Studies on the basis for the properties of fibrin produced from fibrinogen-containing gamma' chains.

Human fibrinogen 1 is homodimeric with respect to its gamma chains (gammaA-gammaA'), whereas fibrinogen 2 molecules each contain one gammaA (gammaA1-411V) and one gamma' chain, which differ by containing a unique C-terminal sequence from gamma'408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1.

Rapid high-resolution electrophoresis of multimeric von Willebrand Factor using a thermopiloted gel apparatus.

Rapid and highly reproducible nonreducing agarose gel electrophoresis (NRAGE) of von Willebrand Factor (vWF) multimers was performed using a thermostated minigel apparatus that monitors and precisely controls internal gel temperature. The substitution of lithium dodecyl sulfate (LiDS) for sodium dodecyl sulfate (SDS) allowed electrophoresis to be performed below the 16 degrees C Krafft point of SDS and facilitated NRAGE of vWF over the entire range of 0-35 degrees C. Internal gel temperature was regulated by a thermocouple probe inserted directly into the gel during electrophoresis which interfaced with a thermopilot that continually measures and adjusts temperature to within +/- 0.

The circulatory half-lives of alpha-profibrin and alpha-fibrin monomer, and comparisons with other fibrin(ogen) derivatives.

Previous studies showed that alpha-fibrin monomer (lacking both A-fibrinopeptides, FPA) is normally cleared from the circulation before it assembles into a clot. Recent studies indicate that substantial quantities of an intermediate, alpha-profibrin lacking only one of the two FPA are produced in the course of conversion of human fibrinogen to fibrin. Since clearance of the alpha-fibrin monomer is saturable and receptor mediated, the extent to which alpha-profibrin or other fibrin(ogen) derivatives might compete for monomer uptake was deemed important.

Direct chemiluminescent immunodetection of proteins in agarose gels.

Chemiluminescent immunodetection of proteins separated by polyacrylamide gel electrophoresis is generally performed only after Western blotting. Agarose gels are adequately permeable to allow immunoprobing directly in the gel. Chemiluminescent substrates had not been applied for direct immunoprobing of agarose gels.

Allosteric effects potentiating the release of the second fibrinopeptide A from fibrinogen by thrombin.

Fibrin formation depends on the release of the two N-terminal fibrinopeptides A (FPA) from fibrinogen, and its formation is accompanied by an intermediate, alpha-profibrin, which lacks only one of the FPA. In this study, we confirm that the maximal levels of alpha-profibrin found over the course of thrombin reactions with human fibrinogen are only half of what would be expected if the first and second FPA were being released independently with equal rate constants. The rapidity of release of the fibrinopeptides by thrombin had been shown to depend on an allosteric transformation that is induced when Na(+) binds to a site defined by the 215-227 residues of thrombin, a transformation that results in the exposure of its fibrinogen-binding exosites transforming the thrombin from a slow to a fast acting form toward fibrinogen.

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.

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.

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.

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.

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.

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.

Thrombin cleavage enhances exposure of a heparin binding domain in the N-terminus of the fibrin beta chain.

Thrombin (IIa)-cleavage of fibrinogen (FBG) to form polymerized fibrin promotes endothelial cell spreading, proliferation, and von Willebrand factor release, requiring the exposure of the beta 15-42 domain. Studies reported here indicate that IIa-cleavage of fibrinopeptide B enhances exposure of a heparin binding domain at the beta 15-42 neo-N-terminus of fibrin. Crossed immunoelectrophoresis showed heparin-induced mobility shifts indicative of complexing with FBG and with N-terminal CNBr fragments of FBG (NDSK) and of fibrin (IIa-NDSK), but not evidence of heparin complexing with FBG lacking B beta 1-42 or with FBG fragments D and E was seen.

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.

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.

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.

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.

Immunoelectrophoretic and immunohistochemical characterizations of fibrinogen derivatives in atherosclerotic aortic intimas and vascular prosthesis pseudo-intimas.

Cadaveric aortic intimas with uncomplicated atherosclerosis were examined to determine the distribution and polypeptide chain composition of fibrinogen-related protein. Immunohistochemical staining showed deposits rich in fibrinopeptides A and B. The deposits were usually disseminated throughout intimas of moderate thickness < 0.

Immunoelectrophoretic characterizations of the cross-linking of fibrinogen and fibrin by factor XIIIa and tissue transglutaminase. Identification of a rapid mode of hybrid alpha-/gamma-chain cross-linking that is promoted by the gamma-chain cross-linking.

Cross-linking of human fibrin by fibrin stabilizing factor (factor XIIIa) and tissue transglutaminase (ti-TG) was examined by immunoprobing electrophoregrams for positive identification of the cross-linked chains. The immunoprobing was carried out by a new, direct staining technique employing composite gels of a porous protein immobilizing matrix (glyoxyl agarose) blended with a removable polyacrylamide filler that eliminates need for Western blotting. We find that the known rapid cross-linking of gamma-chains into gamma 2-dyads by XIIIa is accompanied by co-cross-linking of the gamma 2-dyads with alpha-chains to form hybrid alpha gamma 2-triads.

Electron microscopy of platelet interactions with heme-octapeptide-labeled fibrinogen.

Binding of fibrinogen to ADP-activated platelets was visualized by labeling the molecule with heme-octapeptide (microperoxidase) for direct cytochemical staining. Transmission electron microscopy of the platelet aggregates showed most of the fibrinogen distributed widely over the platelet surface in nonbridging rims of 7- to 9-nm thickness. Short peroxidase-positive bridges (less than 25 nm) were found in clusters in regions of close contact between the platelets, but 50-nm bridging corresponding to the length of the molecule was not seen by this method.

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.

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.

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.