoclonal−Immunoelectron Microscopy and Image Analysis

Abstract

Using a murine antibody (F7) specific for the C-terminal domain of the beta chain of human fibrinogen combined with electron microscopy and image analysis, we show unequivocally that the epitopes for F7 are at the distal nodules of fibrinogen, equidistant from the center of the molecule and arranged not colinearly with the long axis of the molecule but at opposite sides of it, i.e., following twofold symmetry. Thus, given the monoclonality of the immunochemical probe used and the dimeric nature of the fibrinogen molecule, we can conclude that the distal domains of the two beta chains are arranged in the same manner as these epitopes and, therefore, that the fibrinogen molecule has twofold symmetry. This symmetry pattern found here for F7 is the same as that found recently for the platelet fibrinogen receptor binding sites [Weisel, J. W., Nagaswami, C., Vilaire, G., & Bennett, J. S. (1992) J. Biol. Chem. 23, 16637-16643], located almost certainly at the C-terminal end of the gamma chains, and gives further support to the most accurate model of fibrinogen available so far. We discuss the consequences of this symmetry pattern and of the molecular rigidity of fibrinogen in the actual models of fibrin polymerization and platelet aggregation and adhesion.

A Mutant (Arg327→His) GPIIb Associated to Thrombasthenia Exerts a Dominant Negative Effect in Stably Transfected CHO Cells

“…Platelets were broken by sonication at 4° C and subjected to ultracentrifugation at 100,000 X g for 1 h to separate the soluble and particulate fractions. Competitive enzyme immunoas says were used to determine the platelet content of GPIIb-IIIa and fibrinogen, using pure GPIIb, GPIIIa and fibrinogen as standards and monoclonal anti bodies anti GPIIb (M3), anti GPIIIa (P37), and anti fibrinogen (F2) (38,39). Immunoelectroblotting from whole CHO cell lysates was carried out as described before (38) and the total protein content by the method of Markwell etal.…”

“…Cells were harvested using 0.5 mM EDTA in PBS, washed twice with PBS, resuspended at a density of 1 X 106 cells/100 |xl, and incubated with a mono clonal antibody to either GPIIb (M3) (39), GPIIIa (P37 or P97), or a v (M5) (39) at 4° C for 20 min. At the end of this incubation, cells were washed and resus pended in 50 (jl I of PBS containing an appropriate dilution of FITC-conjugated F(ab')2 fragment of rabbit antimouse immunoglobulin (Dako A/S, Denmark), followed by incubation at 4° C during 20 min.…”

The Structure and Biological Features of Fibrinogen and Fibrin

“…They are comprised of two sets of three polypeptide chains termed Aα, Bβ, and γ,1 which are joined together within its N‐terminal E domain by five symmetrical disulfide bridges, one pair at position Aα 28, two pairs between Aα36 and Bβ65, and a third set between the γ8 and γ9 positions 2–4. Because these γ chain bridges are reciprocal (i.e., γ8 to γ9),5 they orient the chains in an antiparallel manner, and this may contribute to the twofold axis of symmetry perpendicular to the long axis that fibrinogen displays 6–8. Other non‐symmetrical interchain disulfide bridges in this region form a so‐called disulfide ring 2,4.…”

“…[2][3][4] Because these γ chain bridges are reciprocal (i.e., γ 8 to γ 9), 5 they orient the chains in an antiparallel manner, and this may contribute to the twofold axis of symmetry perpendicular to the long axis that fibrinogen displays. [6][7][8] Other non-symmetrical interchain disulfide bridges in this region form a so-called disulfide ring. 2,4 The A α chain consists of 610, the B β chain 461, and the major form of γ chain, γ A, 411 residues.…”