Cross-linking site in fibrinogen for alpha 2-plasmin inhibitor

A plasma proteinase inhibitor, alpha 2-plasmin inhibitor (alpha 2PI), is cross-linked with alpha chain of fibrin(ogen) by activated coagulation Factor XIII (plasma transglutaminase). alpha 2PI serves only as a glutamine substrate (amine acceptor) for activated Factor XIII in the cross-linking reaction, and the cross-linking occurs between Gln-2 of the alpha 2PI molecule and a lysine residue (amine donor) of fibrin(ogen) alpha chain, whose position was investigated. alpha 2PI and fibrinogen were reacted by activated Factor XIII. The resulting alpha 2PI fibrinogen A alpha chain complex was separated and subjected to two cycles of Edman degradation using phenyl isothiocyanate for the first cycle and dimethylaminoazobenzene-isothiocyanate for the second cycle. The aqueous phase after the cleavage stage of the second cycle, containing dimethylaminoazobenzene-thiohydantoin-Gln cross-linked with A alpha chain, was subjected to CNBr fragmentation and tryptic digestion. Only one of the peptides was found to have the peak of absorbance at 420 nm, indicating the presence of dimethylaminoazobenzene-thiohydantoin-Gln in that peptide. The peptide was identified as corresponding to residues Asn-290-Arg-348 of A alpha chain by analyses of the NH2-terminal amino acid sequence and amino acid composition. The peptide contains a single lysine at position 303, indicating that Lys-303 of fibrinogen A alpha chain is the lysine residue that forms a cross-link with Gln-2 of alpha 2PI.     

Factor XIII-mediated cross-linking of NH2-terminal peptide of alpha 2-plasmin inhibitor to fibrin

The NH2-terminal 12-residue peptide of alpha 2-plasmin inhibitor, Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Gly-Leu-Lys-NH2 . AcOH, was found to be a good substrate for plasma transglutaminase (activated blood coagulation factor XIII) and rapidly incorporated into fibrin by the enzyme. A high concentration of the peptide inhibited the enzyme-mediated cross-linking of alpha 2-plasmin inhibitor to fibrin probably by competing with the inhibitor for the same site of fibrin alpha-chain.     

Cross-linking of alpha 2-plasmin inhibitor to fibrin catalyzed by activated fibrin-stabilizing factor

During blood coagulation alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked with fibrin by an activated fibrin-stabilizing factor (FSFa) plasma transglutaminase, activated coagulation factor XIII). When alpha 2PI was treated with FSFa in the absence of acceptor amino groups, the inhibitor lost more than 90% of its capacity to be cross-linked to fibrin because of hydrolysis of the gamma-carboxamides of FSFa-susceptible glutamine residues. Chemical modifications of the inhibitor's lysine epsilon-amino groups did not affect the cross-linking capacity of the inhibitor with fibrin, whereas the same chemical modifications in fibrinogen resulted in a remarkable loss of cross-linking capacity. These observations suggest that alpha 2PI plays a role as an acyl donor with its FSFa-susceptible glutamine residues in the cross-linking reaction with fibrin, and fibrin serves as an acyl acceptor with its lysine residues. The number of FSFa-susceptible glutamine residues/molecule of the inhibitor was estimated by measuring the maximum incorporation of [3H]histamine into the inhibitor and by analyzing the distribution of radioactivity in a tryptic digest of [14C]histamine-incorporated alpha 2PI.l It was found that each inhibitor molecule has one glutamine residue that is most susceptible to FSFa. When the radioactive histamine-incorporated inhibitor was reacted with excess amounts of plasmin, a small fragment carrying all the released radioactivity was rapidly released from the NH2-terminal part of the inhibitor moiety of the complex. The NH2-terminal amino acid sequence of the inhibitor was analyzed before and after treatment with FSFa or before and after incorporation of radioactive histamine. The glutamine residue at the second position from the NH2-terminal end was converted to a glutamic acid residue when the inhibitor was treated with FSFa. When the radioactive histamine-incorporated inhibitor ws analyzed, the radioactivity was found predominantly at the second position from the NH2-terminal end. These results indicate that the glutamine residue susceptible to FSFa in alpha 2PI is located next to the NH2-terminal residue.     

Immunoelectrophoretic characterizations of the cross-linking of fibrinogen and fibrin by factor XIIIa and tissue transglutaminase. Identification of a rapid mode of hybrid alpha-/gamma-chain cross-linking that is promoted by the gamma-chain cross-linking.

Cross-linking of human fibrin by fibrin stabilizing factor (factor XIIIa) and tissue transglutaminase (ti-TG) was examined by immunoprobing electrophoregrams for positive identification of the cross-linked chains. The immunoprobing was carried out by a new, direct staining technique employing composite gels of a porous protein immobilizing matrix (glyoxyl agarose) blended with a removable polyacrylamide filler that eliminates need for Western blotting. We find that the known rapid cross-linking of gamma-chains into gamma 2-dyads by XIIIa is accompanied by co-cross-linking of the gamma 2-dyads with alpha-chains to form hybrid alpha gamma 2-triads. Little or no cross-linking of relatively abundant alpha- and gamma-chain monads into hybrid alpha gamma-dydads accompanies formation of the alpha gamma 2-triads. Thus, formation of the gamma 2-dyads accelerates the hybrid cross-linking. This acceleration is viewed as demonstrating a previously unknown mode of cooperative interaction between alpha- and gamma-chains arising from cross-linking of the D-domains of the molecules. This strengthened interaction is not critically dependent on fibrinopeptide-release, because alpha gamma 2-triads are similarly formed when fibrinogen is cross-linked by XIIIa. Also observed in the study with XIIIa was the formation of small amounts of homologous gamma 3 and gamma 4 oligomers which had been predicted by others to contribute to branching of fibrin strands. Unlike XIIIa, ti-TG acts preferentially on alpha-chains rather than gamma-chains as known. As alpha gamma-dyad, not seen in reactions with XIIIa, is produced concurrent with the homologous alpha-chain cross-linking. Also, three different species of alpha 2-dyads were produced by ti-TG, two of which were not seen in reactions with XIIIa. The differences in product formation revealed by the specific staining are viewed as providing criteria for distinguishing products of XIIIa and ti-TG in biologic specimens.     

Gly-Pro-Arg-Pro modifies the glutamine residues in the alpha- and gamma-chains of fibrinogen: inhibition of transglutaminase cross-linking

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl-L-prolyl-L-arginyl-L-proline (GPRP), a tetrapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the alpha- and gamma-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the gamma-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polymerized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [3H]putrescine and [14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [14C]putrescine incorporation into the alpha- and gamma-chains of fibrinogen without inhibiting beta-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [3H]putrescine into both fibrinogen and Fragment D1, without modifying [3H]putrescine incorporation into casein. GPRP also inhibited intermolecular alpha-alpha-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the alpha-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polymerization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the alpha- and gamma-chains of fibrinogen.     

Cross-linking of plasminogen activator inhibitor 2 and alpha 2-antiplasmin to fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen Aalpha chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysine 303 of the Aalpha chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-alphaC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aalpha chain, respectively. PAI-2 or alpha(2)-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aalpha chain. alpha(2)-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the Aalpha chain. Therefore, PAI-2 and alpha(2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis.     

The binding sites on fibrin(ogen) for guinea pig liver transglutaminase are similar to those of blood coagulation factor XIII. Characterization of the binding of liver transglutaminase to fibrin

The present study represents detailed investigations into the nature of interactions between an intracellular "tissue" transglutaminase and a plasma protein, fibrinogen. We demonstrate a specific, saturable, and reversible binding of transglutaminase to fibrin(ogen). The binding was time- and temperature-dependent, was independent of divalent metal ions, did not require the release of either fibrinopeptide A or B, and was partially inhibited by the presence of sodium chloride or plasma proteins, properties similar to Factor XIII binding to fibrin(ogen). Both Factor XIII and liver transglutaminase also shared similar binding sites on fibrinogen, the A alpha- and the B beta-chains. The binding characteristics of liver transglutaminase were thus similar to Factor XIII binding to fibrin, but there were also important differences. Scatchard analyses of the binding data indicated that the affinity of liver transglutaminase (Kd = 4.17 x 10(-7) M) was at least 40-fold weaker compared with the affinity of Factor XIII to fibrinogen. Consequently, a 20-fold molar excess of Factor XIII a-chains specifically and completely inhibited the binding of liver transglutaminase to des-A-fibrinogen. The association between liver transglutaminase and fibrin(ogen) was also critically controlled by the conformational states of the two proteins. Substances capable of altering the conformation of either transglutaminase (such as guanosine 5'-triphosphate) or of fibrinogen (such as the tetrapeptide Gly-Pro-Arg-Pro and Fragment D) disrupted binding. Excess CaCl2 was able to counteract the effects of guanosine 5'-triphosphate on transglutaminase binding to fibrin. In contrast, Factor XIII binding to fibrin was unaffected by either guanosine 5'-triphosphate, CaCl2, or Gly-Pro-Arg-Pro, suggesting a more stable association between the two proteins. The physiologic implications of transglutaminase-fibrin(ogen) interactions are discussed.     

Babesia bovis (argentina): analysis of fibrinogen-like proteins during infection

Fibrinogen and fibrinogen-like proteins (FLP) were isolated from plasma and serum of cattle acutely infected with Babesia bovis. The sizes and chain structures of these proteins were examined and clotting assays performed. The results indicated that the blood was in a hypercoagulable state due mainly to enhanced production of hydrogen bonded fibrin and offset partly by slight inhibition of chain cross-linking. The latter appeared due to a Factor XIII inhibitor. Reduction of A alpha chains of plasma FLP was not evident, nor could lower molecular weight remnants be regularly detected strongly suggesting that fibrin(ogen) lysis rarely occurred. Similarly the size and chain structure of the majority of noncoagulable FLP of serum was consistent with their being the product of coagulation and not fibrinolysis. Only in heavily infected splenectomized cattle were products from lysed cross-linked fibrin detected and these constituted only about 3% of total serum FLP.     

Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology

We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.     

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance.     

Structure of alpha-polymer from in vitro and in vivo highly cross-linked human fibrin.

Peptides derived from plasmic and cyanogen bromide (CNBr) cleavage of highly cross-linked fibrin were isolated and characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analyses, cyanoethylation, and NH2-terminal analyses. Extended plasmic digestions of human fibrin containing four epsilon-(gamma-glutamyl)lysine cross-links per molecule produced a peptide of alpha-chain origin (Mr congruent to 21,000) which was comprised of a small donor peptide cross-linked to the acceptor site peptide from the middle of the alpha-chain. CNBr cleavage of highly cross-linked in vitro fibrin or of fibrin from a spontaneously formed in vivo arterial embolus produced about three cross-linked species of molecular weights 30,000 to 40,000, each of which contained the largest CNBr fragment (Mr = 29,000) from the alpha-chain. The predominant cross-link-containing CNBr fragments derived their donor group from the near COOH-terminal region of the alpha-chain as judged by difference amino acid compositions and NH2-terminal analyses. Additionally, cross-linked fragments of molecular weights 68,000 to 70,000 which appeared to contain two acceptor site peptides (Mr = 29,000) were detected in minor amounts in the CNBr digests of fibrin formed from whole plasma or from purified, plasminogen-free fibrinogen. No larger polymeric cross-linked CNBr fragment was generated from any of the highly cross-linked fibrin preparations examined. A model for the predominant mode of alpha-chain polymerization is proposed.     

Catalytic properties of a calmodulin-regulated transglutaminase from human platelet and chicken gizzard

The kinetic parameters and some enzymatic characteristics of human platelet and chicken gizzard transglutaminases were determined. Activity of the transglutaminases was regulated by calmodulin. These enzymes co-isolated with alpha-actinin and were dissociated from alpha-actinin by gel filtration and absorption onto a calmodulin affinity column. Silver-stained polyacrylamide gels showed that the protein peak eluted by EGTA from this column contained polypeptides of Mr approximately 58,000 and 63,000. The transglutaminases required Ca2+ for incorporation of monodansylcadaverine into casein and actin substrates. Activity was enhanced 3-fold by calmodulin with a biphasic effect, showing stimulation at 10-200 nM and inhibition at concentrations higher than 300 nM. In the presence of 200 nM calmodulin, half-maximal transglutaminase stimulation was obtained with 2.5 microM free [Ca2+]. Chlorpromazine inhibited calmodulin enhancement of the transglutaminases. Activity of the transglutaminases was independent of proteolytic activation, since inhibitors for Ca2+-dependent proteases failed to inhibit filamin cross-linking. For comparison, factor XIIa, a plasma and platelet transglutaminase, required both Ca2+ and thrombin for activation and was insensitive to calmodulin. The cross-linking pattern of fibrin, fibrin monomers, and fibrinogen by the calmodulin-regulated transglutaminases showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, disappearance of fibrinogen alpha-chains with no decrease of beta- and gamma-chains or formation of gamma-gamma dimers. By autoradiography, cross-linked products of 125I-fibrinogen revealed heavily labeled high molecular weight polymers and polypeptides of Mr 98,000, 116,000, and 148,000; the latter appeared to be a transient species. However, when fibrin, fibrin monomers, and fibrinogen were used as factor XIIIa substrates, gamma-gamma dimers and alpha-polymers were formed. Formation of gamma-gamma dimers was slower with fibrinogen than with fibrin. Iodoacetamide blocked activity of factor XIIIa but not of the calmodulin-regulated transglutaminases.     

Dissociation of fibrinogen and fibronectin binding from transglutaminase-mediated cross-linking at the hepatocyte surface

The interaction of fibrinogen and fibronectin with hepatocytes has been dissociated into distinct binding and cross-linking steps. Binding and cross-linking of 125I-labeled ligands were both decreased by transglutaminase inhibitors, but not by heparin or hirudin. Transglutaminase activity was manifest by Ca2+-dependent incorporation of [14C]putrescine into cells. Preferential cross-linking of fibrinogen A alpha over gamma chains, and lack of inhibition by heparin or hirudin indicates the involvement of tissue transglutaminase, and not Factor XIIIa. Hepatic transglutaminase activity, as well as binding and cross-linking of fibrinogen and fibronectin, were maximally supported by Ca2+, partially supported by Mn2+ and Sr2+, and markedly decreased by Mg2+ and Ba2+. In contrast, Co2+ supported binding but not cross-linking or transglutaminase activity, indicating that binding and cross-linking are dissociable events. This conclusion was corroborated by the finding that fibrinogen fragments D95 and D78 both inhibited Ca2+-dependent fibrinogen binding without being cross-linked themselves. Ligand binding in the presence of either cation was localized to the cell surface as evidenced by its trypsin sensitivity. Thus, fibrinogen and fibronectin binding to hepatocytes is independent of transglutaminase activity, whereas cross-linking of these adhesive macromolecules requires an enzymatically active cellular transglutaminase. In addition, fibrinogen binding appears to be mediated by molecular determinants present in fragment D78.     

Mechanism of fibrin-specific fibrinolysis by staphylokinase: participation of alpha 2-plasmin inhibitor

When the extent of plasminogen activation by staphylokinase (SAK) or streptokinase (SK) was measured in human plasma, SAK barely induced plasminogen activation, whereas SK activated plasminogen significantly. When the plasma was clotted with thrombin, the plasminogen activation by SAK was markedly enhanced, but that of SK was little enhanced. Similarly, in a purified system composed of plasminogen, fibrinogen and alpha 2-plasmin inhibitor (alpha 2-PI, alpha 2-antiplasmin), such a fibrin clot increased the activity of SAK significantly. However, when alpha 2-PI was removed from the reaction system, enhancement of the SAK reaction was not observed. In addition, SAK as distinct from SK, showed very little interference with the action of alpha 2-PI. Plasminogen activation by SAK is thus essentially inhibited by alpha 2-PI, but this reaction is not inhibited in fibrin clots. These results suggest that SAK forms a complex with plasminogen, which binds to fibrin and induces fibrinolysis.     

Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase

In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin-pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction may influence the TSG-6/heavy Chain 2-mediated transfer of heavy chains observed during inflammation.     

Polymerization and gelation of fibrinogen in D2O

The solution properties of fibrinogen and the thrombin-induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions. The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis-Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same. From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O. The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O. SDS-gel electrophoresis on fibrin samples, which were cross-linked by factor XIII, yielded results where the cross-linking of the gamma-chain appeared to be the same in D2O and H2O. The alpha-chain cross-linking was somewhat faster in D2O than in H2O. When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin. The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.     

Histaminylation of fibrinogen by tissue transglutaminase-2 (TGM-2): potential role in modulating inflammation

Plasma fibrinogen plays an important role in hemostasis and inflammation. Fibrinogen is converted to fibrin to impede blood loss and serves as the provisional matrix that aids wound healing. Fibrinogen also binds to cytokine activated endothelial cells and promotes the binding and migration of leukocytes into tissues during inflammation. Tissue transglutaminase (TGM-2) released from injured cells could cross-link fibrinogen to form multivalent complexes that could promote adhesion of platelets and vascular cells to endothelium. Histamine released by mast cells is a potent biogenic amine that promotes inflammation. The covalent attachment of histamine to proteins (histaminylation) by TGM-2 could modify local inflammatory reactions. We investigated TGM-2 crosslinking of several biogenic amines (serotonin, histamine, dopamine and noradrenaline) to fibrinogen. We identified histaminylation of fibrinogen by TGM-2 as a preferred reaction in solid and solution phase transglutaminase assays. Histamine caused a concentration-dependent inhibition of fibrinogen cross-linking by TGM-2. Fibrinogen that was not TGM-2 crosslinked bound to un-activated endothelial cells with low affinity. However, the binding was increased by sevenfold when fibrinogen was cross-linked by TGM-2. Histaminylation of fibrinogen also inhibited TGM-2 crosslinking of fibrinogen and the binding to un-activated HUVEC cells by 75–90 %. In summary, the histaminylation of fibrinogen by TGM-2 could play a role in modifying inflammation by sequestering free histamine and by inhibiting TGM-2 crosslinking of fibrinogen.     

The comparative ability of plasma and tissue transglutaminases to use collagen as a substrate

Heat denatured type I and type III calf skin collagen were found to be substrates for guinea pig liver transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13) but not for active plasma factor XIII (factor XIIIa). Liver transglutaminase was shown to catalyse incorporation of 14C-putrescine into subunits of denatured collagen of both types, cross-linking of the latter into high molecular weight polymers and their co-cross-linking to fibrin and fibrinogen. Factor XIIIa is inactive in these respects. None of these reactions was catalysed by liver transglutaminase and plasma factor XIIIa when nondenatured collagens both soluble or in the forms of reconstituted fibrils served as substrates. Some cross-linking of cleavage products of collagen type I (obtained by treatment with collagenase from human neutrophiles) was induced by liver transglutaminase and factor XIIIa. The results indicate that although appropriate glutamine and lysine residues for a epsilon-(gamma-glutamine) lysine cross-linked formation are present in collagen, the native conformation of collagen prevents the action of liver transglutaminase and factor XIIIa.     

C1 inhibitor cross-linking by tissue transglutaminase

C1 inhibitor, a plasma proteinase inhibitor of the serpin superfamily involved in the regulation of complement classical pathway and intrinsic blood coagulation, has been shown to bind to several components of the extracellular matrix. These reactions may be responsible for C1 inhibitor localization in the perivascular space. In the study reported here, we have examined whether C1 inhibitor could function as a substrate for plasma (factor XIIIa) or tissue transglutaminase. We made the following observations: 1) SDS-polyacrylamide gel electrophoresis and autoradiography showed that C1 inhibitor exposed to tissue transglutaminase (but not to factor XIIIa) incorporated the radioactive amine donor substrate [(3)H]putrescine in a calcium-dependent manner; 2) the maximum stoichiometry for the uptake of [(3)H]putrescine by C1 inhibitor was 1:1; 3) proteolytic cleavage and peptide sequencing of reduced and carboxymethylated [(3)H]putrescine-C1 inhibitor identified Gln(453) (P'9) as the single amine acceptor residue; 4) studies with (125)I-labeled C1 inhibitor showed that tissue transglutaminase was also able to cross-link C1 inhibitor to immobilized fibrin; and 5) C1 inhibitor cross-linked by tissue transglutaminase to immobilized fibrin had inhibitory activity against its target enzymes. Thus, tissue transglutaminase-mediated cross-linking of C1 inhibitor to fibrin or other extracellular matrix components may serve as a mechanism for covalent serpin binding and influence local regulation of the proteolytic pathways inhibited by C1 inhibitor.     

Factor XIIIa incorporates thymosin beta4 preferentially into the fibrin(ogen) alphaC-domains

It was shown recently that tissue transglutaminase and presumably plasma transglutaminase, factor XIIIa, can covalently incorporate into fibrin(ogen) a physiologically active peptide, thymosin beta(4) [(Huff et al. (2002) FASEB J. 16, 691-696]. To clarify the mechanism of this incorporation, we studied the interaction of thymosin beta(4) with fibrinogen, fibrin, and their recombinant fragments, the gamma-module (gamma-chain residues 148-411), and the alphaC-domain (Aalpha-chain residues 221-610) and its truncated variants by immunoblot and ELISA. No significant noncovalent interaction between them was detected in the absence of activated factor XIII, while in its presence thymosin beta(4) was effectively incorporated into fibrin and to a lesser extent into fibrinogen. The incorporation at physiological concentrations of fibrin(ogen) and factor XIII was significant with molar incorporation ratios of thymosin beta(4) to fibrinogen and fibrin of 0.2 and 0.4, respectively. Further experiments revealed that although activated factor XIII incorporates thymosin beta(4) into the isolated gamma-module and alphaC-domain, in fibrin the latter serves as the major incorporation site. This site was further localized to the COOH-terminal portion of the alphaC-domain including residues 392-610.