Separation of human plasma fibrin stabilizing factor (factor XIII) from fibrinogen by fibrinogen polymer formation

Human plasma fibrin stabilizing factor (factor XIII) may be separated from fibrinogen through reversible fibrinogen polymer formation at pH 6.6, gamma/2 0.3, 0 degrees C, and subsequent Bio-Gel A 1.5m filtration. Factor XIII activity is eluted after the monomer fibrinogen peak. Polymer fractions from eight preparations, processed in duplicate, contain a mean 0.002 units factor XIII per mg fibrinogen, or about 0.7% the factor XIII content of standard plasma. Factor XIII-free fibrinogen polymers are easily dissociated (greater than 98%) to the monomer form by incubation at 37 degrees C, 18 hours. The fibrinogen preparations utilized were devoid of plasma fibronectin; thus these studies also show that reversible human fibrinogen polymer formation occurs in its absence.     

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.     

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.     

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)     

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.     

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.     

Acidic residues C-terminal to the A2 domain facilitate thrombin-catalyzed activation of factor VIII

Factor VIII is activated by thrombin through proteolysis at Arg740, Arg372, and Arg1689. One region implicated in this exosite-dependent interaction is the factor VIII a2 segment (residues 711-740) separating the A2 and B domains. Residues 717-725 (DYYEDSYED) within this region consist of five acidic residues and three sulfo-Tyr residues, thus representing a high density of negative charge potential. The contributions of these residues to thrombin-catalyzed activation of factor VIII were assessed following mutagenesis of acidic residues to Ala or Tyr residues to Phe and expression and purification of the B-domainless proteins from stable-expressing cell lines. All mutations showed reduced specific activity from approximately 30% to approximately 70% of the wild-type value. While replacement of the Tyr residues showed little, if any, effect on rates of thrombin-catalyzed proteolysis of factor VIII and consequent activation, the acidic to Ala mutations Glu720Ala, Asp721Ala, Glu724Ala, and Asp725Ala showed decreased rates of proteolysis at each of the three P1 residues. Mutations at residues Glu724 and Asp725 were most affected with double mutations at these sites showing approximately 10-fold and approximately 30-fold reduced rates of cleavage at Arg372 and Arg1689, respectively. Factor VIII activation profiles paralleled the results assessing rates of proteolysis. Kinetic analyses revealed these mutations minimally affected apparent V max for thrombin-catalyzed cleavage but variably increased the K m for procofactor up to 7-fold, suggesting the latter parameter was dominant in reducing catalytic efficiency. These results suggest that residues Glu720, Asp721, Glu724, and Asp725 likely constitute an exosite-interactive region in factor VIII facilitating cleavages for procofactor activation.     

Thrombin activity is unaltered by N-terminal truncation of factor XIII activation peptides

In blood coagulation, thrombin helps to activate factor XIII by cleaving the activation peptide at the R37-G38 peptide bond. The residues N-terminal to the scissile bond are important in determining rates of hydrolysis. Solution studies of wild-type and mutant peptides of factor XIII AP (28-37) suggest residues P(4)-P(1) are most critical in substrate recognition. By contrast, the X-ray crystal structure of FXIII AP (28-37) displays all of the residues, P(10)-P(1), interacting with the thrombin active site in a conformation similar to that of fibrinogen Aalpha (7-16) [Sadasivan, C., and Yee, V. C. (2000) J. Biol. Chem. 275, 36942-36948]. Peptides were therefore synthesized with the N-terminal P(10)-P(6) residues removed to further characterize interactions of thrombin with factor XIII activation peptides. The truncations have no adverse effects on thrombin's ability to bind and to hydrolyze the shortened peptides. The wild-type FXIII AP (33-41) V34 sequence actually exhibits a decrease in K(m) relative to the longer (28-41) sequence whereas the cardioprotective FXIII AP (33-41) V34L exhibits a further increase in k(cat) relative to its longer parent sequence. One-dimensional proton line broadening NMR and 2D transferred-NOESY studies indicate that the shortened peptides maintain similar bound conformations as their FXIII AP (28-37) counterparts. Furthermore, the distinctive NOE between the L34 and P36 side chains is preserved. Kinetic and NMR studies thus reveal that the N-terminal portions of FXIII AP (28-37) (V34 and V34L) are not necessary for effective interaction with the thrombin active site surface. FXIII activation peptides bind to thrombin in a manner more like PAR1 than fibrinogen Aalpha.     

Proteolysis at Arg740 facilitates subsequent bond cleavages during thrombin-catalyzed activation of factor VIII

Thrombin activates factor VIII by proteolysis at three P1 residues: Arg372, Arg740, and Arg1689. Cleavage at Arg372 and Arg1689 are essential for procofactor activation; however cleavage at Arg740 has not been rigorously studied. To evaluate the role for cleavage at Arg740, we prepared and stably expressed two recombinant B-domainless factor VIII mutants, R740H and R740Q to slow and eliminate, respectively, cleavage at this site. Specific activity values for the variants were approximately 50 and 20%, respectively, that of wild-type factor VIII. Activation of factor VIII R740H by thrombin showed an approximately 40-fold reduction in the rate of A2 subunit generation, which reflected an approximately 20-fold reduction in cleavage rate at Arg372. Similarly, a approximately 40-fold rate reduction in cleavage at Arg1689 and consequent generation of the A3-C1-C2 subunit were observed. Rate values for A2 and A3-C1-C2 subunit generation were reduced by >700-fold and approximately 140-fold, respectively, in the R740Q variant. These results suggest that initial cleavage at Arg740 affects cleavage at both Arg372 and Arg1689 sites. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed more modest rate reductions (<10-fold) in generating A2 and A3-C1-C2 subunits from either variant, suggesting that factor Xa-catalyzed activation of factor VIII was significantly less dependent upon prior cleavage at residue 740 than thrombin. Overall, these results support a model whereby cleavage of factor VIII by thrombin is an ordered pathway with cleavage at Arg740 facilitating cleavages at Arg372 and Arg1689, which result in procofactor activation.     

Amino acid sequence of the peptide released from bovine factor XIII following activation by thrombin

The amino acid sequence of the peptide released during the conversion of bovine Factor XIII to the active enzyme by thrombin was determined. It contains N-terminal N-acetylserine and a total of 37 residues. The bovine peptide differs from the corresponding human peptide. There are 5 amino acid replacements and one deletion in the human peptide.     

Activation of factor XIII by beta-thrombin]

It was found that similar to alpha-thrombin, beta-thrombin (possessing a high esterase and only a trace coagulating activities) converts plasmic transglutaminase (factor XIII) into its active form, thus promoting stabilization of fibrin. Activation of pure and plasmic preparations of factor XIII after incubation with beta-thrombin was observed in vitro. alpha-Thrombin at concentration corresponding to the trace coagulating activity of beta-thrombin had no activating effects. An intravenous injection of beta-thrombin to animals with aminazine-inhibited anticoagulating system reflectory arc resulted in an increase of factor XIII activity in the same way as was observed in vitro. On the other hand, an intravenous injection of beta-thrombin to intact animals did not increase factor XIII activity, which may be accounted for by a decrease in the level of factor XIII due to activation of the anticoagulating system.     

Binding of alpha-2-antiplasmin with fibrinogen/fibrin and their fragments independent of factor XIII

Possible interaction of alpha-2-antiplasmin with fibrinogen, fibrin and their fragments independent of factor XIII as well as the inhibitor effect on the Glu-plasminogen activation by tissue activator were studied. It was shown that alpha-2-antiplasmin is adsorbed on desAA- and desAABBfibrin films (Kd 69.0 +/- 1.0 nM 68.6 +/- 5.3 nM, respectively). Glu-Plasminogen has no effect on the inhibitor binding with desAABBfibrin. Alpha-2-antiplasmin shows strong affinity for fibrin D-dimer (Kd 65.0 +/- 4.0 nM) and D-fragment of fibrinogen (Kd 119.0 +/- 21.0 nM), but it does not interact with E-fragment. The inhibitor inside the fibrin clot decreases 10 times the activation rate of Glu-plasminogen by the tissue activator both is the presence and without factor XIII at physiological ratio of Glu-plasminogen, tissue activator, fibrin and alpha-2-antiplasmin. Thus we have shown that fibrinogen/fibrin binds alpha-2-antiplasmin independent of the factor XIII. Binding sites of the inhibitor are localized in D-fragment of fibrinogen and/or fibrin D-dimer. Alpha-2-antiplasmin inhibits the Glu-plasminogen activation by tissue activator on fibrin.     

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.     

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)     

Platelet factor XIII is activated by calpain

The action of calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase) on platelet factor XIII has been studied. Calpain I activated platelet factor XIII up to 76% of the maximum level observed with thrombin. Activation was accompanied by the limited proteolysis of the a subunit of platelet factor XIII to produce a 76 kDa fragment which was comparable to the proteolytic product by thrombin. Activation of platelet factor XIII by calpain was inhibited by EDTA, leupeptin, and endogenous calpain-specific inhibitor calpastatin. These findings suggest that calpain is responsible for the intracellular activation of platelet factor XIII.     

Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C

Thrombin-catalyzed activation of Protein C is accelerated by a human endothelial cell surface cofactor. The cofactor occurs also on mouse hemangioma cells (a transformed endothelial cell line), but not on cultured human smooth muscle cells or fibroblasts. The cofactor remains bound to the cell surface during Protein C activation. The cofactor is saturable with respect to both Protein C (Km = 0.72 +/- 0.07 microM) and thrombin (Km = 0.48 +/- 0.05 nM). Diisopropylphosphoryl-thrombin is a competitive inhibitor of the cofactor-dependent reaction with Ki = 0.56 +/- 0.1 nM. Prothrombin Fragment 1, the peptide derived from prothrombin that retains phospholipid binding capacity, does not inhibit activation of Protein C when present in a 7:1 molar excess over Protein C. Platelet Factor 4 (20 microgram/ml) also fails to inhibit Protein C activation. It is concluded that the endothelial cell provides a surface on which Protein C can be activated under physiological conditions.     

Longtime therapy of congenital factor XIII deficiency using factor XIII concentrate]

Factor XIII was determined by enzymatic and immunochemical methods in 3 patients with congenital factor XIII deficiency. Factor XIII activity measured by trans-glutaminase assay was below 1% of normal value in each of these cases. Immunelectrophoresis determination revealed the absence of the functionally active subunit A, whereas subunit S was only slightly diminished (30 to 50% of the normal value). Substitution with factor XIII concentrate caused a parallel increase of factor XIII activity and subunit A concentration. No uptake of factor XIII activity or of subunit. A by platelets could be demonstrated. Despite discontinuous substitution over a period of six years no antibody against factor XIII activity could be demonstrated in one patient with congenital factor XIII deficiency.