scholarly journals Isolation of a fibrin-binding fragment from blood coagulation factor XIII capable of cross-linking fibrin(ogen)

1988 ◽  
Vol 256 (3) ◽  
pp. 1013-1019 ◽  
Author(s):  
C S Greenberg ◽  
J J Enghild ◽  
A Mary ◽  
J V Dobson ◽  
K E Achyuthan
Keyword(s):  
Active Site ◽  
Factor Xiii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Coagulation Factor ◽  
Cysteine Residue ◽  
Factor Xiiia ◽  
Platelet Factor ◽  
Transglutaminase Activity ◽  
A Chain

Purified platelet Factor XIII was radioiodinated and then partially degraded by thrombin or trypsin, and a fibrin-binding fragment was identified by autoradiography and immunoblotting following separation by SDS/polyacrylamide-gel electrophoresis. Limited proteolysis of 125I-Factor XIII by thrombin or trypsin produced an 125I-51 kDa fragment and an unlabelled 19 kDa fragment. The 51 kDa fragment was purified by h.p.l.c. on a TSK-125 gel-filtration column. Partial amino acid sequence analysis of the 51 kDa fragment indicated that it was similar in sequence to the Gly38-Lys513 segment in placental Factor XIII a-chain. More than 70% of the 51 kDa fragment bound to fibrin, whereas the 19 kDa fragment did not bind. The active site was localized to the 51 kDa fragment since this fragment expressed transglutaminase activity, cross-linked fibrin and fibrinogen and incorporated iodo[14C]acetamide into the active-site cysteine residue. Isolation of a fibrin-binding fragment expressing transglutaminase activity demonstrates that each a-chain of the dimeric Factor XIIIa could function independently to cross-link fibrin. The fibrin-binding site could play an important role in localizing Factor XIIIa to the fibrin clot.

scholarly journals Regulation of plasma factor XIII binding to fibrin in vitro

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1028-1034 ◽  
Author(s):  
CS Greenberg ◽  
JV Dobson ◽  
CC Miraglia
Keyword(s):  
Factor Xiii ◽  
Specific Binding ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protamine Sulfate ◽  
Factor Xiiia ◽  
Plasma Sodium ◽  
Platelet Factor ◽  
Plasma Factor

Abstract The binding of plasma factor XIII to fibrinogen or fibrin that has been chemically or enzymatically induced to polymerize was studied. Factor XIII binding was assayed using a 3H-putrescine incorporation assay and an 125I-plasma factor XIII binding assay. More than 80% of the native and radiolabeled plasma factor XIII was bound to fibrin I formed by reptilase in EDTA, citrate, or heparin anticoagulated plasma. Plasma factor XIII and 125I-factor XIII was bound (89.6% to 92.5%) to fibrin II formed by thrombin in either citrate or EDTA anticoagulated plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 125I-plasma factor XIII bound to fibrin I or fibrin II formed by reptilase or thrombin in the presence of EDTA demonstrated the b2- subunit remained bound to the a-chains or thrombin-cleaved a-chains. In the presence of calcium chloride and thrombin, the b2-subunit dissociated and factor XIIIa was bound. Protamine sulfate caused fibrinogen polymerization in the absence of divalent cations and reduced both plasma factor XIII and immunologic fibrinogen levels. Fibrinogen polymerized by protamine sulfate bound plasma factor XIII and the a2-subunit of 125I-platelet factor XIII. Plasma factor XIII was also bound to sonicated non-cross-linked fibrin II in either normal plasma or afibrinogenemic plasma. Plasma levels of several coagulation proteins were unchanged after the addition of reptilase, protamine sulfate, or sonicated fibrin to plasma. These results demonstrate that a specific binding site for the a2-subunit of plasma factor XIII is present on polymerized fibrinogen, fibrin I, and fibrin II. Furthermore, the presence of divalent cations, thrombin-cleavage of plasma factor XIII, and release of fibrinopeptides A or B are not required for plasma factor XIII binding to polymerized fibrinogen and fibrin.

RAPID FORMATION OF LARGE MOLECULAR WEIGHT O-P0LYMERS IN CROSSLINKED FIBRIN INDUCED BY HIGH FACTOR XIII CONCENTRATIONS: ROLE OF PLATELET FACTOR XIII.

1987 ◽  
Author(s):  
C W Francis ◽  
V J Marder
Keyword(s):  
Factor Xiii ◽  
Factor Xiiia ◽  
Freezing And Thawing ◽  
Platelet Factor ◽  
Polymer Formation ◽  
Wide Range ◽  
Sequential Addition ◽  
High Concentrations ◽  
A Chain

Following fibrin polymerization, activated factor XIII stabilizes the clot by catalyzing the formation of specific intermolecular covalent crosslinks between pairs of y chains to form dimers and also among two or more a chains to form polymers. We have identified a series of previously uncharacterized a chain polymers with a wide range of sizes, including some with apparent Mr in excess of several million. Additionally, we establish the role of high concentrations of factor XIII in the extent and rate of α-polymer formation and provide evidence that the factor XIII required can be provided by platelets. Using SDS gel electrophoresis, we find that fibrin prepared from purified fibrinogen or from platelet-deficient plasma contains a series of 21 factor XIIIa crosslinked a chain polymers with Mr from 140,000 to 770,000. The mean Mr difference between individual polymers of 32,000 is consistent with a staggered, overlapping sequential addition of monomers to the growing α-polymer chain. In plasma containing no platelets, α-polymer formation was incomplete with residual α-monomer remaining. Progressively higher platelet counts facilitated more rapid crosslinking of a chains into larger polymers. Intact platelets were not required to promote crosslinking, since platelets lysed by freezing and thawing were also effective. Enrichment of plasma with placental factor XIII in an amount equal to that contained in platelets was as effective as platelets in accelerating the rate of formation and increasing the size of α-chain polymers. We conclude that platelets are a principal source of factor XIII for maximal fibrin stabilization, providing a larger quantity than is available from plasma alone and regulating both the rate and extent of α-polymer formation in thrombi or hemostatic plugs at sites of vascular injury.

Transformation of Cellular Factor XIII into an Active Zymogen Transglutaminase in Thrombin-Stimulated Platelets

1995 ◽  
Vol 73 (04) ◽  
pp. 702-705 ◽  
Author(s):  
László Muszbek ◽  
Gizella Haramura ◽  
János Polgár
Keyword(s):  
Blood Coagulation ◽  
Platelet Activation ◽  
Active Site ◽  
Factor Xiii ◽  
Coagulation Factor ◽  
Transglutaminase Activity ◽  
Activated Platelets ◽  
Monocytes And Macrophages ◽  
Stimulation Of

SummaryThe cellular form of blood coagulation factor XIII (FXIII) is present in platelets, monocytes and macrophages. During long-term stimulation of platelets by thrombin cellular FXIII becomes activated and crosslinks proteins, however, the mechanism of its activation has not been elucidated. It was shown that, contrary to plasma FXIII, the intracellular activation of platelet FXIII does not involve proteolysis. FXIII remained intact in thrombin-activated platelets, i.e., the activation peptide was not removed from the molecule. Part of the zymogen FXIII molecules, however, assumed an active configuration as was demonstrated both by the measurement of transglutaminase activity and by active-site-SH titration. These findings clearly indicate that during platelet activation, when intracellular Ca2+ concentration is raised, a slow non-proteolytic transformation of FXIII zymogen into an active transglutaminase occurs.

scholarly journals Regulation of plasma factor XIII binding to fibrin in vitro

Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1028-1034 ◽  
Author(s):  
CS Greenberg ◽  
JV Dobson ◽  
CC Miraglia
Keyword(s):  
Factor Xiii ◽  
Specific Binding ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protamine Sulfate ◽  
Factor Xiiia ◽  
Plasma Sodium ◽  
Platelet Factor ◽  
Plasma Factor

The binding of plasma factor XIII to fibrinogen or fibrin that has been chemically or enzymatically induced to polymerize was studied. Factor XIII binding was assayed using a 3H-putrescine incorporation assay and an 125I-plasma factor XIII binding assay. More than 80% of the native and radiolabeled plasma factor XIII was bound to fibrin I formed by reptilase in EDTA, citrate, or heparin anticoagulated plasma. Plasma factor XIII and 125I-factor XIII was bound (89.6% to 92.5%) to fibrin II formed by thrombin in either citrate or EDTA anticoagulated plasma. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 125I-plasma factor XIII bound to fibrin I or fibrin II formed by reptilase or thrombin in the presence of EDTA demonstrated the b2- subunit remained bound to the a-chains or thrombin-cleaved a-chains. In the presence of calcium chloride and thrombin, the b2-subunit dissociated and factor XIIIa was bound. Protamine sulfate caused fibrinogen polymerization in the absence of divalent cations and reduced both plasma factor XIII and immunologic fibrinogen levels. Fibrinogen polymerized by protamine sulfate bound plasma factor XIII and the a2-subunit of 125I-platelet factor XIII. Plasma factor XIII was also bound to sonicated non-cross-linked fibrin II in either normal plasma or afibrinogenemic plasma. Plasma levels of several coagulation proteins were unchanged after the addition of reptilase, protamine sulfate, or sonicated fibrin to plasma. These results demonstrate that a specific binding site for the a2-subunit of plasma factor XIII is present on polymerized fibrinogen, fibrin I, and fibrin II. Furthermore, the presence of divalent cations, thrombin-cleavage of plasma factor XIII, and release of fibrinopeptides A or B are not required for plasma factor XIII binding to polymerized fibrinogen and fibrin.

Cytofluorometric Identification of Plasmin-Sensitive Factor XIIIa Binding to Platelets

1988 ◽  
Vol 60 (01) ◽  
pp. 088-093 ◽  
Author(s):  
Julie A Kreager ◽  
Dana V Devine ◽  
Charles S Greenberg
Keyword(s):  
Factor Xiii ◽  
Coagulation Factor ◽  
Fluorescence Analysis ◽  
Factor Xiiia ◽  
Size Factor ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Calcium Dependent ◽  
Sensitive Factor ◽  
Crosslinking Reactions

SummaryWe have investigated the binding of blood coagulation factor XIIIa to thrombin-stimulated platelets using cytofluorometric analysis. Washed thrombin-stimulated platelets bound exoge-nously added factor XIIIa in a calcium-dependent reaction. The expression of endogenous platelet factor XIII was also detected on the surface of thrombin-stimulated platelets. When fluorescence analysis was performed based on particle size, factor XIIIa bound to the surface of greater than 95% of particles which contained more than one platelet, but only 50% of single platelets. The binding of factor XIIIa to thrombin-stimulated platelets was inhibited by plasmin. Plasmin also inhibited thrombin-dependent expression of the factor XIIIa binding site on platelets. Experiments in which thrombin-stimulated platelets were incubated with factor XIIIa in the presence of 125I-dimethyl-casein or 3H-putrescine demonstrated that platelets bear both glutamyl and lysyl substrates for factor XIIIa. Thrombin increased the expression of factor XIIIa substrates by platelets. Plasmin inhibited both the expression of factor XIIIa substrates and degraded them. The binding of factor XIIIa to thrombin-stimulated platelets and the availability of factor XIIIa substrates on the platelet surface could provide a mechanism by which factor XIIIa stabilizes the hemostatic plug by promoting crosslinking reactions between platelet membrane proteins and adhesive glycoproteins. In contrast, plasmin inhibition of factor XIIIa binding and crosslinking could disrupt hemostasis.

Enhanced Affinity of Fibrinogen and Factor XIII Induced by Limited Proteolysis

1979 ◽  
Author(s):  
S.I. Chung ◽  
J.E. Folk ◽  
J.S. Finlayson
Keyword(s):  
Form Factor ◽  
Affinity Chromatography ◽  
Factor Xiii ◽  
Tissue Transglutaminase ◽  
Limited Proteolysis ◽  
Factor Xiiia ◽  
Salt Precipitation ◽  
Salting Out ◽  
Platelet Factor ◽  
Plasma Factor

Previous studies have shown that tissue transglutaminase has strong affinity for fibrinogen. In this study, the binding of protransglutaminase, i.e., plasma Factor XIII(a2b2) and platelet Factor XIII (a2), to fibrinogen was examined by ultracentrifugation, exclusion and affinity chromatography, Immunoelectrophoresis, and salt precipitation. Affinity was detected only by salting out and affinity chromatography. Limited proteolysis to form Factor XIIIa (a′2b2 and a′2) enhanced the affinity for fibrinogen. Limited proteolysis of fibrinogen by thrombin or ancrod enhanced affinity for both a2b2 and a2. Fibrin also bound a′2b2 and a′2. The b2 subunit exhibited no affinity for fibrinogen or fibrin. Thus, in the presence of fibrin(ogen), a′2b2 gave rise to a fibrin(ogen)-a′2 complex, and the b2 subunit was liberated.The enhanced affinity for fibrinogen induced by limited proteolysis of Factor XIII suggests that fibrinogen could serve to adsorb active transglutaminase(s) released or generated in the circulation.

scholarly journals Tb(III)-ion-binding-induced conformational changes in platelet factor XIII

1989 ◽  
Vol 257 (2) ◽  
pp. 331-338 ◽  
Author(s):  
K E Achyuthan ◽  
A Mary ◽  
C S Greenberg
Keyword(s):  
Conformational Changes ◽  
Factor Xiii ◽  
Fluorescence Emission ◽  
Factor Xiiia ◽  
Affinity Column ◽  
Platelet Factor ◽  
Proteolytic Activation ◽  
Transglutaminase Activity ◽  
Molar Excess ◽  
And Function

Ca(II) ions are crucial during proteolytic conversion of Factor XIII zymogen into the active enzyme Factor XIIIa. Factor XIII proteolyzed by thrombin or trypsin in the presence of 5 mM-EDTA resulted in rapid inactivation of transglutaminase activity. Factor XIIIa formed by thrombin or trypsin in the presence of 40 microM-Tb(III) ions, however, was indistinguishable from Factor XIIIa formed in the presence of 2-5 mM-Ca(II) ions with respect to molecular mass and transglutaminase activity. Thrombin treatment of Factor XIII in the presence of 1-5 microM-Tb(III) ions resulted in three fragments (76 kDa, 51 kDa and 19 kDa) with simultaneous loss of transglutaminase activity. Tb(III) ions at concentrations greater than 40 microM made platelet Factor XIII resistant to proteolysis by either thrombin or trypsin. Other lanthanide(III) ions [Ln(III) ions] tested [Ce(III), La(III) and Gd(III) ions] functioned similarly to Tb(III) ions during proteolytic activation of Factor XIII. Ln(III) ions (10-100 microM) were unable to replace the Ca(II) ions required for transglutaminase activity of Factor XIIIa. Tb(III) ions also inhibited in a non-competitive manner the transglutaminase activity of Factor XIIIa (Ki 71 microM) even when measured in the presence of 200-fold molar excess of Ca(II) ions. Factor XIII selectively bound to a Tb(III)-chelate affinity column, and could not be eluted by 100 mM-CaCl2. Binding of Tb(III) ions to Factor XIII was demonstrated by fluorescence emission due to Forster energy transfer. A 10(4)-fold molar excess of CaCl2, but not NaCl, partially quenched Tb(III) fluorescence. Low concentrations (5-20 microM) of Tb(III) ions also inhibited the binding of Factor XIII to des-A-fibrinogen by about 43%, whereas higher concentrations (40-100 microM) promoted binding. Conformational changes in Factor XIII consequent to the binding of Tb(III) ions could be responsible for the observed effects on protein structure and function.

scholarly journals Identification of normal human peripheral blood monocytes and liver as sites of synthesis of coagulation factor XIII a-chain

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 579-582
Author(s):  
LJ Weisberg ◽  
DT Shiu ◽  
PR Conkling ◽  
MA Shuman
Keyword(s):  
Peripheral Blood ◽  
Factor Xiii ◽  
Human Peripheral Blood ◽  
Cdna Probe ◽  
Coagulation Factor ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Normal Human ◽  
Normal Human Peripheral Blood ◽  
A Chain

Factor XIII is the fibrin-stabilizing factor that covalently cross- links fibrin monomers to form a highly organized, stable fibrin clot. The plasma form of factor XIII is a heterodimer, a2b2, consisting of two a-chains and two b-chains; the intracellular form, such as in platelets and placenta, is a dimer, a2, consisting of a-chains only. The catalytic function of factor XIII, a transglutaminase, resides in the a-chain. To address questions regarding sites of synthesis of factor XIII a-chain, an EcoRI restriction fragment from the protein- coding region of the factor XIII a-chain cDNA was used as a probe for Northern blot analysis. The cDNA probe showed hybridization with a single approximately 4.0-kilobase (kb) message in poly (A)+ mRNA prepared from normal human peripheral blood monocytes and normal human liver. The results demonstrate conclusively that factor XIII a-chains are actively synthesized in circulating monocytes and in liver. To our knowledge, these data represent the first demonstration of synthesis of any blood coagulation factor in primary uncultured and unstimulated monocytes or macrophage cells.

scholarly journals In vivo radiolabeling of platelet proteins: a new method with identification of platelet factor XIII

Blood ◽  
1980 ◽  
Vol 55 (4) ◽  
pp. 559-563 ◽  
Author(s):  
DM Rider ◽  
RP McDonagh ◽  
J McDonagh
Keyword(s):  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Platelet Factor ◽  
Intravenous Injections ◽  
Molecular Weights ◽  
The Third

Abstract A method for radiolabeling platelets in vivo was developed in which 3H- arginine was injected into the bone marrow of normal dogs. On the third day after injection, a maximum of 6%--7% of the radioactivity had been incorporated into the total platelet mass. This method of isotope administration resulted in a 50--60-fold increase in maximum uptake of radiolabel by platelets, as compared to values obtained by others using intravenous injections of various radioactive compounds. Tritium- labeled platelets were harvested from the animals and then were washed to remove unbound 3H-arginine. On polyacrylamide gel electrophoresis 7 labeled protein bands, with molecular weights ranging from 29,000to 132,000, were obtained from the platelet-soluble fraction. One 3H- containing protein with a molecular weight of 81,000 was identified immunologically and enzymatically as platelet factor XIII.

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