The Molecular Weights, Mass Distribution, Chain Composition, and Structure of Soluble Fibrin Degradation Products Released from a Fibrin Clot Perfused with Plasmin

Abstract

We used a perfused clot system to study the degradation of cross-linked fibrin. Multiangle laser light scattering showed that plasmin-mediated cleavage caused the release of noncovalently associated fibrin degradation products (FDPs) with a weight-averaged molar mass (M w ) of ϳ6 ؋ 10 6 g/mol. The M w of FDPs is dependent on ionic strength, and the M w observed at 0.15 M NaCl resulted from the self-association of FDPs having M w of ϳ3.8 ؋ 10 6 g/mol. Complete solubilization required the cleavage of ϳ25% of fragment D/fragment E connections, with 48% ␣-, 62% ␤-, and 42% ␥-chains cleaved. These results showed that D-E cleavage cannot be explained by a random mechanism, implying cooperativity. Gel filtration and multiangle laser light scattering showed that FDPs range from 2.5 ؋ 10 5 to 1 ؋ 10 7 g/mol. In addition to fragment E, FDPs are composed of fragments ranging from 2 ؋ 10 5 Da (D-dimer, or DD) to at least 2.3 ؋ 10 6 Da (DX 8 D). FDP mass distribution is consistent with a model whereby FDPs bind to fibrin with affinities proportional to fragment mass. Root mean square radius analysis showed that small FDPs approximate rigid rods, but this relationship breaks down as FDPs size increases, suggesting that large FDPs possess significant flexibility.Fibrinogen is the target protein of the coagulation cascade, and, following vascular injury, fibrin comprises the major protein component of the hemostatic plug. Fibrinogen is a 340-kDa soluble plasma protein consisting of three pairs of disulfidebonded ␣-, ␤-, and ␥-chains arranged as depicted in Fig. 1. The molecule consists of a central globular E region connected by coiled-coil regions to two identical globular D regions. In addition, two other structures comprising approximately the carboxyl-terminal two-thirds of the ␣-chains, designated ␣C regions, have been described (1, 2). The E and D regions in each half-molecule are delineated by a pair of disulfide rings, which link chains ␣ to ␤, ␤ to ␥, and ␥ to ␣ (3). Numerous plasmin-and thrombin-sensitive bonds have been identified and are shown in Fig. 1 (4). Thrombin converts fibrinogen to fibrin by catalyzing the proteolytic removal of both fibrinopeptide A and fibrinopeptide B. These cleavages unmask two polymerization sites in the E region, forming soluble fibrin, to which one D region from each of two fibrin(ogen) molecules can bind (5). The soluble fibrin spontaneously polymerizes to form doublestranded protofibrils, with the fibrin monomers within each strand arranged end to end and the fibrin across strands arranged in a half-staggered overlap, as depicted by the combination of strands a and b in Fig. 2. The protofibrils may further associate laterally to produce fibers, which themselves may associate to form fiber bundles. Collectively, the protofibrils, fibers, and fiber bundles, variously branched, comprise the fibrin clot. As also indicated in Fig. 2, the ␥-chains of adjacent D regions within a strand of a protofibril are covalently crosslinked by isopeptide bonds, the formation of which is catalyzed by f...

A Kinetic Analysis of the Tissue Plasminogen Activator and DSPAα1 Cofactor Activities of Untreated and TAFIa-treated Soluble Fibrin Degradation Products of Varying Size

“…We have recently demonstrated that FDPs released from a perfused clot are composed of noncovalently associated products whose masses range from 250 kDa (the mass of DD/E) to ϳ10,000 kDa (9). Furthermore, our work showed that the majority of the FDPs compose structures much larger than DD/E.…”

“…Preparation of FXIIIa Cross-linked Soluble Fibrin Degradation Products-Soluble FDPs were prepared as described previously (9), except that 1) clots were formed in columns with 9 ml of available volume, 2) the clots were perfused with 0.2 nM plasmin instead of 0.05 nM plasmin, and 3) the FDPs from four perfusions were pooled prior to gel filtration. The pooled FDPs (ϳ25 mg) were subjected to gel filtration on Sephacryl S-1000 and the weight average molecular weight (M w ) of the FDPs in the eluate was determined on-line using multiangle laser light scattering (9).…”

“…Materials-Human fibrinogen, plasminogen (Pgn), prothrombin, and factor XIII (FXIII), were prepared as described previously (9). Plasmin (Pn), thrombin (IIa), and FXIIIa were prepared from the purified zymogens as described (9).…”

“…Plasmin (Pn), thrombin (IIa), and FXIIIa were prepared from the purified zymogens as described (9). Recombinant human thrombin-activable fibrinolysis inhibitor (TAFI) was produced as described by Boffa et al (10).…”

“…The pooled FDPs (ϳ25 mg) were subjected to gel filtration on Sephacryl S-1000 and the weight average molecular weight (M w ) of the FDPs in the eluate was determined on-line using multiangle laser light scattering (9). Samples from the gel filtration column having M w ϭ 0.48 ϫ 10 6 , 1.08 ϫ 10 6 , 1.93 ϫ 10 6 , 3.08 ϫ 10 6 , 3.97 ϫ 10 6 , and 4.94 ϫ 10 6 g/mol were prepared by pooling appropriate fractions, and the samples were concentrated to Ͼ6.5 M by centrifugal concentration as described (9). The concentration of the FDPs refers to the concentration of fragment X equivalents present in the sample.…”