Stable complex of fibrinogen and fibrin.

Fibrin-stabilizing factor acts on monomeric fibrin combined with fibrinogen to form a stable complex that is soluble at physiologic pH and ionic strength. The complex has been isolated by chromatography on agarose gel and characterized by sedimentation rate near 24 S(w.20) at both pH 5.3 and 7.4, by coagulability when treated with thrombin, and by content of fibrinopeptides. Formation of the complex could provide a pathway for solubilization of monomeric fibrin produced in circulating blood.

Fibrinopeptide B and aggregation of fibrinogen.

Removal of fibrinopeptide B from human fibrinogen by reaction with the procoagulant enzyme from copperhead snake venom below 25 degrees C resulted in tight aggregation of the fibrinogen, which, in turn, progressively blocked a concomitant but sluggish release of fibrinopeptide A by the enzyme. When the clots obtained at less than 25 degrees C were warmed, they dissociated into soluble aggregates and monomers. Release of fibrinopeptide A then resumed, and a secondary coagulation followed. The aggregation induced by release of fibrinopeptide B itself involves a plasmin-susceptible segment located just distal to B in the B beta chain of fibrinogen, a segment previously shown to be of little importance in the aggregation induced by release of fibrinopeptide A.

Fibrinogen Cleveland II. An abnormal fibrinogen with defective release of fibrinopeptide A.

An abnormal fibrinogen (fibrinogen Cleveland II) was detected in the plasma of a 23-yr-old white man with a mild bleeding diathesis. The one-stage prothrombin time, thrombin time, and Reptilase time were all prolonged. 16 of 24 tested relatives had the defect, which appeared to be transmitted as an autosomal dominant characteristic. The thrombin time of normal plasma was slightly inhibited by the proband's plasma. The abnormally long thrombin time of fibrinogen Cleveland II was partially corrected by addition of calcium ions. Fibrinogen Cleveland II was indistinguishable from normal fibrinogen by immunoelectrophoresis, DEAE-cellulose column chromatography, or polyacrylamide gel electrophoresis of reduced fibrinogen in sodium dodecyl sulfate. The major defect detected appeared to be impaired release of fibrinopeptide A when fibrinogen Cleveland II was incubated with thrombin. This defect was localized to the NH(2)-terminal disulfide knot portion of the molecule. An abnormality of polymerization of fibrin monomers was also present, but the abnormal fibrin demonstrated relatively normal crosslinking. Despite these defects, fibrinogen Cleveland II achieved a degree of coagulability similar to normal fibrinogen and appeared to incorporate some molecules of fibrin with intact fibrinopeptide A into the clot. The fibrin clot that was formed appeared to be abnormal by electron microscopy. These functional defects and other descriptive characteristics appear to distinguish fibrinogen Cleveland II from other inherited abnormal fibrinogens.

Fibrinogen Bethesda: a congenital dysfibrinogenemia with delayed fibrinopeptide release.

A dysfibrinogenemia (fibrinogen Bethesda) was detected in a 9 yr old male of Mexican-English extraction who had a lifelong history of mild bleeding diathesis. The prothrombin and partial thromboplastin times were moderately prolonged; the thrombin and Reptilase times were markedly prolonged. The plasma fibrinogen level was normal by conventional methods but was markedly reduced by the Clauss method. Results of all other tests for clotting factors, fibrinolysis, antithrombin levels, clot stabilization, and fibrin(ogen) degradation products were normal. The patient's plasma and fibrinogen inhibited the clotting of normal plasma or fibrinogen by thrombin. Family studies revealed that the propositus' mother and two siblings exhibited these abnormalities to a lesser degree and indicated an autosomal dominant inheritance. Fibrinogen Bethesda was similar to normal fibrinogen in the following respects: metabolic turnover time (measured in the propositus' mother); immunodiffusion, ultracentrifugal, electrophoretic (on cellulose acetate or polyacrylamide gel), and chromatographic (on DEAE-cellulose) characteristics; sialic acid content; and aggregation of fibrin monomers. By contrast, fibrinogen Bethesda gave an abnormal immunoelectrophoretic pattern especially when whole plasma (as opposed to purified fibrinogen) was examined, and it showed a pronounced decrease in the rate of fibrinopeptide release by thrombin. This decrease, which was shown to involve both fibrinopeptides A and B, distinguishes fibrinogen Bethesda from previously reported dysfibrinogenemias.

Functional evaluation of an inherited abnormal fibrinogen: fibrinogen "Baltimore".

The rate of clotting and the rate of development and degree of turbidity after addition of thrombin to plasma or purified fibrinogen from a patient with fibrinogen Baltimore was delayed when compared with normal, especially in the presence of low concentrations of thrombin. Optimal coagulation and development of translucent, rather than opaque, clots occurred at a lower pH with the abnormal fibrinogen than with normal. Development of turbidity during clotting of the abnormal plasma or fibrinogen was less than normal at each pH tested, but was maximal in both at approximately pH 6.4. The physical quality of clots formed from fibrinogen Baltimore was abnormal, as demonstrated by a decreased amplitude on thromboelastography. The morphologic appearance of fibrin strands formed from fibrinogen Baltimore by thrombin at pH 7.4 was abnormal when examined by phase contrast or electron microscopy, but those formed by thrombin at pH 6.4 or by thrombin and calcium chloride were similar to, though less compact, than normal fibrin. The periodicity of fibrin formed from fibrinogen Baltimore was similar to normal and was 231-233 A.A study of the release of the fibrinopeptides from the patient's fibrinogen and its chromatographic subfractions verified the existence of both a normally behaving and a defective form of fibrinogen in the patient's plasma. The defective form differed from normal in three functionally different ways: (a) the rate of release of fibrinopeptides A and AP was slower than normal; (b) no visible clot formation accompanied either partial or complete release of the fibrinopeptides from the defective form in 0.3 M NaCl at pH 7.4; and (c) the defective component possessed a high proportion of phosphorylated, relative to nonphosphorylated, fibrinopeptide A, while the coagulable component contained very little of the phosphorylated peptide (AP). The high phosphate content of the defective component did not appear to be the cause of the abnormality, but may be the result of an associated metabolic or genetic phenomenon.

Isolation of aldosterone-stimulating factor (ASF) and its effect on rat adrenal glomerulosa cells in vitro.

A protein fraction has been isolated from normal human urine which upon chronic administration produced hypertension in rats. The hypertension is associated with retention of sodium and increased circulating aldosterone. The protein fraction has been purified to homogeneity, and its molecular weight has been determined to be 26,134 daltons by equilibrium ultracentrifugation. The compound has been identified to be clearly different from ACTH, angiotensin II, and beta-lipotropin. It stimulated aldosterone production from rat glomerulosa cells in vitro in a dose-dependent fashion from 10(-9) to 10(-4)M with a maximum stimulation at 10(-7) where a fourfold increase was obtained during 2 hours of incubation. Removal of some carbohydrate moieties by insoluble neuraminidase caused a twofold increase in aldosterone production in vitro. The protein fraction has been named "aldosterone-stimulating factor" or "ASF." Further studies are in progress to define its physiological role.

Cascade immunoelectrophoresis: combined electrophoretic and solid-phase processing of immunoreactive protein by zonal immobilization.

A new approach to immunochemical analysis of complex mixtures of proteins has been devised through development of a system for (1) sequentially absorbing, desorbing, and cascading antibodies in a profile indicative of both the quantity and electrophoretic characteristics of the antigen, and (2) for carrying out multiple tests of antigenic determinants at sub-picomole levels regardless of the solubility or precipitin forming characteristics of the immunoreactive protein. The procedure employs zonal immobilization in which a novel, aldehyde-rich gel (glyoxyl agarose) is used interchangeably as an inert support for electrophoresis and as an immobilizing matrix to fix the protein for analysis by solid-phase techniques. Proteins may be separated on the gel as with ordinary agarose, and then driven to combine covalently with the gel upon exposure to NaCNBH3. After removing NaCNBH3, the distribution of specified antigens is then established by exposing the gel to antibody an profiling the pattern of antibody uptake by a cross-electrophoretic technique in which the absorbed antibody is (1) desorbed with dodecyl sulfate, then (2) transferred through a gel containing potassium ion to halt migration of the detergent, and (3) displayed by immunoprecipitation with anti-IgG antibodies. To cascade the process further, the IgG may be immobilized and used as a surrogate antigen to bind additional antibody protein. In addition to enhancing sensitivity, the cross-electrophoretic measurement of uptake of antibody by immobilized antigen eliminates dependence on direct immunoprecipitation of the antigen, and may accordingly be of particular advantage with non-precipitin forming antigens and monoclonal antibodies.

Microangiopathic hemolytic anemia in renal allotransplantation. Repart of a successfully treated case and review of the literature.

The development of microangiopathic hemolytic anemia after renal transplantation in a 17 year old white boy is reported, and the literature is reviewed. In this patient microangiopathic hemolytic anemia developed 6 weeks after renal transplantation during a second episode of rejection. Light, fluorescence and electron microscopy demonstrated the renal vascular lesion associated with this syndrome. In contrast to the other four previously reported cases of microangiopathic hemolytic anemia associated with renal allotransplantation, this patient had complete resolution of the microangiopathic hemolytic anemia with heparin therapy and improved allograft function, presumably with diminution of the vascular lesion. He survived a complicated early period after renal transplantation and has shown no recurrence of microangiopathic hemolytic anemia in the 18 months since transplantation. Special red blood cell and fibrinogen studies are discussed.

Characterization of a mode of specific binding of fibrin monomer through its amino-terminal domain by macrophages and macrophage cell-lines.

The studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (alpha-fibrin) or both fibrinopeptides A and B (alpha beta-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX congruent 200-800 molecules/cell, KD congruent to 10(-12) M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX greater than or equal to 10(5) molecules/cell, KD greater than or equal to 10(-6) M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD congruent to 10(-10) M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (beta-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the alpha- and the beta-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with electrophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Three dimensional structure of human fibrinogen under aqueous conditions visualized by atomic force microscopy.

Fibrinogen plays a central role in surface-induced thrombosis. However, the interactions of fibrinogen with different substrata remain poorly understood because of the difficulties involved in imaging globular proteins under aqueous conditions. We present detailed three dimensional molecular scale images of fibrinogen molecules on a hydrophobic surface under aqueous conditions obtained by atomic force microscopy. Hydrated fibrinogen monomers are visualized as overlapping ellipsoids; dimers and trimers have linear conformations predominantly, and increased affinity for the hydrophobic surface compared with monomeric fibrinogen. The results demonstrate the importance of hydration on protein structure and properties that affect surface-dependent interactions.

Low factor XIIIA levels are associated with increased blood loss after coronary artery bypass grafting.

Current hematologic approaches to minimize postoperative bleeding have focused principally on antifibrinolytic agents. To explore whether a need might exist to promote clot stabilization independent of steps that might be taken to prevent lysis, we followed levels of the functional A-chain of factor XIII (fibrin stabilizing factor) immunologically in 19 patients undergoing coronary artery bypass grafting. The levels of factor XIIIA together with alterations in fibrinogen were followed at five stages of operation: (1) initial catheter placement (control), (2) heparinization, (3) initiation of cardiopulmonary bypass, (4) discontinuation of cardiopulmonary bypass, and (5) heparin neutralization with protamine sulfate. Significant (p < 0.05) inverse correlations were observed between postoperative chest-tube drainage volumes and levels of XIIIA at stages 1 through 3, and borderline associations (p < 0.1) were observed for stages 4 and 5. Pronounced losses of factor XIIIA accompanied initiation of cardiopulmonary bypass, when levels fell to 43% +/- 12% (standard deviation) of the control value, significantly below the 59% +/- 9% of the control value expected from hemodilution. By comparison, fibrinogen concentrations fell only to the extent attributable to hemodilution, unaccompanied by substantial degradation as indicated by electrophoretic, functional, and immunologic assays. There was a reversible heparin-induced precipitation of fibrin complexes and fibrinogen dimers from the blood on initiation of hypothermia, but these components returned to the circulation on restoration of normothermia. This precipitation was unrelated to losses of factor XIIIA. The findings warrant inference that XIIIA supplementation in deficient states should be considered as an adjunct to other therapies for postoperative bleeding.

Fibrinopeptide B in fibrin assembly and metabolism: physiologic significance in delayed release of the peptide.

The delayed release of peptide B that accelerates towards the end of fibrin formation unmasks accessory (b-) epitopes for monomer interaction. Ultracentrifuge and chromatographic analysis of the composition and dissociation of soluble complexes formed by monomers in fibrinogen solution indicate that the b-epitope augments aggregation by acting cooperatively with the a-epitope to reinforce rather than cross-bridge oligomer assembly. Monomer/fibrinogen association by coordinated interactions through both epitopes is strengthened by an additional order of magnitude over associations (10(7) and 1.6 X 10(6) M-1) through the a- and b-epitopes individually, without affecting oligomer thickness. It is suggested that the delayed release of B has purpose in allowing early complexes to dissociate for (1) rapid equilibration across interstitial fluids, and for (2) rapid uptake by phagocytic cells which depend on access to the a-epitope for monomer absorption. In late stages of coagulation, stabilization of oligomer assembly imparted by the b-epitope blocks both equilibration of fibrin concentrations and phagocytic clearance of the fibrin to localize deposition.

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.

Multiple peaks induced by domain-specific binding of fibrinogen in anion-exchange high-performance liquid chromatography.

A domain binding model was developed for explaining the multiple peak chromatograms obtained in the high-performance liquid chromatography of pure fibrinogen on a DEAE polymethacrylate column using different gradients of ammonium chloride. The different peaks for fibrinogen result from the binding of either the D or E domain of fibrinogen to the packing material. This was confirmed by comparing the retention times of the chromatograms for fibrinogen, fragment D1 and fragment E. Native and denatured forms of fibrinogen are proposed to be important to fibrinogen's interaction with the column, hiding or exposing the E domain, respectively. Different gradient speeds resolve a different number of peaks for fibrinogen, with slow gradients yielding essentially one peak and fast gradients 10 or more peaks. Temperature studies were done to confirm the model. Different commercial sources of fibrinogen showed different proportions of native and denatured/degraded forms.

Multicolour immuno-staining of fibrinogen polypeptide chains for identification of their derivatives in electrophoregrams.

A novel electrophoretic procedure enabling multiple, direct immunoprobing of electrophoregrams without depending on Western blotting is described, and applied to the identification of the derivatives formed in the early stages of clot stabilization. Multicolour immunostainings for positive identification of cross-linked chains in partially stabilized fibrin clots indicated that the early products of alpha-chain cross-linking by factor XIII are largely hybrids of co-cross-linking of alpha- and gamma-chains rather than alpha-chain polymers suggested from previous studies employing non-specific staining of electrophoregrams. Furthermore, plasma-fibrinogen dimers were found to contain cross-linked alpha-chains with an electrophoretic mobility very near that of gamma-gamma-dyads. A similar product is produced by tissue-transglutaminase, but not by factor XIII.

GPR-phoresis, a novel approach to determining fibrin monomer and other macromolecular derivatives of fibrinogen and fibrin in blood.

An electrophoretic method for determining (i) cross-linked fibrin-complexes, (ii) fibrin-monomer, (iii) fibrinogen-dimers, (iv) normal fibrinogen and (v) degradation products in plasma, has been devised. The technique is based on differences in their migration characteristics in the presence and absence of Gly-Pro-Arg (GPR), a specific inhibitor of fibrin aggregation. In buffer containing 2.5 mM GPR, fibrin monomer and fibrinogen co-migrate anodally, but, unlike fibrinogen which does not depend on GPR for solubility, the fibrin monomers precipitate when they traverse a boundary between buffer containing and buffer lacking GPR. By limiting the GPR to a 2 cm zone of buffer under the conditions employed, the precipitation of fibrin monomer occurs in a sharp band 4 mm anodally to the sample application point. Cross-linked fibrin complexes have slower mobility than fibrin monomer and precipitate in a broad band behind the monomer. Dimeric fibrinogen, like fibrinogen itself but unlike the fibrin complexes, is not constrained to migration within the GPR boundary and passes through it, but behind the band for normal fibrinogen due to sieving by the gel. Fibrinogen and all but low molecular weight degradation products can be specifically precipitated within electrophoregrams by heat denaturation at 47 degrees C. After washing unrelated protein away, the fibrin(ogen) derivatives can be measured by staining with Coomassie blue. Since the method does not depend on immunoprobing for specific staining, it provides an inexpensive and rapid means for differential assessment of the prevalence of the fibrinogen derivatives in disease states and in models of disease, regardless of animal species.

Fibrinogen A alpha and gamma-chain dimers as potential differential indicators of atherosclerotic and thrombotic vascular disease.

Cross-linked fibrin(ogen) dimers are known to be elevated in the plasma of subjects with occlusive vascular disease, and are thought to be fibrin dimers. Immunoelectrophoretic analyses of the dimers, however, indicate that (1) they are predominantly fibrinogen rather than fibrin dimers, and (2) they contain cross-linked A alpha-chains (A alpha-dyads) instead of the gamma-chain dyads that are rapidly formed by factor XIII during blood coagulation. Furthermore, the mobilities of the A alpha-dyads differ from the cross-linked alpha-chain products that accompany the gamma-chain cross-linking by factor XIII. Instead, the mobilities coincide with the distinct A alpha-dyads that are produced by tissue transglutaminase, an intracellular enzyme not normally present in plasma. The intimal fibrinogen deposits in atherosclerotic aortas also possess fibrinopeptide A and cross-linked A alpha-chains. Thus, both the plasma fibrinogen dimers and the intimal fibrinogen deposits appear to derive from the action of released tissue transglutaminase more so than factor XIII. It is proposed that, in the absence of other indications of cytolytic processes, the levels of A alpha-dyads in plasma reflect ongoing cellular injury accompanying atherogenesis. The extent to which gamma-dyads accompany the A alpha-dyads may signal progression of the disease to advanced stages in which ulcerations and occlusive lesions trigger thrombotic complications.

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.

Electrophoretic characterizations of cross-linked fibrinogen derivatives in blood and vascular tissue by zonal immobilization on glyoxyl agarose.

Direct immunoprobing of electrophoregrams of plasma and intimal protein on glyoxyl agarose and composite-gels with polyacrylamide have uncovered novel modes of cross-linking of fibrinogen that differ from those previously characterized. These modes of cross-linking involve the A alpha-chains of fibrinogen and hybrid cross-linking of alpha- and gamma-chains.