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John W. Weisel - One of the best experts on this subject based on the ideXlab platform.

  • Strong Binding of Platelet Integrin αIIbβ3 to Fibrin Clots: Potential Target to Destabilize Thrombi
    Scientific Reports, 2017
    Co-Authors: Peter Höök, Oleg V. Kim, Shixin Xu, Joel S. Bennett, Mark S. Alber, Zhiliang Xu, Rustem I Litvinov, John W. Weisel
    Abstract:

    The formation of platelet thrombi is determined by the integrin αIIbβ3-mediated interactions of platelets with Fibrinogen and Fibrin. Blood clotting in vivo is catalyzed by thrombin, which simultaneously induces Fibrinogen binding to αIIbβ3 and converts Fibrinogen to Fibrin. Thus, after a short time, thrombus formation is governed by αIIbβ3 binding to Fibrin fibers. Surprisingly, there is little understanding of αIIbβ3 interaction with Fibrin polymers. Here we used an optical trap-based system to measure the binding of single αIIbβ3 molecules to polymeric Fibrin and compare it to αIIbβ3 binding to Monomeric Fibrin and Fibrinogen. Like αIIbβ3 binding to Fibrinogen and Monomeric Fibrin, we found that αIIbβ3 binding to polymeric Fibrin can be segregated into two binding regimes, one with weaker rupture forces of 30–60 pN and a second with stronger rupture forces >60 pN that peaked at 70–80 pN. However, we found that the mechanical stability of the bimolecular αIIbβ3-ligand complexes had the following order: Fibrin polymer > Fibrin Monomer > Fibrinogen. These quantitative differences reflect the distinct specificity and underlying molecular mechanisms of αIIbβ3-mediated reactions, implying that targeting platelet interactions with Fibrin could increase the therapeutic indices of antithrombotic agents by focusing on the destabilization of thrombi rather than the prevention of platelet aggregation.

  • homophenotypic aα r16h Fibrinogen kingsport uniquely altered polymerization associated with slower Fibrinopeptide a than Fibrinopeptide b release
    Blood Coagulation & Fibrinolysis, 2007
    Co-Authors: Dennis K Galanakis, Marguerite Neermanarbez, Tomas Scheiner, Agnes Henschen, Doris Hubbs, Chandrasekaran Nagaswami, John W. Weisel
    Abstract:

    We detail for the first time the uniquely altered Fibrin polymerization of homophenotypic Aalpha R16H dysFibrinogen. By polymerase chain reaction amplification and DNA sequencing, our new proposita's genotype consisted of a G>A transition encoding for Aalpha R16H, and an 11 kb Aalpha gene deletion. High-performance liquid chromatography disclosed Fibrinopeptide A release approximately six times slower than its Fibrinopeptide B. Turbidimetric analyses revealed unimpaired Fibrin repolymerization, and abnormal thrombin-induced polymerization (1-7 mumol/l Fibrinogen, > 96% coagulable), consisting of a prolonged lag time, slow rate, and abnormal clot turbidity maxima, all varying with thrombin concentration. For example, at 0.2-3 U/ml, the resulting turbidity maxima ranged from lower to higher than normal control values. By scanning electron microscopy, clots formed by 0.3 and 3 thrombin U/ml displayed mean fibril diameters 42 and 254% of the respective control values (n = 400). Virtually no such differences from control values were demonstrable, however, when clots formed in the presence of high ionic strength (micro = 0.30) or of monoclonal antibeta(15-42)IgG. The latter also prolonged the thrombin clotting time approximately three-fold. Additionally, thrombin-induced clots displayed decreased elastic moduli, with G' values of clots induced by 0.3, 0.7 and 3 thrombin U/ml corresponding to 11, 34, and 45% of control values. The results are consistent with increased des-BB Fibrin Monomer generation preceding and during polymerization. This limited the inherent gelation delay, decreased the clot stiffness, and enabled a progressively coarser, rather than finer, network induced by increasing thrombin concentrations. We hypothesize that during normal polymerization these constitutive des-BB Fibrin Monomer properties attenuate their des-AA Fibrin counterparts.

  • role of the alpha c domains of Fibrin in clot formation
    Biochemistry, 1994
    Co-Authors: Oleg V Gorkun, Agnes Henschen, Yuri Veklich, Leonid Medved, John W. Weisel
    Abstract:

    The role of the carboxyl-terminal portion of the alpha chains of Fibrin (alpha C domains) in clot formation was investigated by transmission and scanning electron microscopy and turbidity studies of clots made from preparations of molecules missing one or both of these domains. Highly purified and entirely clottable preparations of bovine fragment X Monomer, one containing primarily molecules missing a single alpha C domain (fragment X1) and the other consisting of molecules missing both alpha C domains (fragment X2), were used for these experiments. These preparations were characterized by various methods, including the complete determination of the amino- and carboxyl-termini of all peptides and fragments. These preparations formed clots on dilution to neutral pH. In all cases, clots observed by either scanning or transmission electron microscopy were made up of a branched network of fibers, similar to those formed by thrombin treatment of intact Fibrinogen, suggesting that the alpha C domains are not necessary for protofibril and fiber formation or branching. However, both the fiber and clot structure varied with the different fractions, indicating that the alpha C domains do participate in polymerization. The rate of assembly, as indicated by the lag period and maximum rate of turbidity increase, as well as the final turbidity, was decreased with removal of the alpha C domains, suggesting that they accelerate polymerization. preparations of isolated alpha C fragment added to Fibrin Monomer have striking effects on the turbidity curves, showing a decrease in the rate of polymerization in a dose-dependent manner but not complete inhibition. Electron microscopy of Fibrin Monomer desA molecules at neutral pH showed that most of the alpha C domains, like those in Fibrinogen, remain associated with the central region. Thus, it appears that normally with thrombin cleavage of Fibrinogen the effects of the interactions of alpha C domains observed here will be most significant for lateral aggregation.

Sverre Sandberg - One of the best experts on this subject based on the ideXlab platform.

Peter Höök - One of the best experts on this subject based on the ideXlab platform.

  • Strong Binding of Platelet Integrin αIIbβ3 to Fibrin Clots: Potential Target to Destabilize Thrombi
    Scientific Reports, 2017
    Co-Authors: Peter Höök, Oleg V. Kim, Shixin Xu, Joel S. Bennett, Mark S. Alber, Zhiliang Xu, Rustem I Litvinov, John W. Weisel
    Abstract:

    The formation of platelet thrombi is determined by the integrin αIIbβ3-mediated interactions of platelets with Fibrinogen and Fibrin. Blood clotting in vivo is catalyzed by thrombin, which simultaneously induces Fibrinogen binding to αIIbβ3 and converts Fibrinogen to Fibrin. Thus, after a short time, thrombus formation is governed by αIIbβ3 binding to Fibrin fibers. Surprisingly, there is little understanding of αIIbβ3 interaction with Fibrin polymers. Here we used an optical trap-based system to measure the binding of single αIIbβ3 molecules to polymeric Fibrin and compare it to αIIbβ3 binding to Monomeric Fibrin and Fibrinogen. Like αIIbβ3 binding to Fibrinogen and Monomeric Fibrin, we found that αIIbβ3 binding to polymeric Fibrin can be segregated into two binding regimes, one with weaker rupture forces of 30–60 pN and a second with stronger rupture forces >60 pN that peaked at 70–80 pN. However, we found that the mechanical stability of the bimolecular αIIbβ3-ligand complexes had the following order: Fibrin polymer > Fibrin Monomer > Fibrinogen. These quantitative differences reflect the distinct specificity and underlying molecular mechanisms of αIIbβ3-mediated reactions, implying that targeting platelet interactions with Fibrin could increase the therapeutic indices of antithrombotic agents by focusing on the destabilization of thrombi rather than the prevention of platelet aggregation.

Marschall S. Runge - One of the best experts on this subject based on the ideXlab platform.

  • Fibrin-targeted recombinant hirudin inhibits Fibrin deposition on experimental clots more efficiently than recombinant hirudin.
    Circulation, 1994
    Co-Authors: Christoph Bode, M Hudelmayer, P Mehwald, Stefan Bauer, Freitag M, E. Von Hodenberg, John B. Newell, Wolfgang Kübler, E. Haber, Marschall S. Runge
    Abstract:

    BACKGROUND Although the indirect thrombin inhibitor heparin and the more potent direct inhibitor hirudin are useful in preventing thrombosis, a substantial opportunity remains for improving the thrombus selectivity of thrombin inhibitors. METHODS AND RESULTS To explore the effect of targeting an antithrombin to the surface of a clot, we covalently linked recombinant hirudin to the Fab9 (or IgG) of a monoclonal antibody (59D8) that selectively binds to an epitope on Fibrin that becomes exposed only after thrombin cleaves Fibrinopeptide B. Antibody-coupled hirudin bound to an immobilized peptide of the Fibrin beta-chain amino-terminal sequence and inhibited the peptidolytic activity of thrombin more efficiently than free hirudin. Thrombin inhibition dependent on binding to immobilized Fibrin Monomer was enhanced 1100-fold (P CONCLUSIONS Fibrin-targeted hirudin (in comparison with unmodified hirudin) significantly reduces Fibrin deposition on the surface of experimental clots.

D L Heene - One of the best experts on this subject based on the ideXlab platform.

  • Fibrin detected in plasma of patients with disseminated intravascular coagulation by Fibrin specific antibodies consists primarily of high molecular weight factor xiiia crosslinked and plasmin modified complexes partially containing Fibrinopeptide a
    Thrombosis and Haemostasis, 1997
    Co-Authors: S A Pfitzner, Carl-erik Dempfle, Michio Matsuda, D L Heene
    Abstract:

    : Pooled plasma from 40 patients with severe disseminated intravascular coagulation (DIC) secondary to septic conditions was subjected to gel permeation chromatography on Sephacryl S-500 HR after sample pretreatment with KSCN for dissociation of non-covalent Fibrin complexes. Fibrin antigen in eluates was detected by an array of ELISA tests, using two monoclonal antibodies against Fibrin degradation product D-dimer, a monoclonal antibody against an epitope generated by plasmin cleavage of the D-domain, and an antibody against the neo-N-terminus of the alpha-chain of Fibrin exposed by cleavage of Fibrinopeptide A. Tag antibodies were a polyclonal antibody against the Fibrinogen/ Fibrin D-domain, a POD-conjugated version of the monoclonal antibody against Fibrin alpha-chain neo-N-terminus, and a polyclonal antibody against Fibrinopeptide A. Most Fibrin-related material present in the pooled DIC plasma was of higher molecular mass than Fibrinogen. Fibrin polymers were reactive with antibodies against D-dimer, plasmin cleaved D-domain, and Fibrin alpha-chain neo-N-terminus. Part of the polymers reacted with antibodies against Fibrinopeptide A, indicating presence of Fibrinogen or desA-Fibrin Monomer within the covalently linked complex. In conclusion, the primary analytes detected by monoclonal antibodies for D-dimer, plasmin-specific epitopes of Fibrin degradation products, as well as sites exposed by Fibrinopeptide cleavage in plasma from patients with disseminated intravascular coagulation are high molecular weight factor XIIIa-crosslinked Fibrin complexes, containing plasmin-cleaved D-domains, intact Fibrin Monomer units, and Fibrinogen or desA-Fibrin Monomer.

  • binding of a new monoclonal antibody against n terminal heptapeptide of Fibrin alpha chain to Fibrin polymerization site a effects of Fibrinogen and Fibrinogen derivatives and pretreatment of samples with nascn
    Blood Coagulation & Fibrinolysis, 1993
    Co-Authors: Carl-erik Dempfle, M Dollman, Helmut Lill, D Puzzovio, A Dessauer, D L Heene
    Abstract:

    A novel murine monoclonal antibody against the Fibrin alpha-chain N-terminus is presented, which reacts with desAA- and desAABB-Fibrin. In immunoblot procedures, the antibody reacted with Fibrin degradation products X and Y of non-crosslinked Fibrin, and fragment E. No binding was observed to the Fibrin fragment D-dimer, and Fibrinogen fragments D and E. Minor binding to Fibrinogen fragments X, and Y, and desBB-Fibrin were presumably due to minor contamination with (desAA)-Fibrin. A prerequisite for binding was release of Fibrinopeptides A (FpA), the binding site being a Fibrin-specific neo-epitope. No binding was observed to Fibrinogen or to thrombin-treated dysFibrinogen MANNHEIM III (A alpha 16 Arg-->Cys) molecules, which do not release FpA. The antibody bound to abnormal Fibrin molecules prepared from dysFibrinogen MANNHEIM I (A alpha 19 Arg-->Gly), albeit to a lesser extent than to normal Fibrin. Binding of the antibody to the Fibrin epitope was greatly enhanced by denaturation, e.g. by heat, or by treatment with chaotropic ions. Soluble Fibrin in clinical samples is generally found as a complex with Fibrinogen, since polymerization sites 'A' exposed by release of FpA react with complementary binding sites 'a' on the D-domains of other Fibrin and Fibrinogen molecules. Treatment of samples with NaSCN caused dissociation of Fibrin Monomer complexes. Reassociation was prevented by denaturation of both polymerization sites 'A' and 'a'. The antibody in combination with NaSCN-treatment of samples was useful for specific detection of Fibrin Monomer in plasma samples. Measurement was not influenced by Fibrinogen degradation products, whereas Fibrin degradation products at very high concentration caused some underestimation of Fibrin Monomer concentration.