Eugene A. Beck

Plasmic degradation of human fibrinogen I. Structural characterization of degradation products

Abstract 1. 1. Fragments X, Y, D, and E were prepared by plasmic degradation of human fibrinogen and purified by gel filtration and chromatography. Molecular weights of reduced polypeptide chains of fibrinogen and its degradation products were studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea. 2. 2. Plasmic hydrolysis of fibrinogen was interrupted at different digestion stages, and the release of polypeptides from fibrinogen was studied by polyacrylamide electrophoresis of non-reduced and reduced incubation mixtures. 3. 3. In the early phase of plasmic hydrolysis, the β and γ chains of fibrinogen lose small molecular weight peptides at the N-terminal and C-terminal ends, respectively. Large polypeptides are progressively being split from the C-terminal part of the α chain. Fragment X was found to be a heterogenous population of molecules differing in the length of their α chains. 4. 4. On further digestion, one pair of disulfide-bound β and γ chains is released from the fragment X, giving rise to Fragment Y, which in turn splits into another β-γ dimer and Fragment E. Our results suggest that the β-γ dimer corresponds to Fragment D and confirm the partial identity of Fragment E with the disulfide knot.

Studies on factor VIII-related protein: II. Estimation of molecular size differences between factor VIII oligomers

Human factor VIII-related protein was isolated from cryoprecipitate by agarose (Sepharose CL-2B) gel filtration. Electrophoresis on SDS-2% polyacrylamide-0.5% agarose gels revealed size heterogeneity of factor VIII-related protein which was similar to that shown by SDS-1% agarose gel electrophoresis and electron microscopy. The apparent molecular weights were compared with those of crosslinked IgM oligomers and corresponded to values of up to 20 . 10(6) for factor VIII eluting close to the void volume of our gel filtration column. Measurement of mobility intervals on electrophoretic gels suggested a constant size difference between adjacent bands. Smaller aggregates were found in later eluates from Sepharose columns as well as following partial reduction of factor VIII with cysteine. In order to compare the size difference between small and large aggregates of factor VIII-related protein we calibrated the SDS-2% polyacrylamide-0.5% agarose gels with factor VIII which had been crosslinked with dimethyl suberimidate and subsequently disulfied-reduced with 2-metcaptoethanol. By combination of calibration ranges, constant intervals were measured for large and smaller factor VIII aggregates. The interval between any neighboring protein bands, which were immunologically identified as factor VIII-related protein, was equal to the dimer of the basic factor VIII subunit chain. We conclude that factor VIII aggregates correspond to multimers of a dimeric molecule, i.e. pairs of the basic subunit chain.

Inhibition of fibrin polymerization by fragment D is affected by calcium, Gly-Pro-Arg and Gly-His-Arg

Fibrinopeptides A and B were removed from purified human fibrinogen by bovine thrombin, whereas the snake venom protease batroxobin only split fibrinopeptide A from fibrinogen. Aggregation of the resulting desAB- and desA-fibrin monomers was evaluated by recording the turbidity of incubation mixtures. Fibrin assembly was strongly accelerated by increasing the calcium concentration from 10(-5) to 10(-3) M. Fragment D was obtained from fibrinogen by proteolytic degradation with plasmin in the presence of Ca2+. At a 4-fold molar concentration relative to fibrinogen, fragment D dramatically inhibited fibrin polymerization at up to 10(-4) M Ca2+. This anticlotting activity was, however, much less pronounced at 10(-3) M Ca2+. The thrombin clotting time, measured on human plasma, was prolonged by fragment D in a dose-dependent manner. In citrate-containing plasma, the fibrinogen clotting was significantly delayed by an equimolar concentration of fragment D. In barium sulfate-adsorbed oxalated plasma, containing 2.5 mM Ca2+, the same amount of fragment D hardly affected fibrin polymerization. We conclude that fragment D has no important anticlotting effect under physiological conditions. The synthetic peptide Gly-Pro-Arg, corresponding to the amino-terminal sequence of the fibrin alpha-chain, inhibited aggregation of both desA-fibrin and desAB-fibrin at 10(-3) M Ca2+. The inhibition of desAB-fibrin polymerization by Gly-Pro-Arg was abolished at 10(-5) M Ca2+. In addition, Gly-Pro-Arg depressed the anticlotting activity of fragment D at low calcium concentration. An analogue of the amino-terminus of fibrin beta-chain, Gly-His-Arg, strongly accelerated aggregation of desA-fibrin monomers, but only moderately enhanced polymerization of desAB-fibrin monomers at 10(-5) M Ca2+, both in the presence and in the absence of fragment D. This activating effect of Gly-His-Arg was abolished at 10(-3) M Ca2+. It is suggested that the binding of calcium, Gly-His-Arg, and possibly also Gly-Pro-Arg, induces a conformational change in fibrin monomers and thus accelerates the polymerization process.

Plasmic degradation of human fibrinogen. III. Molecular model of the plasmin-resistant disulfide knot in monomeric fragment D.

A mixture of fragments D, derived from fibrinogen by plasmic degradation, was S-reduced and carboxymethylated. Individual chains were separated by gel filtration on Sephadex G-100 and characterized by peptide mapping, N-terminal amino acid analysis, polyacrylamide electrophoresis in sodium dodecyl sulfate, and amino acid composition. It was demonstrated that all D species contain the same alpha- and beta-chain remnants, having mol. wts of 10 000 and 45 000, respectively. Their heterogeneity was shown to be caused by the gradual degradation of the gamma-chain at its C-terminal end. Denatured fragment D was further degraded with plasmin in the presence of 2 M urea. One beta- (mol. wt 17 000) and two gamma-fragments (mol. wts 5000 and 6000) were split from fragment D, in addition to non-characterized small peptides, leaving behind a plasmin-resistant core, designated as fragment d. Fragment d was in turn reduced and carboxymethylated, and the resulting constituent chains were isolated by chromatography on carboxymethyl-cellulose and Sephadex G-100. The reduced alpha-, beta- and gamma-chain remnants of fragment d were found to have been derived from the N-terminal portion of fragment D and have estimated mol. wts of 9000, 24 000 and 13 000, respectively. A tentative scheme for the conversion of an early fragment D into the core fragment d is proposed. Our results conclusively support the model of asymmetric degradation of fibrinogen, according to which 2 mol of monomeric fragment D are produced from 1 mol of fibrinogen.

Cross-linking of human fibrinogen with glutaraldehyde and tetranitromethane.

Abstract Human fibrinogen was allowed to react with chemical cross-linking agents, glutaraldehyde (GA) and tetranitromethane (TNM). Distribution of covalently linked fibrinogen oligometers, as well as the subunit composition of S-reduced reaction products, were examined by SDS-polyacrylamide gel electrophoresis. Intramolecular β-γ and α-β cross-links were exclusively produced at low GA concentration. Enhanced intermolecular cross-linking was observed with increasing concentration of GA. Treatment with TNM resulted in fibrinogen polymers linked together by intermolecular covalent bonds between α-chains. Cross-linking experiments with the plasmic degradation product X indicate that the N-terminal half of the α-chain is involved in GA-cross-linking whereas the C-terminal portions of this chain react with TNM. The described cross-linking patterns were the same when fibrinogen or fibrin reacted with GA or TNM in 0.15 M NaCl, 1 M NaCl and 1 M urea.

Studies on factor VIII-related protein: III. Size distribution and carbohydrate content of human and bovine factor VIII

Human and bovine factor VIII were isolated from cryoprecipitate of fresh frozen plasma by gel filtration on Sepharose CL-2B. The elution diagrams and SDS-agarose electrophoretic analysis of eluted fractions show no significant differences in the size-distribution of factor VIII aggregates between the two species. Agarose gels were stained for carbohydrate by two methods: (1) the dansyl hydrazine reaction following oxidation with periodic acid and (2) staining with fluorescein-labeled concanavalin A. Results of both procedures indicate that in human factor VIII neither the size distribution nor its ristocetin cofactor activity are related to carbohydrate content. Bovine factor VIII contains slightly less sugar than the human preparation as judged from the relative dansyl hydrazine staining intensities. In contrast to human factor VIII, the binding affinity for concanavalin A of bovine factor VIII was gradually decreased with increasing aggregate size. This finding suggests an impaired accessibility of reactive sugar residues in large aggregates of bovine factor VIII.

Studies on factor VIII-related protein: I. Ultrastructural and electrophoretic heterogeneity of human factor VIII-related protein

Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein.

Dissociation of human factor VIII by rhizopus lipase

Abstract Human factor VIII complex, purified from cryoprecipitate by gel filtration on Sepharose CL-2B, does not enter 3 % polyacrylamide gels. However, triglyceride lipase from Rhizopus arrhizus dissociates factor VIII into smaller subunits penetrating the same gels. Dissociated factor VIII retains full ristocetin cofactor activity. Lipase-induced dissociation of factor VIII is inhibited by EDTA, DFP and PMSF, but remains unaffected by specific protease inhibitors. Products of dissociated factor VIII show anomalous behaviour on SDS-polyacrylamide gels. The major lipolytic product migrates corresponding to an approximate molecular weight of 640 000 daltons on 3 % gels, whereas markedly lower molecular weights are estimated from 5 % gels. Following disulfide reduction, all lipolytic products yield a single subunit band (molecular weight 340 000 on 3 % gels), the migration of which is unchanged in comparison with reduced, intact factor VIII complex. Our results suggest that factor VIII may be dissociated by a triglyceride lipase without detectable proteolysis.

Clottability and cross-linking reactivity of fibrin(ogen) following differential release of fibrinopeptides A and B.

Human fibrinogen was treated at pH 6.0, 7.3 and 9.0 with thrombin, batroxobin (thrombin-like fraction of Bothrops atrox venom) or an extract of the venom from Ancistrodon contortrix contortrix. These three enzymes released the NH2-terminal fibrinopeptides A and B at different rates. Thrombin-free, preactivated factor XIII (factor XIIIa) was added to incubation mixtures to stabilize resulting fibrin(ogen) aggregates. Cross-linking of gamma-chains and the size of covalently linked fibrin-fibrinogen oligomers were studied in an early stage of fibrinopeptide cleavage using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Batroxobin (pH 7.3) and thrombin (pH 6.0) preferentially released fibrinopeptide A, and resulting fibrin aggregates became rapidly insoluble. However, when fibrinopeptide B was removed with the contortrix enzyme, soluble cross-linked oligomers appeared initially. The opaque fibrin clots, produced by thrombin (pH 6.0) or contortrix procoagulant fraction (pH 7.3), were found to be devoid of alpha-polymers even after prolonged incubation with factor XIIIa. Our data suggest that the solubility and opacity of fibrin networks are not primarily related to the type of the cross-link (gamma-gamma versus alpha-alpha interactions).

Plasmic degradation of human fibrinogen. IV. Identification of subunit chain remnants in fragment Y.

A method is presented for detection of cross-linking acceptor sites on fibrinogen chains, using monodansyl-cadaverine labeling in the presence of activated fibrin stabilizing factor, and polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Fluorescent gamma-chain monomers and dimers were produced at a considerably faster rate than the labeled alpha-chain derivative. Purified fragments X, Y and D were prepared all from the same plasmic digest of fibrinogen. Following incubation with fibrin stabilizing factor, thrombin and monodansyl-cadaverine, they were reduced with beta-mercaptoethanol and examined by sodium dodecyl sulfate/acrylamide electrophoresis. Three gamma-chains (mol. wts 49 000, 42 000 and 39 000) had reacted with dansyl-cadaverine while no alpha-chain remnant took up the label. Additional protein and carbohydrate staining further facilitated identification of the individual subunit chains. At least three critical peptide bonds, located on alpha, beta- and gamma-chain remnants, must be broken during conversion of fragment Y into D and E. Sequential cleavage results in heterogeneous appearance of reduced subunit chains. As a consequence, there exist several molecular entities of fragment Y, all of which may have the same molecular weight though they represent various products of progressive plasmic digestion. Our results are compatible with the model of asymmetric degradation of fibrinogen, according to which fragment X produces 1 mol of fragment E e and 2 mol of the monomeric fragment D.