Abstract
The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8, to the carboxyl-terminal 150 amino acids of the A alpha chain, binds near the central region of fibrinogen, indicating that the alpha C domains of most molecules are not normally visible because they are on or near the amino-terminal disulfide knot. At pH 3.5, fibrinogen and fibrin monomers appear to be similar, with a projection terminating in a small globular domain from each end of most molecules. In contrast, fragment X monomers, produced by cleavage of the alpha C domains from fibrinogen with plasmin, show no such projections. When fibrin monomer is brought to neutral pH under conditions where polymerization is delayed, individual molecules are still visible showing the alpha C domains as a single additional nodule near the central region. Moreover, analysis of clusters of molecules reveals some intermolecular associations via the alpha C domains. A 40-kDa fragment comprising the alpha C domain has been isolated from a plasmin digest of fibrinogen and characterized by SDS-polyacrylamide gel electrophoresis and determination of amino-terminal amino acid sequences. Electron microscopy of alpha C fragments reveals individual globular structures, as well as oligomeric aggregates. The addition of alpha C fragments to fibrin monomer followed by dilution to neutral pH to initiate polymerization results in lower turbidity, longer lag period, and slower maximum rate of turbidity increase. Also, electron microscopy reveals complexes of alpha C fragments with fibrin monomer at neutral pH. It appears that the free alpha C fragments can bind to the alpha C domains of fibrin, competing with the normal alpha C domain interactions involved in polymerization.
Highlights
From the $Departmentof Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6058, the §Institute of Biochemistry, Academy of Sciences of Ukraine, Kiev 252030, Ukraine, and the7lTNO Institute of Ageing and Vascular Research, Zernikedreef 9, P. 0.Box 430, 2300 A K Leiden, The Netherlands
Amino acids of the Aa chain, binds near the central It is apparent that fibrinogen is exquisitely sensitive to region of fibrinogen, indicatingthat theaC domains of many of the preparative steps for electron microscopy
A 40-kDa fragment comprising the aC domain has accurate model for fibrinogen has been derived from x-ray been isolated from a plasmin digest of fibrinogen and crystallographic and electron microscope studies of crystalline characterized by SDS-polyacrylamide gel electropho- arrays of a modified fibrinogen [7, 8]
Summary
Bovine fibrinogen (96-98% clottable) was prepared from oxalate plasma by salting out with sodium sulfate as described previously [19]. The ionic strength of the digest was adjusted to 2.0 by the addition of NaCl before application to thecolumn; the 40-kDa fragment was eluted in the second peak The fractions in this peak were pooled, dialyzed against 0.125% acetic acid at 4 "C, and lyophilized. As one control for the localization of the aCdomains, fragment X monomer, missing either one (X1-monomer) or both (Xz-monomer) of the aC domains, was prepared These preparations, based on those described by Medved' et al [18],were made by plasmin treatment of fibrinogen followedby ammonium sulfate fractionation, column chromatography, clotting with thrombin, and subsequent dissolution in acetic acid.' Fibrin-, X1-, and X2-monomersamples at acid pH were prepared by dilution of concentrated protein solution (13 mg/ml) with 0.125%acetic acid at pH 3.5 with 30% glycerol to a final concentration of 25 pg/ml. Each experiment was repeated several times, and datapoints were averaged
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