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.     

FXIII polymorphisms, fibrin clot structure and thrombotic risk

Fibrin clot structure is highly dependent on factor XIII activity. Activated FXIII catalyzes the formation of the peptide bonds between the gamma and alpha chains in noncovalently bound fibrin polymers and incorporates various adhesive and antifibrinolytic proteins into the final fibrin clot. In the absence of activated FXIII, clots are unstable and susceptible to fibrinolysis. Several studies have examined the effects of FXIII polymorphisms on final fibrin clot structure and clinical thrombotic risk. The Val34Leu FXIII polymorphism is associated with increased activation by thrombin. In the presence of saturating thrombin concentrations, however, FXIIIa specific enzyme activity is not affected by genetic polymorphisms. Fibrin clots formed in the presence of the FXIII 34Leu polymorphisms do tend to be thinner and less porous, however. The effects of prothrombin concentrations on clot structure have suggested that thinner clots are more resistant to fibrinolysis and associated with increased thrombotic risk. Most clinical studies of 34Leu FXIII carriers, however, have demonstrated a lower incidence of both venous and arterial thrombosis in carriers of the mutant allele compared to Val/Val carriers. One recent study has suggested that the interactions between FXIII phenotype and plasma fibrinogen concentrations significantly influence clinical thrombotic risk.     

Coagulation Factor XIIIa Substrates in Human Plasma: IDENTIFICATION AND INCORPORATION INTO THE CLOT

Coagulation factor XIII (FXIII) is a transglutaminase with a well defined role in the final stages of blood coagulation. Active FXIII (FXIIIa) catalyzes the formation of ϵ-(γ-glutamyl)lysine isopeptide bonds between specific Gln and Lys residues. The primary physiological outcome of this catalytic activity is stabilization of the fibrin clot during coagulation. The stabilization is achieved through the introduction of cross-links between fibrin monomers and through cross-linking of proteins with anti-fibrinolytic activity to fibrin. FXIIIa additionally cross-links several proteins with other functionalities to the clot. Cross-linking of proteins to the clot is generally believed to modify clot characteristics such as proteolytic susceptibility and hereby affect the outcome of tissue damage. In the present study, we use a proteomic approach in combination with transglutaminase-specific labeling to identify FXIIIa plasma protein substrates and their reactive residues. The results revealed a total of 147 FXIIIa substrates, of which 132 have not previously been described. We confirm that 48 of the FXIIIa substrates were indeed incorporated into the insoluble fibrin clot during the coagulation of plasma. The identified substrates are involved in, among other activities, complement activation, coagulation, inflammatory and immune responses, and extracellular matrix organization.     

Multiple Ligands of von Willebrand Factor-binding Protein (vWbp) Promote Staphylococcus aureus Clot Formation in Human Plasma

Staphylococcus aureus secretes coagulase (Coa) and von Willebrand factor-binding protein (vWbp) to activate host prothrombin and form fibrin cables, thereby promoting the establishment of infectious lesions. The D1-D2 domains of Coa and vWbp associate with, and non-proteolytically activate prothrombin. Moreover, Coa encompasses C-terminal tandem repeats for binding to fibrinogen, whereas vWbp has been reported to associate with von Willebrand factor and fibrinogen. Here we used affinity chromatography with non-catalytic Coa and vWbp to identify the ligands for these virulence factors in human plasma. vWbp bound to prothrombin, fibrinogen, fibronectin, and factor XIII, whereas Coa co-purified with prothrombin and fibrinogen. vWbp association with fibrinogen and factor XIII, but not fibronectin, required prothrombin and triggered the non-proteolytic activation of FXIII in vitro. Staphylococcus aureus coagulation of human plasma was associated with the recruitment of prothrombin, FXIII, and fibronectin as well as the formation of cross-linked fibrin. FXIII activity in staphylococcal clots could be attributed to thrombin-dependent proteolytic activation as well as vWbp-mediated non-proteolytic activation of FXIII zymogen.     

The combined effect of fibrin formation and factor XIII A subunit Val34Leu polymorphism on the activation of factor XIII in whole plasma

The first step in the activation of blood coagulation factor XIII (FXIII) is the proteolytic cleavage of the potentially active A subunit (FXIII-A) by thrombin at Arg37-Gly38. Both fibrin formation and FXIII-A Val34Leu polymorphism influence the rate of proteolytic activation of purified factor XIII, however their relative importance and interaction in determining the time of onset and the rate of FXIII activation in whole plasma have not yet been explored. In the present study it was shown that in plasma, fibrin formation preceded the truncation of FXIII-A by thrombin, the activation process took place exclusively on the surface of newly formed fibrin and activated FXIII remained associated with the fibrin clot. The time of fibrin formation closely correlated with the time of FXIII activation, while there was no significant relationship between the time of FXIII activation and FXIII-A Val34Leu genotype. However, in the case of Leu34 variant the lag phase between fibrin formation and FXIII-A truncation was significantly shorter than in the case of Val34 variant. The results suggest that in whole plasma the onset of FXIII activation is determined by fibrin formation, while the rate of activation is modulated by Val34Leu polymorphism.     

Reversible activation of cellular factor XIII by calcium

Factor XIII (FXIII) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets and macrophages. Plasma FXIII is activated by thrombin cleavage (FXIIIa*) and acts in the final stages of blood coagulation cascade. In contrast, the function and activation of cellular FXIII are less characterized. Cellular FXIII relies on a conformational activation of the protein. The nonproteolytic activation of FXIII to FXIIIa° induced by Ca(2+) alone is well known, but up until now it has been discussed under which conditions the process can be induced and whether it can be reversed. Here, we study the nature of the Ca(2+)-induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIa° because they rely on occurrence of enzyme activity or on active site Cys-314 solvent accessibility. Therefore, an analytical HPLC method was developed that separates zymogen recombinant FXIII (rFXIII) from rFXIIIa°. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca(2+) concentration, buffer, and salt components. Moreover, it is established that Ca(2+) activation of rFXIII is fully reversible, and only 2-5 mm CaCl(2) is sufficient to retain full rFXIIIa° activity. However, below 2 mm CaCl(2) the rFXIIIa° molecule deactivates. The deactivated molecule can subsequently undergo a new activation round. Furthermore, it is demonstrated that thermal stress of freeze-dried rFXIII can induce a new predisposed form that activates faster than nonstressed rFXIII.     

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.     

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.     

Platelet vinculin: a substrate of activated factor XIII

In addition to plasma, Factor XIII of blood coagulation (FXIII) is also present in the cytosol of platelets, monocytes and macrophages. However, its intracellular function has not yet been revealed. Activated Factor XIII (FXIIIa) is a transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) of highly restricted substrate specificity with only a few known protein substrates. In this report, we showed that FXIIIa can link dansylcadaverine, radiolabelled histamine and putrescine to vinculin. Quantitative determinations revealed that in the vinculin molecule a single glutamine residue can serve as acyl donor for the incorporation of small-molecular-weight amines. Vinculin could not be crosslinked to another vinculin molecule. It could be covalently bound, however, to fibrinogen, which indicates that the acyl donor glutamine residue can be engaged in an epsilon-(gamma-glutamyl)lysyl crosslink formation. Since it has been shown that platelet actin and myosin, two main components of cytoskeleton, are also substrates for FXIIIa, and that vinculin is associated to the cytoskeleton during platelet activation, the involvement of FXIII in the stabilization of cytoskeleton at certain phases of cellular function is a likely possibility.     

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)     

Thrombin hydrolysis of V29F and V34L mutants of factor XIII (28-41) reveals roles of the P(9) and P(4) positions in factor XIII activation

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The more easily activated factor XIII V34L has been correlated with protection from myocardial infarction. V34L and V29F factor XIII mutant peptides were designed to further characterize substrate binding to thrombin. HPLC kinetic studies have been carried out on thrombin hydrolysis of FXIII activation peptide (28-41), FXIII (28-41) V34L, FXIII (28-41) V29F, and FXIII (28-41) V29F V34L. The V34L mutations lead to improvements in both K(m) and k(cat) whereas the V29F mutation primarily affects K(m). Interactions of the peptides with thrombin have been monitored by 1D proton line broadening NMR and 2D transferred NOESY studies. The results were compared with previously published X-ray crystal structures of thrombin-bound fibrinogen Aalpha (7-16), thrombin receptor PAR1 (38-60), and factor XIII (28-37). In solution, the (34)VVPR(37) and (34)LVPR(37) segments of the factor XIII activation peptide serve as the major anchor points onto thrombin. The N-terminal segments are proposed to interact transiently with the enzyme surface. Long-range NOEs from FXIII V29 or F29 toward (34)V/LVPR(37) have not been observed by NMR studies. Overall, the kinetic and NMR results suggest that the factor XIII activation peptide binds to thrombin in a manner more similar to the thrombin receptor PAR1 than to fibrinogen Aalpha. The V29 and V34 positions affect, in different ways, the ability of thrombin to effectively hydrolyze the activation peptide. Mutations at these sites may prove useful in controlling factor XIII activation.     

Regulation of formation of factor XIIIa by its fibrin substrates

Thrombin-catalyzed release of activation peptide (AP) from plasma factor XIII was studied to characterize the regulation of this initial step in the activation of factor XIII zymogen (fibrin-stabilizing factor). High-performance liquid chromatography was used to monitor the kinetics of release of AP. Non-cross-linked polymeric fibrins I and II (polymerized des-A- and des-A,B-fibrinogens), physiological substrates of factor XIIIa, were shown to be potent promoters of thrombin-catalyzed release of activation peptide from factor XIII. These promoters are proposed to act by complexing factor XIII and reducing the apparent Km for thrombin-catalyzed release of AP. Since thrombin-catalyzed release of AP is inefficient in the absence of polymerized fibrin, this mode of regulation should minimize formation of factor XIIIa prior to the formation of its fibrin substrates. The promoting activity of polymeric fibrin was rapidly lost when catalytically competent factor XIIIa was allowed to form. This observation suggested the possibility that factor XIIIa catalyzed cross-linking of fibrin inactivates fibrin as a promoter for the thrombin-catalyzed release of AP from factor XIII. Consistent with this view, the thiol reagent S-methyl methanethiosulfonate inactivated factor XIIIa, blocked cross-linking of fibrin, and protected against loss of its promoter activity. This mode of feedback regulation of the activation process by catalytically active factor XIIIa may serve to ensure against continued generation of factor XIIIa after its fibrin substrates have been cross-linked.     

Effect of FXIII on monocyte and fibroblast function.

Factor XIII is a plasma transglutaminase that participates in the final stage of the coagulation cascade. Thrombin-activated FXIII (FXIIIa) catalyzes the formation of covalent crosslinks between gamma-glutamyl and epsilon-lysyl residues on fibrin molecules to yield the mature clot. In addition to its role in hemostasis, FXIIIa was previously shown by us to stimulate endothelial cells to exhibit pro-angiogenic activity. In this work, we studied the effect of FXIIIa on other cells that participate in angiogenesis and tissue repair, such as monocytes and fibroblasts. FXIIIa significantly enhanced migration and proliferation, and inhibited apoptosis of monocytes and fibroblasts. Similar to our previous observations with endothelial cells, the stimulating effect of FXIIIa on monocytes and fibroblasts was elicited via its binding to alpha (v)beta (3) integrin leading to cJun upregulation and TSP-1 downregulation. Since monocytes and fibroblasts are essential components of the tissue repair process, the results of this study, together with the proangiogenic activity of FXIIIa, further substantiate a significant role of FXIII in tissue repair.     

Probing interactions between the coagulants thrombin, Factor XIII, and fibrin(ogen)

Thrombin cleaves fibrinopeptides A and B from fibrinogen leading to the formation of a fibrin network that is later covalently crosslinked by Factor XIII (FXIII). Thrombin helps activate FXIII by catalyzing hydrolysis of the FXIII activation peptides (AP). In the current work, the role of exosites in the ternary thrombin-FXIII-fibrin(ogen) complex was further explored. Hydrolysis studies indicate that thrombin predominantly utilizes its active site region to bind extended Factor XIII AP (FXIII AP 33-64 and 28-56) leaving the anion-binding exosites for fibrin(ogen) binding. The presence of fibrin-I leads to improvements in the K(m) for hydrolysis of FXIII AP (28-41), whereas peptides based on the cardioprotective FXIII V34L sequence exhibit less reliance on this cofactor. Surface plasmon resonance measurements reveal that d-Phe-Pro-Arg-chloromethylketone-thrombin binds to fibrinogen faster than to FXIII a(2) and dissociates from fibrinogen more slowly than from FXIII a(2). This system of thrombin exosite interactions with differing affinities promotes efficient clot formation.     

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background

Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis.

Methodology/Principal Findings

We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis.

Conclusions/Significance

We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.     

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.     

Mapping of factor XIII solvent accessibility as a function of activation state using chemical modification methods

The transglutaminase Factor XIII (FXIII) catalyzes the formation of covalent cross-links between adjacent noncovalently associated fibrin chains in blood coagulation. The resulting covalently cross-linked hard clot is much more mechanically stable and resistant to proteolytic degradation. FXIII is activated by the serine protease thrombin in the presence of calcium ions. Protein modification experiments involving the labeling of cysteine and lysine side chains of the enzyme were performed before and after activation of the enzyme in an effort to gain further insight into structural changes occurring during the activation of FXIII. The experiments revealed differences in the labeling patterns of nonactivated and activated FXIII. These differences result from the exposure or sequestration of specific cysteine or lysine residues when the enzyme is activated, either physiologically with thrombin or nonproteolytically by exposure to calcium. Of note is the acetylation of Lys 73 and Lys 221 upon activation. Both of these residues lie within possible substrate recognition regions of FXIII. The active site Cys 314 is consistently alkylated in the activated enzyme, as is Cys 409, located near the dimer interface. Within the beta-barrel 2 domain of FXIII, Cys 695 becomes alkylated in activated FXIII. Within the same domain, an acetylated Lys (677 or 678), which is observed in the zymogen, cannot be found in the activated enzyme. The results provide a more extensive view of FXIII activation than has been previously available.     

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.     

ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats

The affinities of Factor XIII (FXIII), Factor XIIIa (FXIIla), and cellular transglutaminase (Tg) for fibrinogen (Fgn), fibrin (Fbn), and fibronectin (Fn) were compared using a solid-phase binding assay. Initial rates of binding were as follows: FXIII bound Fbn 3-fold more than Fgn. FXIII did not bind Fn till 20 min. Increasing the ligands concentrations and binding time, resulted in weak binding of FXIII to Fn. FXIIla bound Fbn 2-fold more than Fgn and 28-fold more than Fn. Tg bound Fn 130-fold more than either Fgn or Fbn. At equilibrium, the extent of binding was determined to be as follows: FXIII bound Fbn 3–15-fold more than Fgn and 8-fold more than Fn. FXIIIa bound Fgn and Fbn equally and 12–25-fold more than Fn. FXIIla bound Fgn or Fbn 2-fold and 25-fold greater than FXIII-Fbn and FXIII-Fgn interactions, respectively. Tg bound about equally to Fgn and Fbn and 10–20-fold less than Fn. The K _d s for FXIIla binding to Fn, Fgn, and Fbn were 100, 23, and 19 nM, respectively. The K _d for Tg binding to Fn was 6.5 nM. The binding hierarchies are: [Tg-Fn]>[FXIIIa-Fgn]=[FXIIIa-Fbn]>[FXIII-Fbn]>[FXIIIFgn]=[FXIIIa-Fn]>[Tg-Fbn]=[Tg-Fgn]>[FXIII-Fn]. Such hierarchies could regulate the cross-linkings by FXIIIa and Tg during hemostasis, wound healing, and cell adhesion.Abbreviations Tg cellular transglutaminase - FXIII coagulation factor XIII - FXIIla factor XIIIa (thrombin-activated FXIII) - Fgn human plasma fibrinogen - Fn human plasma fibronectin - Fbn human plasma fibrin (thrombin-cleaved fibrinogen) - ECM extracellular matrix     

Cross-linking of cold-insoluble globulin by fibrin-stabilizing factor.

Cold-insoluble globulin (CI globulin) was purified from human plasma and identified on the basis of its sedimentation coefficient, electrophoretic mobility, and concentration in normal plasma. CI globulin was distinguished from antihemophilic factor (AHF) by amino acid analysis, position of elution from 4% agarose, and electrophoretic migration in polyacrylamide gels in the presence of sodium dodecyl sulfate without prior reduction. CI globulin and AHF could not be distinguished by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction and probably have very similar subunit molecular weights. CI globulin apparently consists of two polypeptide chains, each of molecular weight 2.0 x 10(5), held together by disulfide bonds. CI globulin was a substrate for activated fibrin-stabilizing factor (FSF, blood coagulation factor XIII). FSF catalyzed the incorporation of a fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, into CI globulin and also catalyzed the cross-linking of CI globulin into multimers, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction. In the presence of fibrin, cross-linking of CI globulin by FSF occurred without the formation of CI globulin multimers. Instead, polypeptides with apparent molecular weights of 2.6 x 10(5) and 3.0 x 10(5) were seen. The formation of these polypeptides coincided with the loss of the alpha chain of fibrin and CI globulin. The polypeptides were not seen when fibrin alone was cross-linked. The formation of the polypeptides was greater in fine clots than in coarse clots, and greater in clots incubated at 0 degrees than in clots incubated at 37 degrees. In clots made from purified fibrinogen, CI globulin, and FSF, the concentration of CI globulin in the clot liquor was greater if either FSF or calcium ion was omitted and cross-linking did not take place. These observations suggest that CI globulin is enzymically cross-linked to one of the chains of fibrin, most likely the alpha chain, and is thus covalently incorporated into the fibrin clot. CI globulin is very similar to a protein in the plasma membrane of fibroblasts. The cross-linking of CI globulin to itself and to fibrin may typify reactions also involving the fibroblast membrane protein.