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)     

Role of calcium ion in the generation of factor XIII activity

The involvement of calcium ion in the activation of both plasma factor XIII (alpha 2 beta 2) and platelet factor XIII (alpha 2) was investigated. The second-order dependence of the rate constant for exposure of the active-site thiol group of alpha-thrombin-cleaved plasma factor XIII (alpha 2'beta 2) on the concentration of calcium ion suggested that the binding of two calcium ions is required for transformation of the alpha 2'beta 2 tetramer to enzymatically active factor XIIIa. Fibrinogen, previously reported to lower the calcium ion concentration required for efficient activation of alpha 2'beta 2 [Credo, R. B., Curtis, C. G., & Lorand, L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4234-4237], was found in the present study to increase the rate of exposure of the active-site thiol group. Whereas calcium ion is required for exposure of the active-site thiol group in cleaved plasma factor XIII (alpha 2'beta 2), exposure of an active-site thiol group in cleaved platelet factor XIII (alpha 2') occurs in the absence of calcium ion. The rate constant (2.2 x 10(5) M-1 s-1) for alpha-thrombin-catalyed exposure of the active-site thiol group of platelet factor XIII zymogen (alpha 2) in the presence of calcium ion was greater than the rate constant (0.7 x 10(5) M-1 s-1) determined in the absence of calcium ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

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.     

Tb(III)-ion-binding-induced conformational changes in platelet factor XIII.

Ca(II) ions are crucial during proteolytic conversion of Factor XIII zymogen into the active enzyme Factor XIIIa. Factor XIII proteolyzed by thrombin or trypsin in the presence of 5 mM-EDTA resulted in rapid inactivation of transglutaminase activity. Factor XIIIa formed by thrombin or trypsin in the presence of 40 microM-Tb(III) ions, however, was indistinguishable from Factor XIIIa formed in the presence of 2-5 mM-Ca(II) ions with respect to molecular mass and transglutaminase activity. Thrombin treatment of Factor XIII in the presence of 1-5 microM-Tb(III) ions resulted in three fragments (76 kDa, 51 kDa and 19 kDa) with simultaneous loss of transglutaminase activity. Tb(III) ions at concentrations greater than 40 microM made platelet Factor XIII resistant to proteolysis by either thrombin or trypsin. Other lanthanide(III) ions [Ln(III) ions] tested [Ce(III), La(III) and Gd(III) ions] functioned similarly to Tb(III) ions during proteolytic activation of Factor XIII. Ln(III) ions (10-100 microM) were unable to replace the Ca(II) ions required for transglutaminase activity of Factor XIIIa. Tb(III) ions also inhibited in a non-competitive manner the transglutaminase activity of Factor XIIIa (Ki 71 microM) even when measured in the presence of 200-fold molar excess of Ca(II) ions. Factor XIII selectively bound to a Tb(III)-chelate affinity column, and could not be eluted by 100 mM-CaCl2. Binding of Tb(III) ions to Factor XIII was demonstrated by fluorescence emission due to Forster energy transfer. A 10(4)-fold molar excess of CaCl2, but not NaCl, partially quenched Tb(III) fluorescence. Low concentrations (5-20 microM) of Tb(III) ions also inhibited the binding of Factor XIII to des-A-fibrinogen by about 43%, whereas higher concentrations (40-100 microM) promoted binding. Conformational changes in Factor XIII consequent to the binding of Tb(III) ions could be responsible for the observed effects on protein structure and function.     

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)     

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.     

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.     

Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide

Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.     

An equilibrium study of metal ion binding to human plasma coagulation factor XIII.

1. The binding of Ca2+ to plasma coagulation Factor XIII from man and from cow caused a small decrease in the intrinsic fluorescence of the protein with a dissociation constant of 0.1 mM. A similar decrease was observed with the thrombin-activated Factors (Factors XIIa). The decrease in protein fluorescence was also caused by both Ni2+ and Mn2+ but not by Mg2+. 2. 45Ca2+ binding was directly demonstrated by equilibrium dialysis. Ca2+ at 0.2 mM bound to Factor XIII (a2b2) and Factor XIIIa (a'2b2) but not to isolated b2-protein. A tight-binding site for Ca2+ is associated with the a-subunits. 3. The Ca2+ essential for the enzyme activity of Factor XIII from man, pig and cow can be replaced by Ni2+, Cu2+, La3+, Mn2+, Fe3+, Y3+, Co2+, Sr2+ or Tb3+, but not by Mg2+.     

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)     

Protein-protein interactions in blood clotting. The use of polarization of fluorescence to measure the dissociation of plasma factor XIIIa.

1. Human plasma Factor XIII (the precursor of fibrin-glutamine-fibrin-lysine endo-gamma-glutamyltransferase) was randomly labelled by incubation with fluorescein isothiocyanate. The biochemical properties of the system were unaltered by the label. The polarization of the fluorescein fluorescence attached to the plasma protein was measured and the following conclusions were reached. 2. Factor XIII (a'2b2) does not dissociate in the protein-concentration range 10-500 microgram/ml either with or without added Ca2+. 3. Factor XIIIa (a'2b2) does not dissociate in the absence of Ca2+ in the protein-concentration range 10-500 microgram/ml. 4. Additions of Ca2+ to Factor XIIIa result in a decreased polarization of fluorescence as the tetramer dissociates. The decrease in polarization was the same amplitude at protein concentrations 10-500 microgram/ml and Ca2+ concentrations 2-66 mM and indicates that the overall process is essentially irreversible. The decrease in polarization consisted of fast and slow exponential phases. Both the rate of the fast phase and the proportion of the reaction it represented increased with Ca2+ concentration. 5. A comparison of the rate of dissociation measured by fluorescence polarization and the rate of appearance of enzyme activity in the presence of a protein substrate suggests that the Factor XIII is autoactivated by a soluble a-subunit-containing molecular forming a tight complex with the substrate.     

Isolation of a fibrin-binding fragment from blood coagulation factor XIII capable of cross-linking fibrin(ogen).

Purified platelet Factor XIII was radioiodinated and then partially degraded by thrombin or trypsin, and a fibrin-binding fragment was identified by autoradiography and immunoblotting following separation by SDS/polyacrylamide-gel electrophoresis. Limited proteolysis of 125I-Factor XIII by thrombin or trypsin produced an 125I-51 kDa fragment and an unlabelled 19 kDa fragment. The 51 kDa fragment was purified by h.p.l.c. on a TSK-125 gel-filtration column. Partial amino acid sequence analysis of the 51 kDa fragment indicated that it was similar in sequence to the Gly38-Lys513 segment in placental Factor XIII a-chain. More than 70% of the 51 kDa fragment bound to fibrin, whereas the 19 kDa fragment did not bind. The active site was localized to the 51 kDa fragment since this fragment expressed transglutaminase activity, cross-linked fibrin and fibrinogen and incorporated iodo[14C]acetamide into the active-site cysteine residue. Isolation of a fibrin-binding fragment expressing transglutaminase activity demonstrates that each a-chain of the dimeric Factor XIIIa could function independently to cross-link fibrin. The fibrin-binding site could play an important role in localizing Factor XIIIa to the fibrin clot.     

Influence of cysteine proteinase inhibitors on platelet and plasma components of blood coagulation system

Factor XIIIa plays an important role in stabilization of formed fibrin clot during blood coagulation. Recent studies proved that factor XIIIa affects formation of coated platelets, which are highly procoagulant and characterized by a high level of alpha-granular proteins on their surface and expose surface phosphatidylserine after platelet activation. The ability of newly found cysteine proteinase inhibitors (CPIs) from plants to affect thiol group of the factor XIIIa active centre was recently discovered. Here, the effect of CPIs on the formation of coated platelets and activity of plasma components during blood coagulation process was investigated. It was found that CPIs dose-dependently decreased the fraction of coated platelets in the total platelet population during platelet activation and decreased endogenous thrombin potential (ETP) by 40% for thrombin generation in platelet-rich as well as in platelet-poor plasma. Such decrease of ETP could not be explained by the CPIs influence on factor XIIIa. Investigation of the effects of these inhibitors on factor Xa and thrombin activity has shown that CPIs dose-dependently inhibited their activity and might cause an ETP decrease. Thus, the obtained data indicated that CPIs affected both platelet and plasma components of blood coagulation system.     

Platelet factor XIII. The collagen receptor?

We have studied the binding of collagen fibers with platelet proteins using affinity chromatography on collagen-Sepharose. Only a few proteins from a platelet lysate were trapped by this column. When denatured collagen (gelatin) was used as the affinity ligand, the major protein did not bind and was identified as platelet Factor XIII by polyacrylamide gel electrophoresis, immunoprecipitation, and enzymic activity. This is a zymogen form of transglutaminase, which corresponds to the "a" subunit of the coagulation factor in plasma. Immunoglobulins specific for platelet Factor XIII obtained from antiserum raised against plasma Factor XIII were able to initiate platelet aggregation by themselves, in strong contrast to nonspecific antibodies. This specific immunoglobulin-mediated platelet aggregation required the presence of Ca2+. It was inhibited by aspirin and prostacyclin, but not by specific inhibitors for other agonists. These data suggest the possibility that the zymogen form of Factor XIII is located on the surface of platelets and may play a key role as the receptor for collagen-induced platelet aggregation.     

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.     

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.     

Change in factor XIII activity in plasma during thrombocyte aggregation]

On addition to rat plasma, platelet-enriched, of active factor XIII (XIIIa), there occurred along with increase in the aggregation induced by ADP, a reduction in the activity of factor XIII in the plasma. Addition to the plasma of inactive factor XIII failed to influence either the degree of aggregation, or the change in the activity of factor XIII in the plasma in comparison with control samples. In platelet aggregation induced by thrombin, addition of factor XIII was accompanied by a marked fall of its activity in the plasma. In comparison with control, the extent of aggregation in this case decreased. The observed differences in the character of aggregation coursing in the presence of factor XIIIa when different aggregating agents (ADP and thrombin) were used were apparently due to the interaction of active factor XIII with thrombin added to the plasma in the capacity of an aggregating agent.