Shadow-cast electron microscopy of fibrinogen with antibody fragments bound to specific regions.

Abstract

Specimens of human fibrinogen mixed with Fab fragments of antibodies that were specific for various portions of the fibrinogen molecule were tungsten shadow-cast and examined by electron microscopy. Typical trinodular fibrinogen molecules were observed when Fab fragments were omitted or when fragments from nonimmune sera were used. In the experimental fibrinogen-Fab preparations, a significant number of molecules were found with an extra nodule. In the case of Fab fragments from antibodies directed to fragment E, the additional nodule was attached to the central sphere of the fibrinogen molecule. Simi A in length. The model they developed on the basis oftheir micrographs consisted oftwo terminal spheres, each about 60 A in diameter, connected to a slightly smaller central sphere, the diameter ofwhich was estimated to be 50 A. The connections between the spheres were unresolvable and were assumed to be rods less than 15 A thick. In the ensuing period, electron microscopy changed rapidly, and shadow-casting procedures gave rise to negative-staining techniques. The latter led to a number ofreports indicatingthat fibrinogen was not a trinodular rod but was in fact-a large bloated or hollow ball (2-4). Indeed, the physicochemical properties of the molecule could be interpreted in different ways; primarily because of the difficulty in distinguishing asymmetry from hydration, and either of these extreme models could be accommodated satisfactorily. Subsequently, a series ofreports appeared that purported to show that the trinodular structure ofHall and Slayter was an artifact ofthe procedure used (5, 6).On the other hand, a considerable amount of biochemical support for the Hall and Slayter model has accumulated over the years, beginning withthe nature ofthe core fragments produced by limited proteolysis (7,8) and continuing with the actual kinetics of generation of those fragments, which included asymmetric intermediates of the sort that would be expected fromThe publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.cleavages between the spheres (9). A number of other experiments also provided additional, although less direct, support for a triglobular model (10-13).Recently, shadow-casting procedures have been revived for the study of fibrinogen, and once again trinodular structures of the sort shown by Hall and Slayter have been viewed (14,15). Furthermore, Fowler and Erickson (15) were even able to show a trinodular structure by negative-staining techniques, and Estis and Haschemeyer (16) observed a similar structure in unstained preparations. Moreover, a plethora of detailed biochemical data exist, all of which, including the complete amino acid sequence of the molecule and the exact arrangement of disulfide bonds that bridge the six subunit chains (17)(18)(19)(20)(21)(22), are consistent with the Hall and Slayter model. The picture that has emerged is a po...

Natively unfolded regions of the vertebrate fibrinogen molecule

“…There have been conflicting reports over the years about the whereabouts of the αC domain as determined by various electron microscope approaches, some of which contend that the αC domains are associated with the central domain,32, 33 and others, mostly utilizing antibodies (or FAB fragments) directed against peptides in the αC region, which find the immuno‐reactive material in varied and more distal locations 34, 35. In the latter experiments, only the antibodies (or FAB fragments), and not the αC regions themselves, appeared to be shadowed.…”

Fibrinogen Seattle Releases Half the Normal Amount of Fibrinopeptide B

“…Over 70 heritable dysfibrinogens have been discovered [21 ] since the initial report by di Imperato and Dettori [26], The char acterization and categorization of the be havioral aberrations in these qualitatively abnormal fibrinogens assume an added significance now that more of the struc ture of the normal human molecule is known [7,18,30,38,43,44],…”

Surface Markers of Fibrinogen and Its Physiologic Derivatives Revealed by Antibody Probes

“…2The two carboxyterminal D domains are represented by the D fragment derived by plasmic cleavage [21,75,98,102,103,109] with the most representational frag ment derived by cleavage in the presence of calcium [98]. The remaining two domains are the Aα 48,000 domains, which represent the carboxyterminal twothirds of the Aα chain [52,55,130], separate from the domainal confines of the D domain [18,25,120]. Physicochemical studies of thermal denaturation of fibrinogen by Donovan and Mihalyi [24] clearly de lineate the existence of two major domains pos sessing ordered structure, i.e., the E and D domains.…”

“…5. If the trinodular model represents one of the conformations of fibrinogen and the postulated coiled coils do indeed exist and serve to connect the central E domain to the lateral D domains, there Norton and Slayter, 1981Price et al, 1981Gollwitzer et al, 1975Nossel et al, 1974Nossel et al, 1974Blombäck et al, 1976Harfenist et al, 1975Telford et al, 1980Price et al, 1981Sobel et al, 1981Sobel et al, 1981Cierniewski et al, 1977 must be a profound "end-to-end" interaction of the proximal to the distal coiled coils if conformed as schematically depicted in Figure 7.…”

“…Among the most strongly supported static models at present is the trinodular model. This model, first described by Hall and Slayter [50], has been ob served by electron microscopy of surface-associated fibrinogen by other workers as well [36,37,91,120,134]. It must be considered that this conformation may be influenced, selected or imparted by contact with the surface used in preparation for ultrastructural analysis.…”