Plasmic Degradation of Crosslinked Fibrin

Abstract

A B S T R A C T Crosslinked fibrin was digested by plasmin, and three soluble complexes larger than DD/E were purified and characterized. After gel filtration chromatography, the purified complexes were shown to have molecular weights of 465,000, 703,000, and 850,000, as determined by equilibrium sedimentation. Each of the complexes was dissociated into two or more fragments by SDS-polyacrylamide gel electrophoresis. The structure of these subunit fragments was deduced from determinations of their molecular weights and polypeptide chain composition and from known sites of plasmin cleavage of fibrin. Fragments larger than DD have been identified that contain intact yry crosslinks as well as fragments resulting from cleavages at or near this site. The former include DY (mol wt 247,000), YY (mol wt 285,000), DXD (mol wt 461,000), and YXD (mol wt 500,000); and the latter include fragments XD (mol wt 334,000) and XY (mol wt 391,000). A schematic model was developed to explain the structure of the large noncovalently bound complexes based on their molecular weight and observed component fragments. Our scheme supports the twostranded half-staggered overlap model as the basic unit of fibrin structure, in which each complex consists of fragments from two adjacent complementary antiparallel fibrin strands. The smallest derivative, complex 1, is the DD/E complex; complex 2 contains apposed DY and YD fragments, and complex 3 consists of fragments DXD and YY. Complex 4 is less wellcharacterized, but its intact structure is projected to consist of YXD and DXY fragments from adjacent fibrin strands. Each complex is heterogeneous in subunit composition, reflecting additional plasmin cleavages within and/or adjacent to its theoretical boundaries. Since most ofthe protein initially released into solution from degrading fibrin is as complexes larger than DD/E,

Identification of covalently linked trimeric and tetrameric D domains in crosslinked fibrin.

“…The evidence supporting this conclusion includes (i) NaDodSO4/PAGE and crossed immunoelectrophoresis of digests of crosslinked fibrin demonstrated two plasmin-resistant D-containing core fragments of =300 and =365 kDa, respectively; (ii) TEM images and STEM mass analyses revealed structures corresponding in shape and size to trimeric and tetrameric forms of fragment D. Their existence is inconsistent with the assumption that every crosslinked degradation product larger than D dimer must contain one or more covalently linked E domains (36,(43)(44)(45) and raises the question as to whether certain E-containing digest components, especially those that are about the same size as D trimers or D tetramers (e.g., YY, 290 kDa; XY, 385 kDa), have been overestimated or, under certain conditions, misidentified. The discovery oftrimeric and tetrameric y-chain remnants, and the corresponding forms of crosslinked D fragments, indicates that e-(y-Glu)Lys bridging of these chains differs from the reciprocal bridging pattern that characterizes D dimers (Fig.…”

“…In contrast, the intermolecular e-(y-Glu)Lys y-chain bonds result in degradation products unique to crosslinked fibrin, of which the bimolecular fragment, "D dimer," is the most abundant and best characterized (32, 37-39). Crosslinked core derivatives larger than D dimer are known to exist (36,(40)(41)(42)(43)(44)(45), each of which is postulated (36, 43-45) to contain one or more E domains (e.g., DY, YY, DXD). …”

Elevated levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with mortality in acute lung injury

“…Deposition of fibrin in the alveolar space is the net result of an alteration in the balance of coagulation and fibrinolytic proteases [plasmin and urokinase type plasminogen activators (u-PA)] and antiproteases [plasminogen activator inhibitor-1 (PAI-1) and ␣ 2 -antiplasmin] and the availability of plasmaderived fibrinogen (31,35). Upon fibrinogen influx into the alveolar compartment, coagulation/fibrinolysis proteases/antiproteases act to generate insoluble crosslinked fibrin and soluble plasmic-cleavage fragments (13,14,22,25,35). The significance of fibrin turnover in sepsis is underscored by the recent trial demonstrating an improvement in mortality upon administration of the anticoagulant, activated protein C (3,21).…”

An isopeptide bond splitting enzyme from Hirudo medicinalis similar to γ‐glutamyl transpeptidase

“…The isopeptidic crosslinked amino acids produced in man by factor XIIIa remain intact after the proteolysis of the clot by plasmin and are excreted with the urine [11,12]. If this is the general fate of Glu Lys(N 6 -γ-glutamyllysine), a possible function of the new enzyme could be the salvage of lysin and glutamic acid during the long storage of the blood in the gut of the leech.…”