The fibrin intermediate, its place in the fibrinogen-fibrin transformation.

Our preceding study indicated that, in course of coagulation of human fibrinogen by thrombin, substantial production of the fibrin intermediate (alpha-profibrin) lacking only one fibrinopeptide A (FPA) precedes the formation of alpha-fibrin monomer lacking both FPAs. The plateau concentration of alpha-profibrin (20% of initial fibrinogen) appearing in reactions indicated, however, that the second FPA is released four times faster than the first. The study reported here confirms those findings, and provides new insight into the significance of differing rate constants for the production of alpha-profibrin and its conversion to alpha-fibrin. The intermediate could be isolated in a distinct electrophoretic band by electrophoresing partial thrombin digests at high concentrations. Its identity was verified by digesting it with CNBr and by demonstrating that its N-terminal domain, the NDSK fragment, both lacks an FPA and contains an FPA, unlike the NDSKs of the bands from fibrin which contained no FPA or the fibrinogen band that lacked no FPA. The single step isolation also enabled us to confirm the 15-20% plateau level of alpha-profibrin in course of thrombin reactions, well below the 37% maximum that would be expected if release of the first and second FPA proceeded independently with no difference in rate. The 37% maximum is observed in reactions with atroxin, and it is suggested that the abundant production of alpha-profibrin underlies the therapeutic utility of atroxin as a defibrinating agent. Gel chromatography procedures were optimized for isolation of alpha-profibrin/fibrin mixtures free of fibrinogen, the final step of which involves literal use of agarose gel as a filter to remove fibrin aggregates from the fibrinogen free fractions (aggregates are left behind in gel filtration, rather than their moving ahead in gel chromatography). Unlike human fibrinogen, rabbit fibrinogen does not yield much alpha-profibrin in course of its conversion to fibrin, less than 10% as determined by electrophoresis and comparison with abundant production with atroxin. This low production of alpha-profibrin conformed with conclusions from our early studies on the generalized Shwartzman reaction in rabbits, and we now infer that the low production of alpha-profibrin and rapid conversion to fibrin by rabbit fibrinogen underlies the unparalleled susceptibility of these animals toward fibrinoid formation in the generalized Shwartzman reaction.

Confirmation of mendelian properties of heterodimeric fibrinogen molecules in a heterozygotic dysfibrinogenemia, "fibrinogen Amarillo," using gprphoresis to differentiate semifibrin molecules from fibrinogen and fibrin.

The fibrinogen molecule consists of two sets of Aalpha, Bbeta, and gamma chains assembled into a bilateral disulfide linked (Aalpha, Bbeta, gamma)2 structure. Cleavage of the two A-fibrinopeptides (FPA, Aalpha1-16) from normal Aalpha chains with arginine at position 16 (RFPA) by thrombin or the venom enzyme atroxin transforms fibrinogen into self-aggregating fibrin monomers (alpha, Bbeta, gamma)(2). Mutant Aalpha16R-->H fibrinopeptide (HFPA) cannot be cleaved from fibrinogen by atroxin. Many studies on heterozygous dysfibrinogenemias with this mutation suggested that incorporation of the mutant chains into the molecules was ordered in a manner yielding only (1) homodimeric normal (RFPARFPA) atroxin-coagulable molecules and (2) homodimeric abnormal (H(FPA)HFPA) atroxin-incoagulable molecules in equal quantities. Although heterodimeric molecules (RFPAHFPA) could not be found in studies on the intact protein, Meh et al. demonstrated their existence by showing that CNBr digests of fibrinogens from atroxin-treated Aalpha16R-->H heterozygotic dysfibrinogenemias consistently yielded N-terminal fragments (NDSKs) with partially resolved electrophoretic bands predominantly in between the NDSKs of fibrinogen and alpha-fibrin. An opportunity to confirm and better quantify the heterodimers arose with the recent development of a method (GPRphoresis) for identifying molecules lacking only one FPA, which is applied here in study of a newly presenting case of an Aalpha16R-->H dysfibrinogenemia, "fibrinogen Amarillo." GPRphoresis uses electrophoretic shifts, staged with GPRP-NH(2) to separate the self-aggregating fibrin monomers lacking both FPAs from weakly aggregating "semifibrin" molecules lacking one FPA An antifibrin alpha17-23 antibody is used to measure and differentiate the semifibrin from fibrinogen with FPA fully intact. Applying GPRphoresis to atroxin digests of fibrinogen Amarillo clearly demonstrated RFPARFPA, RFPAHFPA, and HFPAHFPA molecules in nearly perfect Mendelian 1:2:1 proportions. In turn, the high levels of the semifibrin in the terminal atroxin digests provide genetic phenotypic evidence supporting fidelity of the GPRphoresis method.

Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody.

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

Isolation and characterization of the fibrin intermediate arising from cleavage of one fibrinopeptide A from fibrinogen.

The thrombin-catalyzed cleavage of N-terminal fibrinopeptide A (FPA) from the two Aalpha-chains of fibrinogen exposes aggregation sites with the critical sequence GPR located just behind FPA. It is well known that exposure of both GPR sites transforms fibrinogen into self-aggregating, fully coagulable alpha-fibrin monomers, but the fibrin precursor with one site exposed and one FPA intact has eluded description. The formation of this "alpha-profibrin" in the course of thrombin reactions and its distribution among both the aggregating and non-aggregating components of the reactions are characterized here by immunoprobing electrophoretic and gel chromatographic separations using monoclonal antibodies specific for FPA and for exposed GPR sites. These analyses show alpha-profibrin to be a non-aggregating derivative indistinguishable from fibrinogen in solutions that are rich in fibrinogen relative to dissolved fibrin. But alpha-profibrin forms soluble complexes with alpha-fibrin monomer under conditions in which it and fibrin predominate over fibrinogen. It was isolated as a complex with fibrin by gel chromatography of cryoprecipitates and then separated from the fibrin either by electrophoretic gel shifts induced with a peptide analog of the GPR aggregation site or by chromatographic gel shifts induced with monoclonal anti-FPA antibody. The weak aggregation of alpha-profibrin with itself and with fibrinogen conforms with prior indications that coupled interactions through the paired GPR sites on fibrin monomers are pivotal to their aggregation. It is suggested that alpha-profibrin may be a hypercoagulable fibrin precursor because it is converted to alpha-fibrin monomer faster than fibrinogen converts to monomer.

Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases.

A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.

Preparative electrophoresis on linear polyacrylamide-agarose composite gels.

A preparative method for isolating centigram quantities of high molecular weight polypeptide chains with high resolution and recovery uses linear polyacrylamide/agarose composite (LPAC) gels as electrophoretic media from which the polypeptides can be easily extracted. The composites are prepared in a manner yielding linear copolymers of acrylamide and 1-allyloxy-2,3-propanediol within 2% agarose gels. After electrophoresis in sodium dodecyl sulfate (SDS), protein bands were rapidly visualized for excision by briefly immersing the gel in cold 0.1 M KCl which precipitates the protein-associated SDS. The gel slices are then freeze-thawed to disrupt the agarose matrix and promote syneresis of fluid upon centrifugation. The polypeptides are then separated from the polyacrylamide in the supernatant solution by precipitating with either acidic isopropanol, trichloroacetic acid, ammonium sulfate or other general protein precipitants. As determined with polypeptide chains of fibrinogen and its cross-linked derivatives, recoveries were virtually complete (95.4% +/- 2.2%), and were independent of molecular weights over the range tested (10(4) --10(6)).

Visualization of apo B, fibrinogen/fibrin, and fibronectin in the intima of normal human aorta and large arteries and during atherosclerosis.

Apolipoprotein B (apo B), fibrinogen/fibrin, blood platelets, factor VIII-related antigen of the blood coagulation system, and smooth muscle cells (SMC) were identified in the intima of normal and atherosclerotic human aorta and large arteries by the indirect immunofluorescence technique. Fibrinogen/fibrin was revealed by a monoclonal antibody (monAb) against the C-terminal region of human fibrinogen A alpha-chain. Fibronectin was visualized by monAb to the cellular form and against an epitope shared by different fibronectin subunit variants. In normal intima, fatty streaks, small amounts of fibrinogen/fibrin together with large amounts of apo B were observed. Fibronectin detected by two types of monAb was not found in extracellular matrix (ECM), whereas cellular fibronectin encircled SMC. According to the data obtained, fibrinogen/fibrin accumulates in plaques as a result of intramural thrombus incorporation, blood insudation, intramural haemorrhage, and in or around cells, apparently macrophages.