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.     

Thrombospondin is a substrate for blood coagulation factor XIIIa

Thrombospondin (TSP) is released from alpha granules of activated platelets, binds to platelet surfaces, and copolymerizes with fibrin. In the present experiments, we investigated the action of factor XIIIa (plasma transglutaminase) on TSP. Factor XIIIa catalyzed incorporation of [14C]putrescine into soluble TSP and ligation of TSP to itself and to fibrin intermediates. Proteolytic digestion of [14C]putrescine-labeled TSP with trypsin or thrombin yielded a labeled disulfide-bonded core of 90 or 120-130 kilodalton (kDa) subunits, labeled fragments of less than 10 kDa, and an unlabeled 30-kDa heparin-binding fragment, indicating the presence of multiple factor XIIIa reactive glutaminyl residues located in several domains of the molecule. TSP became ligated in fibrin clots formed from amidinated fibrinogen, i.e., fibrin that could not contribute lysyl residues to factor IIIa catalyzed cross-links. The disulfide-bonded core of TSP formed upon thrombin digestion copolymerized with fibrin as efficiently as intact TSP. However, a lower proportion of the disulfide-bonded core became ligated. These results indicate that TSP, both in clots and in solution, contributes glutaminyl and lysyl residues to factor XIIIa catalyzed ligation. Cross-linking may be important in stabilizing interactions among TSP, fibrinogen, or fibrin and other molecules in hemostatic plugs.     

Structural features of glutamine substrates for human plasma factor XIIIa (activated blood coagulation factor XIII)

The action of human plasma factor XIIIa (thrombin-activated blood coagulation factor XIII) and guinea pig liver transglutaminase on purified caseins, fibrin, the derivatized gamma chain of fibrin, and a number of synthetic glutamine peptides, and peptide derivatives is reported. There are wide variations in the properties of the individual proteins and peptides as substrates for amine incorporation by the two transglutaminases. beta-Casein and several of its derivatives are excellent substrates for factor XIIIa. However, beta-casein is a relatively poor substrate for the liver enzyme. The primary site of amine incorporation by factor XIIIa in beta-casein was identified as glutamine 167. This was accomplished by labeling with fluorescent amine followed by proteolytic digestion and identification of labeled peptides. An 11-residue peptide and a 15-residue peptide, each containing 1 glutamine residue and each modeled after the primary site of amine incorporation in beta-casein, were prepared. A 13-residue peptide modeled after the primary crosslinking site in fibrin gamma chain was also prepared. Each of these polypeptides proved to be an efficient substrate for factor XIIIa and displayed significantly better substrate properties than a number of small glutamine peptide derivatives that are good substrates for liver transglutaminase.     

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 V is a substrate for the transamidase factor XIIIa

Coagulation Factor V (Mr = 330,000), upon cleavage by thrombin, produces Factor Va, which is composed of two subunits with Mr values of 94,000 and 74,000, along with two activation fragments possessing no known function. Studies were undertaken to assess the ability of the transamidase Factor XIIIa to covalently incorporate the lysine analogs [3H]putrescine and dansylcadaverine into the thrombin-cleaved (activated) and unactivated forms of human and bovine Factor V. The incorporation of either probe into thrombin-activated Factor V proceeded at an initial rate approximately twice that for unactivated Factor V. The extent of the incorporation of [3H]putrescine or dansylcadaverine into activated or unactivated human Factor V was identical; 4 mol of either probe per mol of Factor V. In the case of bovine Factor V, however, while 4 mol of probe were bound per mol of the unactivated pro-cofactor, 5 mol of either lysine analog were covalently linked to 1 mol of thrombin-cleaved Factor V. Polyacrylamide gel fluorography, immunoaffinity chromatography, and immunoprecipitation identified the largest activation fragment of human Factor V (Mr = 150,000) and bovine Factor V (Mr = 120,000) to contain the sites of incorporation of the covalently bound probes. High molecular weight, apparently covalent polymers of Factor V were produced by the action of Factor XIIIa on activated and unactivated human or bovine Factor V. The absence of either probe in the reaction mixtures did not appear to allow an enhancement of protein polymerization.     

Staphylococcus aureus fibronectin-binding protein serves as a substrate for coagulation factor XIIIa: evidence for factor XIIIa-catalyzed covalent cross-linking to fibronectin and fibrin

In this study, we have investigated the interactions of a Staphylococcal recombinant fibronectin-binding protein A (rFnbA) with fibronectin, fibrinogen, and fibrin. Using analytical size-exclusion chromatography, we evaluated the stoichiometry of reversible binding of FnbA to fibronectin and demonstrated that, in solution, it can accommodate at least two molecules of fibronectin. Results of ELISA experiments demonstrated that rFnbA binds with equally high affinity to both immobilized fibrinogen and fibrin. When included into a thrombin-induced fibrin polymerization reaction, rFnbA strongly inhibited fibrin assembly in a dose-dependent manner. In this study, we have shown that rFnbA can act as a substrate for coagulation factor XIIIa. Factor XIIIa catalyzes the incorporation of amine donor (dansylacadaverine) and amine acceptor (peptide patterned on the N-terminal sequence of fibronectin) synthetic probes into rFnbA, suggesting that it serves as a bifunctional substrate containing reactive glutamine and lysine residues. We have demonstrated that the reversible complex formed by rFnbA and fibronectin or rFnbA and fibrin is covalently stabilized by the transglutaminase action of factor XIIIa. Incubation of rFnbA in the presence of either of its ligands and factor XIIIa results in the introduction of intermolecular epsilon-(gamma-glutamyl)lysine isopeptide bond(s) and the formation of high molecular mass heteropolymers. These findings suggest a novel mechanism by which pathogenic Staphylococcus aureus may utilize the transglutaminase activity of factor XIIIa for attachment to soluble proteins, cell surfaces, and matrixes.     

Identification of factor XIIIA-reactive glutamine acceptor and lysine donor sites within fibronectin-binding protein (FnbA) from Staphylococcus aureus

Staphylococcal fibronectin-binding protein (FnbA) is a surface-associated receptor responsible for the reversible binding of bacteria to human fibronectin and fibrin(ogen). Recently we have shown that FnbA serves as a substrate for coagulation factor XIIIa and undergoes covalent cross-linking to its ligands, resulting in the formation of heteropolymers (Matsuka, Y. V., Anderson, E. T., Milner-Fish, T., Ooi, P., and Baker, S. (2003) Staphylococcus aureus fibronectin-binding protein serves as a substrate for coagulation factor XIIIa: Evidence for factor XIIIa-catalyzed covalent cross-linking to fibronectin and fibrin, Biochemistry 42, 14643-14652). Factor XIIIa also catalyzes the incorporation in FnbA of fluorescent probes dansylcadaverine and glutamine-containing synthetic peptide patterned on the NH(2)-terminal segment of fibronectin. In this study, the above probes were utilized for site-specific labeling and identification of reactive Gln and Lys residues targeted by factor XIIIa in rFnbA. Probe-decorated rFnbA samples were subjected to trypsin or Glu-C digestion, followed by separation of labeled peptides using reversed phase HPLC. Sequencing and mass spectral analyses of isolated probe-modified peptides have been employed for the identification of factor XIIIa-reactive Gln and Lys residues. Analysis of dansylcadaverine-labeled peptides resulted in the identification of one major, Gln103, and three minor, Gln105, Gln783, and Gln830, amine acceptor sites. The labeling procedure with dansyl-PGGQQIV probe revealed that Lys157, Lys503, Lys620, and Lys762 serve as amine donor sites. The identified reactive glutamine acceptor and lysine donor sites of FnbA may participate in transglutaminase-mediated cross-linking reactions resulting in the covalent attachment of pathogenic Staphylococcus aureus to human host proteins.     

Incorporation of thrombospondin into fibrin clots

Thrombospondin is a major platelet glycoprotein which is released from platelets during blood coagulation. We examined the interaction of thrombospondin with polymerizing fibrin. Thrombospondin, purified from human platelets and labeled with 125I, became incorporated into clots formed from both plasma and purified fibrinogen. Plasma clots contained somewhat less thrombospondin than clots formed from equivalent concentrations of fibrinogen. In plasma clots and fibrin clots formed in the presence of factor XIII, thrombospondin was cross-linked in the clot; thrombospondin in the supernatant remained largely monomeric. Cross-linking of thrombospondin by factor XIII, however, only slightly increased the amount of thrombospondin which was incorporated into the clot. In contrast, incorporation of 125I-fibronectin into clots was dependent upon cross-linking. Most of the incorporation of 125I-thrombospondin occurred during fibrin polymerization as judged by parallel studies of the incorporation of 125I-fibrinogen. The amount of thrombospondin incorporated into a clot was directly related to thrombospondin concentration and was only weakly dependent on fibrinogen concentration. Incorporation was not saturated at thrombospondin:fibrin (mol/mol) ratios as high as 2/1. Thrombospondin, however, modified the final structure of fibrin clots in a concentration-dependent manner as monitored by opacity. When tryptic digests of 125I-thrombospondin were studied, the 270-kilodalton core became incorporated into fibrin whereas the 30-kilodalton heparin binding fragment was excluded. These results indicate that thrombospondin specifically co-polymerizes with fibrin during blood coagulation and may be an important modulator of clot structure.     

Human complement C3 is a substrate for transglutaminases. A functional link between non-protease-based members of the coagulation and complement cascades

In this study, we report the finding of functional cross-talk between two non-protease components of the complement and coagulation cascades. We show that complement C3, a central component of the complement system, is associated with the fibrin clot and that C3 becomes covalently cross-linked to other proteins during coagulation. Enzymatic incorporation of dansylcadaverine and dansyl-PGGQQIV into C3 by coagulation factor XIIIa and tissue transglutaminase demonstrated that C3 is a transglutaminase substrate. This suggested that coagulation factor XIIIa covalently cross-links C3 to clot components during coagulation. Using mass spectrometry, we verified that C3 indeed is covalently associated with the fibrin clot in a ratio of 0.05:1 relative to the known coagulation factor XIIIa substrate α2-antiplasmin.     

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.     

Sequence location of a putative transglutaminase cross-linking site in human vitronectin.

We attempted to locate the glutamine residue in human vitronectin, susceptible to cross-linking by transglutaminases. Vitronectin was incubated with 14C-labelled putrescine and plasma factor XIIIa and, after reduction and alkylation, the vitronectin was digested with trypsin. HPLC of the digest followed by scintillation counting revealed one major and two minor radioactivity labelled peaks. Sub-digestion with Staphylococcus aureus protease, sequence analysis and mass-spectrometry of the resulting peptides demonstrated that Gln-93 of vitronectin had incorporated putrescine. Additionally, Gln-73, Gln-84 and Gln-86 were found to be minor sites for incorporation.     

Specific binding of blood coagulation factor XIIIa to thrombin-stimulated platelets

We studied the binding of 125I-platelet and plasma Factor XIII (125I-Factor XIII) to human platelets. When 125I-Factor XIII was incubated with gel-filtered platelets, calcium chloride (5 mM) and thrombin (1 unit/ml) at 37 degrees C, saturable binding was observed. Half-maximal binding occurred at 1 min. Binding was inhibited 93% by a 100-fold molar excess of unlabeled ligand but not by other purified proteins. Greater than 87% of platelet-bound radioactivity migrated as thrombin-cleaved a-chains (a'-chains) in sodium dodecyl sulfate-polyacrylamide gels indicating that Factor XIIIa but not Factor XIII binds to platelets. 125I-Factor XIIIa does not bind to unstimulated platelets. When platelet secretion was blocked, binding was markedly inhibited. 125I-Factor XIIIa bound minimally to platelets stimulated with agonists other than thrombin. Thus, binding is dependent on platelet activation, as well as modification of platelets by thrombin. 125I-Factor XIIIa bound to gamma-thrombin-stimulated platelets, at concentrations which did not clot fibrinogen. Therefore, Factor XIIIa is not bound to fibrin associated with platelets. Binding was only partially reversible. Approximately 12,000 molecules of Factor XIIIa were bound per platelet. 125I-Factor XIIIa bound normally to platelets from patients with severe Glanzmann's thrombasthenia indicating that 125I-Factor XIIIa does not bind to platelet glycoproteins IIb or IIIa, or platelet-bound fibrinogen. Chymotrypsin treatment of platelets inhibited 125I-Factor XIIIa binding by 78% without inhibiting secretion. Methylamine and putrescine, Factor XIIIa substrates, and N-ethylmaleimide, an active site inhibitor, did not inhibit binding. Factor XIIIa bound to platelets was enzymatically active and catalyzed [3H]putrescine incorporation into platelet proteins. The specific binding of Factor XIIIa to platelets suggests it may play a role in physiologic reactions involving platelets.     

Transglutaminase-like activity participates in cell wall biogenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Transglutaminases (TGases) catalyze the cross-linking between protein molecules by formation of an amide bond between γ-carboxyamide group of glutamine and the ε-amine group of lysine under deamination of glutamine. We have demonstrated the participation of transglutaminase-like activity in the isolated cell walls and in the process of cell wall regeneration in protoplasts of the yeast Saccharomyces cerevisiae. A radioactive TGase substrate [³H]putrescine was incorporated into the isolated cell walls and into the TCA-insoluble fraction in regenerating protoplasts. The incorporation was increased by adding exogenous artificial substrate of TGase N,N’-dimethylcasein and was inhibited by TGase inhibitor cystamine and/or EDTA. These results suggest the existence of a TGase-type reaction involved in the formation of covalent cross-links between glycoprotein molecules during cell wall construction in S. cerevisiae.     

Measurement of blood coagulation Factor XIIIa formation in plasma containing glycyl-L-prolyl-L-arginyl-L-proline

A method to directly measure the formation of blood coagulation Factor XIIIa in platelet-poor plasma unmodified by heat is described. The synthetic peptide glycyl-L-prolyl-L-arginyl-L-proline, a fibrin-polymerization inhibitor, was used to prevent clotting of platelet-poor plasma. Plasma was diluted to a final concentration of 2.5% (v/v) in 0.1 M Tris-HCl, pH 8.5, buffer containing 25% glycerol, 5 mM calcium chloride, and 0.25 mM glycyl-L-prolyl-L-arginyl-L-proline and then activated by thrombin (20 U/ml) for 15 min. The Factor XIIIa-catalyzed incorporation of [3H]putrescine into Hammersten casein was used to measure Factor XIIIa formation. The assay detected Factor XIIIa in 2.5 to 50 microliter of thrombin-treated plasma. When purified Factor XIII was added to Factor XIII-deficient plasma, there was complete recovery of the Factor XIII added. Glycyl-L-prolyl-L-arginyl-L-proline did not inhibit Factor XIIIa activity in thrombin-treated plasma or purified platelet Factor XIIIa. Glycerol stabilized Factor XIIIa activity in thrombin-treated plasma and buffer for 60 min. The presence of fibrinogen in plasma did not modify the assay results. The time course of thrombin-catalyzed Factor XIIIa formation in platelet-poor plasma containing glycyl-L-prolyl-L-arginyl-L-proline was directly measured using the assay.     

Biotinylated peptides containing a factor XIIIa or a tissue transglutaminase-reactive glutaminyl residue that block protein cross-linking phenomena by becoming incorporated into amine donor sites.

Biotinylated peptides Biot-Gln-Gln-Ile-Val and Biot-epsilon-Aca-Gln-Gln-Ile-Val were shown to act as acceptor substrates for amines in reactions catalyzed by both tissue transglutaminase and coagulation factor XIIIa. Moreover, the peptides could be employed for specifically blocking the potential amine donor sites of protein substrates participating in biological cross-linking with these enzymes. The presence of the biotin label allowed for ready detectability of the marked donor substrates during the cross-linking of crystallins in lens homogenate by the intrinsic transglutaminase and that of the alpha chains of human fibrin by factor XIIIa.     

Fibrinoligase-catalyzed cross-linking of myosin from platelet and skeletal muscle.

Fibrinoligase (thrombin- and calcium-activated Factor XIII) from human plasma catalyzes the incorporation of dansylcadaverine and [¹⁴C]putrescine into myosin, prepared from either human platelets or rabbit skeletal muscle. At least 9 mol of amine is incorporated per mole of myosin of either type when the enzyme is used under saturating conditions. Both heavy and light chains of the platelet and muscle myosins incorporate dansylcadaverine and [ ¹⁴C]putrescine. However, in quantitative terms, the incorporation into the light chains of either type is much less than into the heavy chains. Profound fluorescent changes occurred when dansylcadaverine was bound to myosin. Highly cross-linked platelet and muscle myosin polymers form in the absence of added amines, indicating the presence of both acceptor and donor sites. ATPase activity was not altered by cross-linking of 50–60% of myosin. The nature of the cross-link in myosin was found to be a γ-glutamyl-?-lysine bond, with an average of 19 mol of dipeptide per mole of platelet myosin.     

Lipoprotein (a) is a substrate for factor XIIIa and tissue transglutaminase.

The mechanisms which mediate deposition of lipoprotein (a) (Lp(a)), an atherogenic lipoprotein particle, onto the vessel wall and cell surfaces are unknown. An irreversible deposition of Lp(a) may require the presence of enzymes that catalyze its binding to surface-oriented structures. Transglutaminases catalyze cross-linking of proteins as well as incorporation of primary amines into protein substrates. We studied whether tissue transglutaminase and/or activated Factor XIII (plasma derived or recombinant FXIIIa) incorporate primary amines into Lp(a). In the presence of Ca2+, Factor XIIIa and tissue transglutaminase catalyze incorporation of monodansylcadaverine or [14C]putrescine into purified Lp(a) in a specific and time-dependent manner. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that monodansylcadaverine became incorporated into the apo(a) portion of Lp(a). Lp(a) purified from five different donors showing different apo(a) phenotypes were substrates for tissue transglutaminases (TG). Western blot analysis confirmed that apo(a) was the major monodansylcadaverine carrying protein moiety of Lp(a). Tissue TG also extensively cross-linked the apo(a) portion of the Lp(a) particle. Characterization of the specificity of tissue TG showed that fibronectin, alpha 2-plasmin inhibitor, and apo(a) could be readily labeled with monodansylcadaverine by tissue TG, but other proteins including low density lipoprotein, IgG, alpha 1-proteinase inhibitor, and albumin showed poor or no reactivity. Direct comparison of Lp(a) with low density lipoprotein showed that apoB 100 was a poor substrate for transglutaminases. Recombinant apolipoprotein (a) proved to be an excellent substrate for TGs in that 1 mol of recombinant apolipoprotein (a) incorporated as much as 15 mol of [14C]putrescine, which corresponded to five times the amount of amine incorporated into Lp(a). The susceptibility of Lp(a) to transglutaminases suggests a mechanism whereby the interaction of Lp(a) with surface receptors and other surface oriented structures could be enzymatically altered.     

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.     

Interactions of factor XIII with fibrin as substrate and cofactor.

Factor XIIIa (a2') is a homodimeric transglutaminase that is formed via limited alpha-thrombin-catalyzed proteolysis of the platelet (a2) or plasma (a2b2) factor XIII zymogen in a reaction that results in proteolytic removal of a 37-aminoacyl residue peptide from the N-terminus of the a chains and exposure of the active-site thiol group in the resulting a' chains of factor XIIIa. In this study, we characterized interactions of factor XIII and factor XIIIa with fibrin, a natural substrate for factor XIIIa and a cofactor for the alpha-thrombin-catalyzed activation of plasma factor XIII. The carbamylmethyl derivatives of the active-site thiol group of platelet factor XIII (CMa2) and factor XIIIa (CMa2') were prepared, and their interactions with fibrin were measured. The enzyme-like derivative (CMa2') which contained nicked a' chains bound more tightly to fibrin (Kd = 2.1 microM) than did CMa2 (Kd = 14 microM), the platelet zymogen-like derivative with intact a chains, but the binding of each was weaker than the binding of plasma factor XIII zymogen (a2b2) to fibrin (Kd = 0.20 microM) under the same conditions. Saturation of fibrin with plasma factor XIII zymogen (a2b2) did not affect the binding of CMa2' to fibrin, suggesting that the plasma factor XIII zymogen (a2b2) and the active-site-modified form of factor XIIIa (CMa2') bind to separate, noninteracting sites of fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitronectin is a substrate for transglutaminases

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.