Immune labeling of the D and E regions of human fibrinogen by electron microscopy.

Abstract

Human fibrinogen was digested with trypsin to yield core fragments D and E, and antibodies were made against the isolated fragments. The Fab' fragments derived from these antibodies were mixed with native fibrinogen, resulting in the formation of soluble immune complexes. These were rotary shadowed with platinum or negatively contrasted with uranyl acetate and examined by electron microscopy. Fab' from anti-D immunoglobulin was found to be attached to the outer nodules of fibrinogen with a frequency of 79% prior to affinity purification and 91% afterward. Fab' from anti-E immunoglobulin attached to the central nodule with a frequency of 78% prior to affinity purification and 82% afterward. The evidence clearly identifies fragment D produced by plasmin or trypsin digestion of fibrinogen with the outer nodules and the single fragment E, with the central nodule.The generally accepted view of fibrinogen structure is the trinodular model (1). This model is supported by results from a number of laboratories (2-6). Fibrinogen is composed of three pairs (Aa, BJ3, and y) of polypeptide chains of known sequence. The primary structure is consistent with the model of three globular domains joined by largely helical regions (7), the lengths of which are very close to those suggested by the trinodular model.Fibrinogen is cleaved by plasmin or trypsin to yield characteristic "core fragments". Marder et aL (8) proposed a scheme, which is widely accepted, for the proteolysis of fibrinogen to core fragments D and E via high molecular weight intermediates X and Y; their results indicated that two D fragments and one E fragment are generated from each fibrinogen molecule. D and E fragments have been demonstrated to be immunologically distinct (9) and to differ in molecular weight and in physicochemical characteristics (10). However, D and E fragments tend to associate noncovalently (11), which has increased the difficulty of completely separating the two fragments.It has been suggested that the D and E fragments may correspond to the three globular domains observed by electron microscopy, based on indirect evidence (12). Preparation of immunologically distinct antisera affords a means of testing the hypothesis that the D and E fragments represent the cleaved outer and central fibrinogen nodules, respectively, by using immunolabeling electron microscopy (13). Therefore, univalent Fab' fragments from pepsin-digested immunoglobulin directed. against either D or E fragments were complexed with native human fibrinogen and the complexes were isolated by gel filtration. Electron micrographs of metal replicas of the complexes were then scored for binding of Fab' to specific regions of fibrinogen. The results of this analysis support the prediction. that the D and E fragments have their origin in the ends and the center of fibrinogen. MATERIALS AND METHODSPreparation of Fibrinogen. The procedure was as described (14) with minor deviations. Fibrinogen was isolated from outdated human plasma by two glycine precipitations at 2.1 M fol...

Deep-etch visualization of proteins involved in clathrin assembly.

“…10 portrays the AP-2 molecule as being composed of three discrete globular domains connected by thinner protease-sensitive strands. This model is similar to that for fibrinogen (6,17,32), although the relative sizes of the three globular domains are clearly different. The AP-2 is also considerably less asymmetric than fibrino- the structural observations in this report and from previous biochemical analyses (10,33).…”

Immunoelectron microscopic analysis of the binding of monoclonal antibodies to molecular variants of human placental alkaline phosphatase

“…As for the circular immune complex, the mAbs seemed to bind two of four electron-transparent regions, suggesting that each subunit is composed of two adjacent regions mimicking a dumbbell shape. Direct visualization of the immune complexes in the electron microscope has proven to be a very useful method to locate the antibody binding site on macromolecules, such as ribosomal protein (Wabl, 1974), fibrinogen (Price et al, 1981;Norton & Slayter, 1981), or proteoglycan (Buckwalter et al, 1982).…”

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

“…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.…”