We synthesized three fibrinogen variants, BbetaE397A, BbetaD398A, and BbetaD432A, with substitutions at positions identified in crystallographic studies as critical for binding the "B" peptide, Gly-His-Arg-Pro-amide (GHRPam), to the "b" polymerization site. We examined thrombin- and batroxobin-catalyzed polymerization by turbidity measurements and found that BbetaE397A and BbetaD398A were impaired while BbetaD432A was normal. Changes in polymerization as a function of calcium were similar for variant and normal fibrinogens. We determined crystal structures of fragment D from the variant BbetaD398A in the absence and presence of GHRPam. In the absence of peptide, the structure showed that the alanine substitution altered only specific local interactions, as alignment of the variant structure with the analogous normal structure resulted in an RMSD of 0.53 A over all atoms. The structure also showed reduced occupancy of the beta2 calcium-binding site that includes the side chain carbonyl of BbetaD398, suggesting that calcium was not bound at this site in our polymerization studies. In the presence of peptide, the structure showed that GHRPam was not bound in the "b" site and the conformational changes associated with peptide binding to normal fragment D did not occur. This structure also showed GHRPam bound in the "a" polymerization site, although in two different conformations. Calcium binding was associated with only one of these conformations, suggesting that calcium binding to the gamma2-site and an alternative peptide conformation were induced by crystal packing. We conclude that BbetaE397 and BbetaD398 are essential for the "B:b" interaction, while BbetaD432 is not.
Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: A␣ R16C and ␥ G165R. As seen previously with other heterozygous A␣ R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to A␣ 16 C. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the ␣-helix content in the variant D3 fragment. It is concluded that the A␣-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the ␥-chain induced a conformational change in the D3 fragment. IntroductionFibrinogen, a soluble plasma glycoprotein of 340 kd, is made up of 2 copies of 3 different polypeptide chains (A␣ 2 , B 2 ,␥ 2 ), linked together with 29 interchain and intrachain disulfide bridges. The molecule is organized in a dimeric fashion consisting of a central E domain containing the amino termini of all 6 polypeptide chains and 2 outer D domains. In the final stage of blood coagulation, thrombin cleaves the fibrinopeptides A and B in a sequential manner from the amino termini of the A␣-and B-chains. Cleavage occurs between residues R16 (single-letter amino acid abbreviations) and G17 of the A␣-chain and residues R14 and G15 of the B-chain, exposing the A and B polymerization sites. Resultant monomers join together to form 2-stranded, halfstaggered protofibrils. It has been shown that protofibrils result from longitudinal D-D interactions and noncovalent contacts between the A and the a sites. The a site is formed by residues 329, 330, 340, and 364 in the carboxy-terminal part of the ␥-chain. 1,2 Subsequently, the growing fibrils aggregate in a lateral fashion to form fibers that increase progressively in thickness and develop branch points. The evolving network is finally stabilized with covalent bonds by the activated factor XIII, resulting in a clot resistant to mechanical disruption.Dysfibrinogenemia is a heritable disorder characterized by structural mutations in any of the 3 polypeptide cha...
Functional biochemical properties of 5 batches of the fibrinogen component of a fibrin glue produced by the ZLB Central Laboratory, Bern, each consisting of 4 different in-process samples (taken after the first and second precipitation step, lyophilization, and dry-heat treatment) were studied in vitro. We focused our attention on the effect of the anti-viral treatment of the lyophilized product by dry heat for 1 h at 100 degrees C. A slight reduction in maximal turbidity of all heat-treated samples was observed during the clotting assay compared to nontreated samples. Treatment with dry heat did not result in generation of fibrinogen fragments that might accelerate tissue-plasminogen-activator (t-PA)-enhanced plasminogen to plasmin conversion. The time course of fibrin cross-linking by factor XIII showed no differences between heated and unheated samples. This result indicates that exposure of the fibrinogen component to severe heat neither reduced activity of factor XIIIa nor affected the correct alignment of cross-linking sites in polymerized fibrin. Incubation of fibrinogen with thrombin, plasminogen, and t-PA resulted in a slightly enhanced degradation of fibrin derived from the heat-treated samples. The amount of residual moisture, determined to be within the range of 0.6-2.1% before heat treatment, did not influence clotting, cross-linking, and fibrinolysis parameters. In conclusion, the virus inactivation treatment by dry heat for 1 h at 100 degrees C induces no significant alterations of the in vitro biochemical properties of the fibrinogen component of this fibrin glue.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.