Gly-Pro-Arg-Pro modifies the glutamine residues in the alpha- and gamma-chains of fibrinogen: inhibition of transglutaminase cross-linking

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl-L-prolyl-L-arginyl-L-proline (GPRP), a tetrapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the alpha- and gamma-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the gamma-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polymerized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [3H]putrescine and [14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [14C]putrescine incorporation into the alpha- and gamma-chains of fibrinogen without inhibiting beta-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [3H]putrescine into both fibrinogen and Fragment D1, without modifying [3H]putrescine incorporation into casein. GPRP also inhibited intermolecular alpha-alpha-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the alpha-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polymerization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the alpha- and gamma-chains of fibrinogen.     

Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase

In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin-pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction may influence the TSG-6/heavy Chain 2-mediated transfer of heavy chains observed during inflammation.     

Dissociation of fibrinogen and fibronectin binding from transglutaminase-mediated cross-linking at the hepatocyte surface

The interaction of fibrinogen and fibronectin with hepatocytes has been dissociated into distinct binding and cross-linking steps. Binding and cross-linking of 125I-labeled ligands were both decreased by transglutaminase inhibitors, but not by heparin or hirudin. Transglutaminase activity was manifest by Ca2+-dependent incorporation of [14C]putrescine into cells. Preferential cross-linking of fibrinogen A alpha over gamma chains, and lack of inhibition by heparin or hirudin indicates the involvement of tissue transglutaminase, and not Factor XIIIa. Hepatic transglutaminase activity, as well as binding and cross-linking of fibrinogen and fibronectin, were maximally supported by Ca2+, partially supported by Mn2+ and Sr2+, and markedly decreased by Mg2+ and Ba2+. In contrast, Co2+ supported binding but not cross-linking or transglutaminase activity, indicating that binding and cross-linking are dissociable events. This conclusion was corroborated by the finding that fibrinogen fragments D95 and D78 both inhibited Ca2+-dependent fibrinogen binding without being cross-linked themselves. Ligand binding in the presence of either cation was localized to the cell surface as evidenced by its trypsin sensitivity. Thus, fibrinogen and fibronectin binding to hepatocytes is independent of transglutaminase activity, whereas cross-linking of these adhesive macromolecules requires an enzymatically active cellular transglutaminase. In addition, fibrinogen binding appears to be mediated by molecular determinants present in fragment D78.     

The comparative ability of plasma and tissue transglutaminases to use collagen as a substrate

Heat denatured type I and type III calf skin collagen were found to be substrates for guinea pig liver transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13) but not for active plasma factor XIII (factor XIIIa). Liver transglutaminase was shown to catalyse incorporation of 14C-putrescine into subunits of denatured collagen of both types, cross-linking of the latter into high molecular weight polymers and their co-cross-linking to fibrin and fibrinogen. Factor XIIIa is inactive in these respects. None of these reactions was catalysed by liver transglutaminase and plasma factor XIIIa when nondenatured collagens both soluble or in the forms of reconstituted fibrils served as substrates. Some cross-linking of cleavage products of collagen type I (obtained by treatment with collagenase from human neutrophiles) was induced by liver transglutaminase and factor XIIIa. The results indicate that although appropriate glutamine and lysine residues for a epsilon-(gamma-glutamine) lysine cross-linked formation are present in collagen, the native conformation of collagen prevents the action of liver transglutaminase and factor XIIIa.     

Reversible cross-linking of alpha 2-plasmin inhibitor to fibrinogen by fibrin-stabilizing factor

alpha 2-Plasmin inhibitor, a primary inhibitor of fibrinolysis, is cross-linked to fibrin by plasma transglutaminase (glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13, activated fibrin-stabilizing factor) when blood coagulation takes place. alpha 2-Plasmin inhibitor was found also to be cross-linked to fibrinogen by plasma transglutaminase. The inhibitor was corss-linked exclusively to the A alpha-chain of fibrinogen, and the cross-linking reaction proceeded very rapidly. The reaction was almost completed before the formation of the gamma-chain dimers of fibrinogen which precedes cross-linking polymerization of the A alpha-chain of fibrinogen. The maximum level of inhibitor cross-linking achieved was approx. 30% of the inhibitor present at the start of the reaction. The level of cross-linking of the inhibitor was not changed when the cross-linking reaction was preceded by dimerization of fibrinogen. The cross-linking reaction was found to be a reversible one, since the cross-linked complex of the inhibitor and fibrinogen was partly dissociated to each of its components when the complex was incubated with plasma transglutaminase. These results suggest that the self-limiting nature of the cross-linking reaction between alpha 2-plasmin inhibitor and fibrin(ogen) is due to the reaction equilibrium favoring dissociation of the complex, and not due to the development of structural hindrance in polymerizing fibrin(ogen).     

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.     

Immunoelectrophoretic characterizations of the cross-linking of fibrinogen and fibrin by factor XIIIa and tissue transglutaminase. Identification of a rapid mode of hybrid alpha-/gamma-chain cross-linking that is promoted by the gamma-chain cross-linking.

Cross-linking of human fibrin by fibrin stabilizing factor (factor XIIIa) and tissue transglutaminase (ti-TG) was examined by immunoprobing electrophoregrams for positive identification of the cross-linked chains. The immunoprobing was carried out by a new, direct staining technique employing composite gels of a porous protein immobilizing matrix (glyoxyl agarose) blended with a removable polyacrylamide filler that eliminates need for Western blotting. We find that the known rapid cross-linking of gamma-chains into gamma 2-dyads by XIIIa is accompanied by co-cross-linking of the gamma 2-dyads with alpha-chains to form hybrid alpha gamma 2-triads. Little or no cross-linking of relatively abundant alpha- and gamma-chain monads into hybrid alpha gamma-dydads accompanies formation of the alpha gamma 2-triads. Thus, formation of the gamma 2-dyads accelerates the hybrid cross-linking. This acceleration is viewed as demonstrating a previously unknown mode of cooperative interaction between alpha- and gamma-chains arising from cross-linking of the D-domains of the molecules. This strengthened interaction is not critically dependent on fibrinopeptide-release, because alpha gamma 2-triads are similarly formed when fibrinogen is cross-linked by XIIIa. Also observed in the study with XIIIa was the formation of small amounts of homologous gamma 3 and gamma 4 oligomers which had been predicted by others to contribute to branching of fibrin strands. Unlike XIIIa, ti-TG acts preferentially on alpha-chains rather than gamma-chains as known. As alpha gamma-dyad, not seen in reactions with XIIIa, is produced concurrent with the homologous alpha-chain cross-linking. Also, three different species of alpha 2-dyads were produced by ti-TG, two of which were not seen in reactions with XIIIa. The differences in product formation revealed by the specific staining are viewed as providing criteria for distinguishing products of XIIIa and ti-TG in biologic specimens.     

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.     

Characterization of thrombospondin as a substrate for factor XIII transglutaminase

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

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

Calmodulin Affinity for Brain Coated Vesicle Proteins

A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of 125I-labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin-C. Scatchard analysis revealed one high-affinity, low-capacity binding site, KD = 3.9 +/- 0.6 nM, Bmax = 16.3 +/- 2.4 pmol/mg, and one low-affinity, high-capacity binding site, KD = 102 +/- 15.0 nM, Bmax = 151 +/- 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 microgram calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X-100-treated extract of coated vesicles was passed through a Sepharose 4B-calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+-dependent manner. A 30,000 Mr protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+-Mg2+]-ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca2+-Mg2+]-ATPase complex, the other coated vesicle calmodulin binding site.     

Staphylococcus aureus fibronectin-binding protein serves as a substrate for coagulation factor XIIIa: evidence for factor XIIIa-catalyzed covalent cross-linking to fibronectin and fibrin

In this study, we have investigated the interactions of a Staphylococcal recombinant fibronectin-binding protein A (rFnbA) with fibronectin, fibrinogen, and fibrin. Using analytical size-exclusion chromatography, we evaluated the stoichiometry of reversible binding of FnbA to fibronectin and demonstrated that, in solution, it can accommodate at least two molecules of fibronectin. Results of ELISA experiments demonstrated that rFnbA binds with equally high affinity to both immobilized fibrinogen and fibrin. When included into a thrombin-induced fibrin polymerization reaction, rFnbA strongly inhibited fibrin assembly in a dose-dependent manner. In this study, we have shown that rFnbA can act as a substrate for coagulation factor XIIIa. Factor XIIIa catalyzes the incorporation of amine donor (dansylacadaverine) and amine acceptor (peptide patterned on the N-terminal sequence of fibronectin) synthetic probes into rFnbA, suggesting that it serves as a bifunctional substrate containing reactive glutamine and lysine residues. We have demonstrated that the reversible complex formed by rFnbA and fibronectin or rFnbA and fibrin is covalently stabilized by the transglutaminase action of factor XIIIa. Incubation of rFnbA in the presence of either of its ligands and factor XIIIa results in the introduction of intermolecular epsilon-(gamma-glutamyl)lysine isopeptide bond(s) and the formation of high molecular mass heteropolymers. These findings suggest a novel mechanism by which pathogenic Staphylococcus aureus may utilize the transglutaminase activity of factor XIIIa for attachment to soluble proteins, cell surfaces, and matrixes.     

Biotinylated peptides containing a factor XIIIa or a tissue transglutaminase-reactive glutaminyl residue that block protein cross-linking phenomena by becoming incorporated into amine donor sites.

Biotinylated peptides Biot-Gln-Gln-Ile-Val and Biot-epsilon-Aca-Gln-Gln-Ile-Val were shown to act as acceptor substrates for amines in reactions catalyzed by both tissue transglutaminase and coagulation factor XIIIa. Moreover, the peptides could be employed for specifically blocking the potential amine donor sites of protein substrates participating in biological cross-linking with these enzymes. The presence of the biotin label allowed for ready detectability of the marked donor substrates during the cross-linking of crystallins in lens homogenate by the intrinsic transglutaminase and that of the alpha chains of human fibrin by factor XIIIa.     

Displacement of alpha-actinin from the NMDA receptor NR1 C0 domain By Ca2+/calmodulin promotes CaMKII binding

Ca2+ influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor triggers activation and postsynaptic accumulation of Ca2+/calmodulin-dependent kinase II (CaMKII). CaMKII, calmodulin, and alpha-actinin directly bind to the short membrane proximal C0 domain of the C-terminal region of the NMDA receptor NR1 subunit. In a negative feedback loop, calmodulin mediates Ca2+-dependent inactivation of the NMDA receptor by displacing alpha-actinin from NR1 C0 upon Ca2+ influx. We show that Ca2+-depleted calmodulin and alpha-actinin simultaneously bind to NR1 C0. Upon addition of Ca2+, calmodulin dislodges alpha-actinin. Either the N- or C-terminal half of calmodulin is sufficient for Ca2+-induced displacement of alpha-actinin. Whereas alpha-actinin directly antagonizes CaMKII binding to NR1 C0, the addition of Ca2+/calmodulin shifts binding of NR1 C0 toward CaMKII by displacing alpha-actinin. Displacement of alpha-actinin results in the simultaneous binding of calmodulin and CaMKII to NR1 C0. Our results reveal an intricate mechanism whereby Ca2+ functions to govern the complex interactions between the two most prevalent signaling molecules in synaptic plasticity, the NMDA receptor and CaMKII.     

Susceptibility of platelet alpha-actinin to a Ca2+-activated neutral protease

Purified alpha-actinin from human platelets was digested with Ca2+-activated protease from muscle. The alpha subunit (Mr = 100 kDa) was degraded into a unique polypeptide b of slightly lower molecular mass. In fresh platelets, only the a subunit was detected by immunoblotting techniques, while in out-dated platelets, both a and b polypeptides were present. Since a similar conversion of a to b occurs in vitro as in whole platelets, it can be assumed that, in platelets, alpha-actinin is cleaved by the endogenous Ca2+-activated protease.     

Autophosphorylation of Calmodulin-Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity

Previous studies have purified from brain a Ca2+/calmodulin-dependent protein kinase II (designated CaM-kinase II) that phosphorylates synapsin I, a synaptic vesicle-associated phosphoprotein. CaM-kinase II is composed of a major Mr 50K polypeptide and a minor Mr 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin-dependent manner. Recent studies have demonstrated that the 50K component of CaM-kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM-kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [alpha-32P]8-azido-ATP was used to demonstrate that the 60K and 50K polypeptides of SJ-associated CaM-kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin-dependent manner. Experiments using 32P-labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I-calmodulin binding sites. These results suggested that the autophosphorylation of CaM-kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I-calmodulin overlay techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM-kinase II polypeptides bound more calmodulin (50-70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM-kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaM-kinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM-kinase II. The CaM-kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300-1,000 nM). These findings show that the degree of autophosphorylation of CaM-kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.     

Proteins of the human placental microvillar cytoskeleton. alpha-Actinin.

The human placental syncytiotrophoblast microvilli are supported by an underlying cytoskeleton consisting mainly of actin microfilaments. The major proteins associated with the actin have Mr values of 105 000, 80 000 and 68 000. The 105 000-Mr protein is recognized by an antibody preparation raised to purified chicken gizzard alpha-actinin. Electron microscopy has shown that the human placental protein has dimensions similar to those reported for muscle alpha-actinin. About half of the placental microvillar alpha-actinin is released from the cytoskeleton in the presence of Ca2+. This effect occurs at concentrations of Ca2+ greater than 0.3 muM and has been used as the basis of a method for the purification of the placental alpha-actinin. This sensitivity to Ca2+ is not affected by trifluoperazine and is therefore likely to be a property of the alpha-actinin as such rather than being mediated via calmodulin.     

Isolation and characterization of cDNA clones to mouse macrophage and human endothelial cell tissue transglutaminases.

The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of both tissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophage tissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (greater than 80%). Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology (75% identity) was found in the active site region.     

Soluble fibrin consists of fibrin oligomers of heterogeneous distribution

Soluble fibrin is observed in patients with intravascular coagulation and represents an intermediary product of conversion of fibrin monomers into a fibrin clot whereby the presence of fibrinogen may suppress fibrin clot formation. The interactions between fibrin and fibrinogen and the occurrence of fibrin oligomers in soluble fibrin were studied by sucrose density ultracentrifugation. Different concentrations of soluble fibrin, prepared by mixing 125I-fibrin (24 nM - 1.5 microM) with a constant concentration of 131I-fibrinogen (6 microM) were analyzed at 37 degrees C in stable linear sucrose density gradients containing a uniform concentration of unlabelled fibrinogen (6 microM) and calcium ions in order to mimic the physiological situation. At any fibrin concentration, 125I-fibrin sedimented faster than 131I-fibrinogen through 5-30% (w/v) sucrose gradients. Sedimentation rates of fibrin increased from 9 S to 23 S depending on the initial fibrin concentration. The relative amount of residual fibrin monomer not incorporated into oligomers was calculated from the sedimentation profiles. At any fibrin concentration, the portion of free monomer was always more than twofold higher for batroxobin-generated (desAA-) fibrin than for thrombin-generated (desAABB-) fibrin. Apparent association constants for desAABB-fibrin were 3-10 times higher than those for desAA-fibrin indicating a stronger interaction between monomers of the former type of fibrin. In the presence of excess fibrinogen the predominant species in soluble desAA-fibrin were monomers and dimers, whereas dimers, trimers and higher-molecular-mass oligomers were present in soluble desAABB-fibrin. Strong interactions between both types of fibrin were demonstrated from their cosedimentation, whereby the size of these copolymers were shown to be governed by the oligomer size of the desAABB-fibrin type. These results provide evidence for the occurrence of differently sized oligomers of fibrin in soluble fibrin and for the concept of a cooperative polymerization process between both types of fibrin devoid of any stable complexes between fibrin and fibrinogen.     

Vitronectin is a substrate for transglutaminases

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.