Factor XIIIa-Catalyzed Cross-Linking of Recombinant αC Fragments of Human Fibrinogen†

A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation.

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Clottability and Cross-Linking Reactivity of Fibrin(ogen) Following Differential Release of Fibrinopeptides A and B

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648078 ◽

1976 ◽

Vol 36 (03) ◽

pp. 582-592 ◽

Cited By ~ 12

Author(s):

M Furlan ◽

T Seelich ◽

E. A Beck

Keyword(s):

Early Stage ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Factor Xiiia ◽

Cross Linking ◽

Human Fibrinogen ◽

Prolonged Incubation ◽

Fibrin Networks ◽

Covalently Linked ◽

Bothrops Atrox

SummaryHuman fibrinogen was treated at pH 6.0, 7.3 and 9.0 with thrombin, batroxobin (thrombinlike 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 γ-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 α-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 (γ-γ versus α-α interactions).

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A Hamster Antibody to the Mouse Fibrinogen Chain Inhibits Platelet-fibrinogen Interactions and FXIIIa-mediated Fibrin Cross-linking, and Facilitates Thrombolysis

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616532 ◽

2001 ◽

Vol 86 (10) ◽

pp. 1047-1056 ◽

Cited By ~ 17

Author(s):

Markéta JirouȈsková ◽

Susan Smyth ◽

Bohdan Kudryk ◽

Barry Coller

Keyword(s):

Factor Xiiia ◽

Cross Linking ◽

Murine Models ◽

Clot Retraction ◽

Human Fibrinogen ◽

Hemostatic System ◽

Fibrinogen Binding ◽

C Terminus ◽

New Information ◽

Tissue Plasminogen

SummaryMurine models employing genetically altered mice have the potential to provide important new information about the hemostatic system, but before such data can be extrapolated to humans it is necessary to define the similarities and differences between murine and human hemostasis. After establishing the similarities of murine fibrinogen to human fibrinogen in its pattern of proteolysis in response to plasmin and its cross-linking by factor XIIIa, we studied a new hamster monoclonal antibody (mAb) 7E9 that reacts with the γ chain of mouse fibrinogen. This antibody inhibits platelet adhesion to fibrinogen, platelet-mediated clot retraction, platelet aggregation, and FXIIIa-mediated cross-linking of fibrin; it also facilitates tissue plasminogen activator (tPA)-mediated lysis of fibrin formed either in the absence or presence of platelets. These data provide evidence that the C-terminus of mouse fibrinogen γ chain, like that of human fibrinogen, is involved in fibrinogen binding to platelets and FXIIIa-mediated cross-linking of fibrin. Our data raise the possibility that a therapeutic agent that targets the C-terminus of the γ chain in human fibrinogen might have broad antithrombotic and profibrinolytic effects.

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Localization of the α-Chain Cross-Linking Sites in Human Fibrinogen

10.1055/s-0039-1687459 ◽

1979 ◽

Author(s):

B. Hessel ◽

G. Savidge ◽

B. Blombäck

Keyword(s):

Experimental Data ◽

Calcium Ions ◽

Whole Blood ◽

Cyanogen Bromide ◽

Degradation Products ◽

Factor Xiiia ◽

Antigenic Determinants ◽

Cross Linking ◽

Human Fibrinogen ◽

The Cross

Antisera were raised in rabbits against the fragments Hi2-DSK and N-DSK obtained from cyanogen bromide treatment of human fibrinogen. Sensitive radioimmunoassay techniques were developed to quantitate substances sharing the same antigenic determinants as these fragments. We have found that the cross-linking process in fibrin formation involves the Hi2-DSK region which has its origin in the Aα-chain of fibrinogen. This relationship is based upon the following experimental data: - Fibrin samples produced in the presence and absence of Factor Xllla and Ca++ and thus cross-Linked and non cross-linked respectively were treated with cyanogen bromide. The resulting degradation products were chroraatographed on Sephadex G-100, and radioimmunoassays for Hi2-DSK and N-DSK were performed on the eluates. The elution profiles of Hi2-DSK and N-DSK antigenic determinants demonstrated that Hi2-DSK eluated after N-DSK in non cross-linked fibrin samples but in the cross-linked samples Hi2-DSK eluated before N-DSK. These results demonstrate that the Hi2-DSK region is involved in the cross-linking process in the presence of Factor XIIIa and calcium ions. Fibrin samples produced in the coagulation of whole blood showed a similar cross-linking profile.Supported by The Bank of Sweden Tercentenary Foundation.

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Tridegin, a Novel Peptidic Inhibitor of Factor XIIIa from the Leech, Haementeria ghilianii, Enhances Fibrinolysis In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656085 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0959-0963 ◽

Cited By ~ 18

Author(s):

Lisa Seale ◽

Sarah Finney ◽

Roy T Sawyer ◽

Robert B Wallis

Keyword(s):

Potent Inhibitor ◽

Platelet Rich Plasma ◽

Factor Xiiia ◽

Cross Linking ◽

Fibrinolytic Enzymes ◽

Small Polypeptide ◽

Tissue Plasminogen ◽

Protein Cross Linking

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

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Cross-linking of human fibrinogen with glutaraldehyde and tetranitromethane

Thrombosis Research ◽

10.1016/0049-3848(75)90086-9 ◽

1975 ◽

Vol 7 (6) ◽

pp. 827-838 ◽

Cited By ~ 12

Author(s):

Miha Furlan ◽

Eugene A. Beck

Keyword(s):

Cross Linking ◽

Human Fibrinogen

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Delayed intermolecular ??-chain cross-linking by factor XIIIa in fibrinogen Asahi characterized by a ??-Met-310 to Thr substitution with an N-glycosylated ??-Asn-308

Blood Coagulation & Fibrinolysis ◽

10.1097/00001721-199010000-00039 ◽

1990 ◽

Vol 1 (5) ◽

pp. 557-560 ◽

Cited By ~ 9

Author(s):

K. Yamazumi ◽

K. Shimura ◽

H. Maekawa ◽

S. Muramatsu ◽

S. Terukina ◽

...

Keyword(s):

Factor Xiiia ◽

Cross Linking

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Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽

10.1182/blood.v83.4.1006.1006 ◽

1994 ◽

Vol 83 (4) ◽

pp. 1006-1016 ◽

Cited By ~ 29

Author(s):

AD Cox ◽

DV Devine

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Activation ◽

Factor Xiiia ◽

Cross Linking ◽

Platelet Surface ◽

Activated Platelets ◽

Fibrinogen Binding ◽

A Chain ◽

Functional Receptor

Abstract Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

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