Promotion of thrombin-catalyzed activation of factor XIII by fibrinogen

High-performance liquid chromatography was used to analyze the kinetics of the thrombin-catalyzed release of the activation peptide from the factor XIII zymogen (fibrin-stabilizing factor). The specificity constant (kcat/Km) for this reaction, measured at factor XIII concentrations much below Km, was (0.13-0.16) X 10(6) M-1 s-1 at pH 7.4, mu = 0.15, and 37 degrees C. Separate estimates, obtained from the dependence of the initial rates of release of the activation peptide on the concentration of factor XIII, gave values of 10 (+/- 3) s-1 for kcat and 84 (+/- 30) microM for Km, in terms of ab protomers of the zymogen. The thrombin-mediated release of the activation peptide was dramatically enhanced in the presence of fibrinogen. Furthermore, the time course of release, in relation to that of fibrinopeptide A, suggested that some des-A-fibrinogen species (e.g., alpha 2B beta 2 gamma 2) may be the true activator for promoting the cleavage of the Arg-36 peptide bonds in the a subunits of factor XIII. This observation suggests that generation of factor XIIIa and its substrate (fibrin) is coordinated so that thrombin-mediated zymogen activation proceeds efficiently only after the process of clotting has been initiated by the removal of fibrinopeptide A from fibrinogen.     

Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII

Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.     

Alpha-thrombin-catalyzed activation of human platelet factor XIII: relationship between proteolysis and factor XIIIa activity

The kinetics of activation of platelet factor XIII, an a-subunit dimer, were characterized by determining rate constants for activation peptide (AP) release, generation of activity, and exposure of the active-site thiol group. The specificity constant (kappacat/Km) for alpha-thrombin-catalyzed AP release, 1.2 x 10(5) M-1s-1, was found to be similar to that for AP release from the tetramer plasma factor XIII (a2b2) [Janus, T.J., Lewis, S. D., Lorand, L., & Shafer, J. A. (1983) Biochemistry 22, 6269-6272], implying that the b subunits of plasma factor XIII do not hinder alpha-thrombin-catalyzed cleavage of AP from the a subunit. Platelet factor XIIIa activity was generated at a rate approximately twice the rate of AP release. This difference in rates was shown to be consistent with a reaction pathway for activation of platelet factor XIII wherein full factor XIIIa activity is generated when one AP is removed from the dimeric zymogen so that removal of the second AP has no detectable effect on catalytic activity. In accord with this conclusion, the rate constant for exposure of the active-site thiol group, as measured by the incorporation of [1-14C]-iodoacetamide, was about twice that observed for the removal of AP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of thrombospondin as a substrate for factor XIII transglutaminase

Thrombin activation of platelets induces the release of a high molecular weight glycoprotein, thrombospondin. On treatment with factor XIII transglutaminase and [3H]putrescine, thrombospondin undergoes specific incorporation of this labeled amine, with 2-3 mol of putrescine being incorporated per mol of thrombospondin. Analysis of plasmin digests of [3H]putrescine-thrombospondin showed that the Mr 53,000-core peptide contains the glutamine site for amine incorporation. In the absence of amine substrate, thrombospondin was found to provide both donor (glutamine) and acceptor (lysine) sites for intermolecular cross-links by factors XIIIa, and high molecular weight protein complexes were formed. Homopolymers of thrombospondin were also observed by electron microscopy. Thrombin-cleaved thrombospondin has more cross-linking sites accessible for [3H]putrescine incorporation or for cross-linkage to itself than does the uncleaved native protein. Examination of thrombospondin cross-linkage in the presence of other protein substrates (fibronectin, collagen, fibrinogen, and von Willebrand factor) for factor XIIIa, resulted in reduced thrombospondin polymer formation. Electron microscopy and autoradiography of fibrin clots formed in the presence of 125I-thrombospondin showed an association of thrombospondin with fibrin fibrils. However, confirmation that this association involves covalent epsilon-(gamma-glutamyl)lysyl cross-links between thrombospondin and fibrin was not obtained.     

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)     

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.     

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.     

Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII

Steady-state kinetic parameters were compared for the action of alpha- and gamma-thrombin on the physiologically important thrombin substrates fibrinogen and factor XIII at 37 degrees C, pH 7.4, and 0.14 M NaCl. gamma-Thrombin, an alpha-thrombin derivative proteolytically cleaved at R-B73 and K-B154, was observed to catalyze the release of fibrinopeptide A (FPA) from fibrinogen with a specificity constant (kcat/Km) of 5 X 10(3) M-1 s-1. This value was approximately 2400-fold lower than the specificity constant for the corresponding alpha-thrombin-catalyzed reaction. The low specificity constant was attributed to an increase in Km and a decrease in kcat for gamma-thrombin-catalyzed release of FPA from fibrinogen. Conversion of alpha-thrombin to gamma-thrombin also resulted in an approximately 800-fold reduction in the specificity constant for thrombin-catalyzed release of fibrinopeptide B (FPB) from fibrin I, as well as a loss in discriminatory power. Whereas alpha-thrombin preferentially released FPA from intact fibrinogen, gamma-thrombin released FPA and FPB from intact fibrinogen at similar rates. In contrast to the large difference in specificity constants observed for alpha- and gamma-thrombin catalysis with fibrin(ogen) as substrate, the specificity constant (2.6 X 10(4) M-1 s-1) observed for gamma-thrombin-catalyzed release of activation peptide from factor XIII was only 5-fold lower than the corresponding value for the alpha-thrombin-catalyzed reaction. Additionally, the promotion of factor XIII activation by fibrin characteristic of the alpha-thrombin-catalyzed reaction did not occur in the gamma-thrombin-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of gamma A/gamma ' fibrinogen in plasma factor XIII activation

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, gammaA/gammaA fibrin, or a minor variant form, gammaA/gamma' fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, gammaA/gamma' fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin gammaA and gamma' chains and a significantly more rapid cross-linking of fibrin alpha chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, gammaA/gamma' fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.     

Inhibition of factor XIII activation by an anti-peptide monoclonal antibody.

As the final enzyme in the coagulation cascade, activated fibrin stabilizing factor or factor XIII catalyzes the intermolecular cross-linking of fibrin chains. To study this enzyme in plasma, we derived a monoclonal antibody (MAb 309) against a peptide sequence (NH2-G-V-N-L-Q-E-F-C-COOH) in the thrombin activation site of factor XIII. Radioimmunoassays indicate that MAb 309 binds specifically to both platelet and plasma factor XIII. Peptide inhibition studies demonstrate that the MAb binds equally well to the factor XIII (FXIII) zymogen and the active form of FXIII (FXIIIa). In immunoblots of whole platelet lysates, MAb 309 binds only to FXIII and does not cross-react with other proteins. In saturation binding studies, the antibody shows a binding avidity of (1.75 +/- 0.35) x 10(9) M-1. MAb 309 also inhibited 99% of apparent FXIIIa activity in a standard transglutaminase assay. SDS-PAGE analysis of fibrin clots showed that MAb 309 inhibited fibrin gamma-gamma cross-linking. Moreover, MAb 309 accelerated the lysis of plasma clots, consistent with inhibition of fibrin-fibrin and fibrin-alpha 2-antiplasmin cross-linking. Immunoblotting experiments revealed that MAb 309 affected apparent FXIIIa activity by inhibiting the thrombin activation of the FXIII zymogen. In addition to its utility as a specific probe for the FXIII a-subunit, the strategy used to obtain MAb 309 may be used to generate MAbs that inhibit the activation of other coagulation factor zymogens.     

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.     

Characterization of the fibrin polymer structure that accelerates thrombin cleavage of plasma factor XIII

The effect of plasmin-derived fibrin(ogen) degradation products on alpha-thrombin cleavage of plasma Factor XIII was studied to identify the fibrin polymer structure that promotes Factor XIIIa formation. Fibrin polymers derived from fibrinogen and Fragment X enhanced the rate of thrombin cleavage of plasma Factor XIII in plasma or buffered solutions. The concentrations of fibrinogen and Fragment X that promoted half-maximal rates of Factor XIIIa formation were 5 and 40 micrograms/ml, respectively. Fragments Y, D, E, D-dimer, and photooxidized fibrinogen did not enhance thrombin cleavage of Factor XIII. Although purified Fragment D1 inhibited fibrin gelation, the soluble protofibrils promoted thrombin activation of Factor XIII. Noncrosslinked fibrin fibers failed to enhance thrombin cleavage of Factor XIII. In conclusion, soluble fibrin oligomers function to promote thrombin cleavage of plasma Factor XIII during blood clotting.     

Expression of functional human coagulation factor XIII A-domain in plant cell suspensions and whole plants

Coagulation factor XIII, a zymogen present in blood as a tetramer (A2B2) of A- and B-domains, is one of the components of many "wound sealants" which are proposed for use or currently in use as effective hemostatic agents, sealants, and tissue adhesives in surgery. After activation by alpha-thrombin cleavage, coagulation factor XIII A-domain, a transglutaminase, is formed and catalyzes the covalent cross-linking of the alpha- and gamma-chains of linear fibrin to form homopolymers, which can quickly stop bleeding. We have successfully expressed the A-domain of factor XIII in both plant cell cultures and whole plants. Transgenic plant cell culture allows a rapid method for testing production feasibility while expression in whole plants demonstrates an economic production system for recombinant human plasma-based proteins. The expressed factor XIII A-domain had a similar size as that of human plasma-derived factor XIII. Crude plant extract containing recombinant factor XIII A-domain showed transglutaminase activity with monodansylcadaverine and casein as substrates and cross-linking activity in the presence of linear fibrin. The expression of factor XIII A-domain was not affected by plant leaf position.     

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.     

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.     

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.     

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.     

Cross-Linking of Fibronectin to C-Terminal Fragments of the Fibrinogen α-Chain by Factor XIIIa

Fibronectin binds specifically to fibrin and is covalently cross-linked to the fibrin α chain by activated factor XIII (XIIIa). This reaction is important for wound healing. Here we investigate XIIIa-catalyzed cross-linking of fibronectin and some of its fragments to a recombinant fragment representing the COOH-terminal 30kDa of the fibrin α chain (αC30K:His 368–Val 610). Only fibronectin and those fragments containing an intact NH₂-terminus were able to form cross-linked complexes. As many as 10 of the 17 lysines in αC30K can serve as amine donors in this reaction. Analysis of the rate of XIIIa-catalyzed cross-linking of fibronectin NH₂-terminal peptides and fragments with αC30K revealed that the presence of the first type I “finger” module accelerates the cross-linking reaction; addition of fingers 2–5 had no further effect.     

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.     

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.