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.     

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.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.     

Employing mutants to study thrombin residues responsible for factor XIII activation peptide recognition: a kinetic study

In the last stages of coagulation, thrombin helps to activate Factor XIII. The resultant transglutaminase introduces covalent cross-links into fibrin thus promoting clot stability. To better understand the roles of individual thrombin residues in recognition and hydrolysis of the Factor XIII activation peptide, mutations within thrombin's aryl and apolar binding site were explored. The thrombin mutants W215A, E217A, W215A/E217A, L99A, and I174A were examined through HPLC kinetics against the substrates FXIII (28-41) V34 AP and FXIII (28-41) V34L AP. Several mutants responded differently to FXIII (28-41) V34 AP vs the cardioprotective V34L AP. W215 provides an important platform for binding and directing FXIII APs for proper hydrolysis. Loss of this platform leads to decreases in kinetics, particularly to the kcat of FXIII V34L AP. E217 also plays a supporting role, but the E217A mutation is not as detrimental as W215A. W215A/E217A is unfavorable for both activation peptides and its coupling effect has been characterized. This mutant can readily bind the peptides but cannot orient them for effective hydrolysis. Kinetic studies with I174A indicate that this thrombin residue is more crucial for interactions with the larger V34L AP segment. The L99A mutation causes deleterious effects to binding and hydrolysis of both APs. The V34L, however, is able to partially compensate for the loss perhaps by increasing contact within the aryl and apolar sites. Understanding how specific FXIII and thrombin residues participate in binding and control hydrolysis may lead to the design of coagulation enzymes whose degree of activation and optimal target site can be controlled.     

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.     

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)     

Cross-linking of salmon fibrinogen and fibrin by factor XIII and transglutaminase

Unlike mammalian species, salmon plasma contains 2 cross-linking enzyme systems: Factor XIII and a transglutaminase which appears to be similar in its action to that described by Folk and Chung. Also, salmon plasma contains an exceedingly active protease which possesses the ability to rapidly destroy fibrinogen clottability even when the plasma is stored at a temperature of ?20°C.     

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.     

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.     

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

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

b-chains prevent the proteolytic inactivation of the a-chains of plasma factor XIII

While the transglutaminase activity is associated exclusively with the thrombin-cleaved a chains of plasma Factor XIII, there is little information regarding the role of the b-chains. The present investigations were undertaken to clarify the role of the b-chains during proteolytic activation of plasma factor XIII a-chains. The a-chains of platelet Factor XIII (a2) were extremely sensitive to alpha-thrombin proteolysis, especially in the presence of 5 mM EDTA, resulting in two major fragments with molecular masses 51 +/- 3 kDa and 19 +/- 4 kDa. Furthermore, fibrin enhanced the alpha-thrombin proteolysis of thrombin-cleaved platelet Factor XIII a-chains in presence of CaCl2 or EDTA, resulting in several peptide fragments with molecular masses from 51 +/- 3 kDa to 14 +/- 4 kDa. By contrast, thrombin-cleaved a-chains of plasma Factor XIII (a2b2) were not further degraded by alpha-thrombin in presence of 5 mM EDTA. Even in the combined presence of 5 mM EDTA and 0.1 mg/ml fibrin, alpha-thrombin proteolysis of plasma Factor XIIIa was limited to the formation of a 76 kDa fragment (= Factor XIIIa), a 51 +/- 3 kDa fragment and trace amounts of a 14 +/- 4 kDa species. Platelet Factor XIII proteolyzed by 500 nM alpha-thrombin in presence of 5 mM EDTA expressed less than 20% of enzymatic activity obtained when platelet Factor XIII was activated in presence of 5 mM CaCl2. In contrast, plasma Factor XIII activated by 500 nM apha-thrombin in presence of 5 mM EDTA expressed nearly 65% of original transglutaminase activity. Likewise, when plasma Factor XIII was proteolyzed by 100-1000 nM gamma-thrombin in presence of 5 mM CaCl2 or 5 mM EDTA, maximal transglutaminase activity was observed. However, when platelet Factor XIII was similarly treated with gamma-thrombin in presence of 5 mM EDTA, only one-half the original transglutaminase activity was obtained. The b-chains thus appear to mimic the function of Ca2+ in preserving transglutaminase activity of thrombin-cleaved a-chains. The b-chains of plasma Factor XIII were not degraded by either alpha- or gamma-thrombin treatment, in presence of 5 mM EDTA or 5 mM CaCl2. Both platelet and plasma Factor XIII a-chains were degraded by trypsin to fragments with molecular masses of 51 +/- 3 kDa and 19 +/- 4 kDa in presence of 5 mM CaCl2 and to fragments with molecular masses of 19 +/- 4 kDa and lower, in presence of 5 mM EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resistance of factor XIII to degradation or activation by plasmin

The effect of plasmin on the subunit polypeptides of factor XIII has been investigated. purified human plasma (a2b2) and platelet (a2) zymogens and the enzyme (a2) were incubated with plasmin at plasmin: factor XIII ratios of 0.03-0.5 casein units per mg protein. Under conditions in which plasmin readily digested fibrinogen and casein, it had no effect on either a2b2 or a2. There was no evidence for cleavage of peptide bonds in the zymogens, and all the potential catalytic activity was retained after prolonged incubation. Similarly a2*, either in the presence or absence of b subunit, was also unaffected by plasmin incubation. 90% of the activity was recovered after incubation of factor XIII with plasmin. b subunit was also not degraded. Additionally, no evidence was obtained that plasmin could activate factor Xiii. These results indicate that in purified systems there is no significant interaction between plasmin and factor XIII.     

Examining thrombin hydrolysis of the factor XIII activation peptide segment leads to a proposal for explaining the cardioprotective effects observed with the factor XIII V34L mutation

In the blood coagulation cascade, thrombin cleaves fibrinopeptides A and B from fibrinogen revealing sites for fibrin polymerization that lead to insoluble clot formation. Factor XIII stabilizes this clot by catalyzing the formation of intermolecular cross-links in the fibrin network. Thrombin activates the Factor XIII a(2) dimer by cleaving the Factor XIII activation peptide segment at the Arg(37)-Gly(38) peptide bond. Using a high performance liquid chromatography assay, the kinetic constants K(m), k(cat), and k(cat)/K(m) were determined for thrombin hydrolysis of fibrinogen Aalpha-(7-20), Factor XIII activation peptide-(28-41), and Factor XIII activation peptide-(28-41) with a Val(34) to Leu substitution. This Val to Leu mutation has been correlated with protection from myocardial infarction. In the absence of fibrin, the Factor XIII activation peptide-(28-41) exhibits a 10-fold lower k(cat)/K(m) value than fibrinogen Aalpha-(7-20). With the Factor XIII V34L mutation, decreases in K(m) and increases in k(cat) produce a 6-fold increase in k(cat)/K(m) relative to the wild-type Factor XIII sequence. A review of the x-ray crystal structures of known substrates and inhibitors of thrombin leads to a hypothesis that the new Leu generates a peptide with more extensive interactions with the surface of thrombin. As a result, the Factor XIII V34L is proposed to be susceptible to wasteful conversion of zymogen to activated enzyme. Premature depletion may provide cardioprotective effects.     

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.     

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.     

Calcium-induced dissociation of human plasma factor XIII and the appearance of catalytic activity

1. The Ca(2+) dependence of the activity of plasma Factor XIII(a) was studied by using the continuous assay based on the incorporation of dansylcadaverine into dephosphorylated acetylated beta-casein (beta-substrate). The K(m) for Ca(2+) is about 0.170mm. 2. At low concentrations of Ca(2+) there was a lag in attaining the steady-state rate. The size of the lag was decreased and eventually abolished if the enzyme was preincubated with a high concentration of Ca(2+) before assay. The concentration of Ca(2+) required to decrease the lag phase by 50% in 10min depended on the protein concentration: at 0.87mg of protein/ml it required 17mm-Ca(2+) and at 0.44mg/ml it needed 10mm-Ca(2+). 3. The concentrations of Ca(2+) required either to abolish the lag phase in the appearance of enzyme activity or to activate the essential thiol for reaction with 5,5'-dithiobis-(2-nitrobenzoate) in 10min incubation were similar at the same protein concentration. This indicated that Ca(2+) induces a conformation change that is responsible for both phenomena. A model is proposed that links this conformation change to the dissociation of the tetrameric enzyme. 4. This was supported by the observation that the addition of excess of b chains to the Factor XIII(a) (a'(2)b(2)) increased the concentration of Ca(2+) required to expose the reactive thiol, and inhibited the Ca(2+)-dependent aggregation of a' chains. 5. Platelet Factor XIII(a) (a'(2)) was inhibited by 5,5'-dithiobis-(2-nitrobenzoate) in the absence of Ca(2+), and no lag phases were observed in attaining the steady-state rate at low Ca(2+) concentrations, thus confirming the model for the activation of the plasma enzyme. 6. The Ca(2+) dependence of platelet Factor XIII(a) indicated that Ca(2+) has an additional role in the enzyme mechanism of the plasma enzyme, perhaps being involved in substrate binding. 7. The dependence of the stability of plasma Factor XIII(a) on Ca(2+) and protein concentration indicates that the decay in activity is related to the tetramer dissociation. 8. beta-Substrate decreased the Ca(2+) concentration required for (1) abolition of the lag phase and (2) enzyme inhibition by thiol reagents. The effect on the former is greater than on the latter. 9. The role of the b chains of the plasma Factor and the evolutionary significance of the plasma and platelet Factors are considered.