The role of ß-barrels 1 and 2 in the enzymatic activity of factor XIII A-subunit.

Essentials The roles of ß-barrels 1 and 2 in factor XIII (FXIII) are currently unknown. FXIII truncations lacking ß-barrel 2, both ß-barrels, or full length FXIII, were made. Removing ß-barrel 2 caused total loss of activity, removing both ß-barrels returned 30% activity. ß-barrel 2 is necessary for exposure of the active site cysteine during activation. SUMMARY: Background Factor XIII is composed of an activation peptide segment, a ß-sandwich domain, a catalytic core, and, finally, ß-barrels 1 and 2. FXIII is activated following cleavage of its A-subunits by thrombin. The resultant transglutaminase activity leads to increased resistance of fibrin clots to fibrinolysis. Objectives To assess the functional roles of ß-barrels 1 and 2 in FXIII, we expressed and characterized the full-length FXIII A-subunit (FXIII-A) and variants truncated to residue 628 (truncated to ß-barrel 1 [TB1]), residue 515 (truncated to catalytic core [TCC]), and residue 184 (truncated to ß-sandwich). Methods Proteins were analyzed by gel electrophoresis, circular dichroism, fluorometric assays, and colorimetric activity assays, clot structure was analyzed by turbidity measurements and confocal microscopy, and clot formation was analyzed with a Chandler loop system. Results and Conclusions Circular dichroism spectroscopy and tryptophan fluorometry indicated that full-length FXIII-A and the truncation variants TCC and TB1 retain their secondary and tertiary structure. Removal of ß-barrel 2 (TB1) resulted in total loss of transglutaminase activity, whereas the additional removal of ß-barrel 1 (TCC) restored enzymatic activity to ~ 30% of that of full-length FXIII-A. These activity trends were observed with physiological substrates and smaller model substrates. Our data suggest that the ß-barrel 1 domain protects the active site cysteine in the FXIII protransglutaminase, whereas the ß-barrel 2 domain is necessary for exposure of the active site cysteine during activation. This study demonstrates the importance of individual ß-barrel domains in modulating access to the FXIII active site region.

Probing thrombin's ability to accommodate a V34F substitution within the factor XIII activation peptide segment (28-41).

In blood coagulation, thrombin helps to activate factor XIII (FXIII) by cleaving the activation peptide (AP) at the R37-G38 peptide bond. The common polymorphism V34L yields a FXIII that is more easily activated than the wild type enzyme. Peptides based on the FXIII (28-41) (28TVELQGVVPRGVNL41) sequence serve as an important model system to evaluate the substrate specificity of thrombin and thus how to regulate FXIII activation. Our previous kinetic and nuclear magnetic resonance (NMR) studies have suggested that the P4-P1 amino acids on this FXIII segment provide key anchors to the thrombin active site surface. Furthermore, the most effective amino acid to have at the P4 position is a leucine. In the current work, a peptide containing V34F was examined to probe the ability to accommodate an aromatic residue at this position. Kinetic parameters for thrombin-catalyzed hydrolysis of FXIII AP (28-41) V34F are comparable with that of the wild type V34. One-dimensional proton line-broadening studies reveal that the 34FVPR37 segment encompassing the P4-P1 positions makes the most contact with the thrombin surface. Two-dimensional transferred-nuclear overhauser effect spectroscopy (NOESY) studies indicate that when the peptide is bound to thrombin, the F34 aromatic ring is oriented to promote P4-P2 interactions with P36. This characteristic has been viewed as a hallmark for V34L. An ability to generate this interaction may promote the ability of FXIII AP (28-41) V34F to remain a viable substrate for thrombin.

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.

New general approach for determining the solution structure of a ligand bound weakly to a receptor: structure of a fibrinogen Aalpha-like peptide bound to thrombin (S195A) obtained using NOE distance constraints and an ECEPP/3 flexible docking program.

A new approach incorporating flexible docking simulations and NMR data is presented for calculating the bound conformation of a ligand that interacts weakly with an enzyme. This approach consists of sampling directly the conformation of a flexible ligand inside a receptor active site containing surrounding flexible loops. To make this sampling efficient, a ligand-growing procedure has been adopted. Optimization of the ECEPP/3-plus-NOE constraint function is carried out by using a collective variable Monte Carlo minimization technique. Numerous energy minimizations are made possible for such a large system by using a Bezier splines energy grid technique. This new flexible docking approach was applied to determine the structure of a fibrinogen Aalpha-like peptide (7DFLAEGGGVRGPRV20) bound to an active site mutant of thrombin [thrombin(S195A)]. Structure calculations of the bound ligand, using 2D-transferred NOESY distance constraints in the DIANA program, showed that the N-terminal portion of the peptide (D7-R16) involves a chain reversal, whereas the C-terminal portion (G17-V20) adopts a fold that exists in several different orientations. In addition, the ECEPP/3 flexible docking package was used to assess the conformational variability of the ligand and surrounding 60D-insertion loop of thrombin. Amino acid residues (17-20) of the peptide interact with a region of the enzyme that exhibits broad specificity, with a preferred direction between the 60D-insertion loop and Pro37 of thrombin.

Structural examination of the influence of phosphorylation on the binding of fibrinopeptide A to bovine thrombin.

Upon addition of thrombin, fibrinopeptides A and B are cleaved off from the N-termini of four chains of fibrinogen (Aalpha Bbeta gamma)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aalpha chain, cleavage occurs most prevalently at the Arg16-Gly17 peptide bond. About 25-30% of the human fibrinogen Aalpha chains are phosphorylated in nature at the position of Ser3, but the function for this modification is not understood. Previous NMR studies indicated that the N-terminal portion (1ADSGE5) of unphosphorylated fibrinopeptide A does not interact with the surface of bovine thrombin. Kinetic and NMR studies have now been carried out to assess whether phosphorylation at Ser3 allows the N-terminal segment (1ADSGEGDFLAEGGGVR16) to become anchored on the thrombin surface, leading to formation of a catalytically more efficient enzyme-substrate complex. Kinetic results indicate that phosphorylation leads to an approximately 65% increase in substrate specificity (kcat/Km) toward hydrolysis of fibrinogen Aalpha(1-20). 31P NMR studies reveal that the phosphorylated group does interact with thrombin, and 1H line broadening studies suggest that phosphorylation does promote binding of amino acids 1-5. Two-dimensional transferred nuclear Overhauser effect spectroscopy studies of bound fibrinopeptide A(1-16 Ser3P) indicate that phosphorylation allows new through-space interactions involving amino acid residues 1ADSGE5 to be observed. Computational docking of the peptide onto the X-ray structure of thrombin suggests that the phosphate may interact with basic residues at the rim of the heparin binding site of thrombin. As a result, the phosphate may serve as an anionic linker between the fibrinopeptide and the enzyme thrombin.

Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin.

Bovine prethrombin-2 has been produced in Escherichia coli using a T7 expression system. The expressed prethrombin-2 formed intracellular inclusion bodies which were solubilized by reversible sulfonation of the cysteines in the presence of 7 M guanidine hydrochloride. Sulfonated prethrombin-2 was refolded in the presence of 4 M guanidine hydrochloride, using oxidized and reduced glutathione as the redox couple. The folded protein was purified by heparin affinity chromatography and activated to thrombin with Echis carinatus snake venom. The resulting thrombin was also purified by heparin affinity chromatography. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline by recombinant thrombin (kcat = 123 +/- 10 s-1 and Km = 2.91 +/- 0.3 microM). These values are in good agreement with those determined for wild-type thrombin (kcat = 97 +/- 8 s-1 and Km = 2.71 +- 0.25 microM). From the thrombin-mediated release of fibrinopeptide A from fibrinogen, kcat/Km was found to be the same for recombinant (17.3 +/- 1.2 microM-1 s-1) and wild-type (16.7 +/- 2.0 microM-1 s-1) thrombin. These results, taken together with circular dichroism spectra and the elution position of prethrombin-2 from a heparin affinity resin, indicate that prethrombin-2 was folded into a conformation similar to that of the wild-type protein. In addition, since E. coli produces deglycosylated enzymes, these findings suggest that the carbohydrate on the B chain of wild-type thrombin does not affect the amidolytic and fibrinolytic activities of thrombin. Finally, this expression system can be used to prepare mutants of prethrombin-2 for future structure-function studies involving thrombin and its substrates; some preliminary results of this type are presented here.

Intrauterine fetal death: mediconursing and psychosocial considerations for the childbearing patient.

The purpose of this article is to provide the perinatal nurse with a current review of the pregnancy complication, intrauterine fetal death. The causes, associated complications, and mediconursing management of this pregnancy complication are reviewed. Additionally, by use of the Snyder childbearing trajectory model, the psychosocial effects of intrauterine fetal death are discussed. The presentation of the Snyder model is intended to provide the nurse with a conceptual framework through which psychosocial nursing interventions can be derived.

Activation and inhibition of protein kinase C isozymes alpha and beta by Gd3+.

Gd3+ was evaluated as a probe for Ca2+ sites on protein kinase C (PKC) by studying its ability to replace Ca2+ in activation of PKC isozymes II (beta) and III (alpha) in the lipid systems phosphatidylserine/1,2-dioleoyl-sn-glycerol (PS/DO) and diheptanoylphosphatidylcholine (PC7)/DO. PKC beta was stimulated by Ca2+ or Gd3+ in PS/DO whereas activity in PC7/DO was independent of these metals. Thus, it is suggested that Gd3+ replaces Ca2+ at a site involving metal-lipid interactions. High concentrations of Ca2+ or Gd3+ inhibited activity in both lipid systems. Analysis of the Gd3+ inhibition in the PC7/DO system suggests that it is due to formation of GdATP, which competes at the MgATP site. Activity of PKC alpha was dependent on low concentrations of Ca2+ in both lipid systems. The ability of Gd3+ to substitute for Ca2+ could not be evaluated in the PS system due to the inability to completely remove contaminating Ca2+ without chelating buffers. Successful reduction of contaminating Ca2+ was achieved in the PC7 system but Gd3+ failed to substitute for Ca2+ in activating PKC alpha and only caused inhibition. This is consistent with binding of Gd3+ to a Ca2+ site at or near the active site of the enzyme rather than to a site on the lipid. These results indicate that interactions between PKC and Gd3+ are complex, involving occupation of more than one class of sites. Conditions for separately evaluating the individual sites can be manipulated by selection of isozyme and lipid system.

High-affinity Ca(2+)- and substrate-binding sites on protein kinase C alpha as determined by nuclear magnetic resonance spectroscopy.

Water proton nuclear magnetic resonance (NMR) relaxation rates were used to identify metal sites on protein kinase C (PKC) isozymes alpha and beta using paramagnetic Gd3+ as a probe. The paramagnetic effect of Gd3+ on water proton relaxation was enhanced with PKC isozymes alpha and beta in the presence of diheptanoylphosphatidylcholine/1,2-dioleoyl-sn-glycerol (PC7/DO). The data are consistent with a single class of metal-binding sites on PKC beta and two classes of sites on PKC alpha: a single high-affinity site with a KD for Gd3+ of 0.2 microM and a larger class of sites with a lower affinity for Gd3+. Titration with Ca2+ abolished the observed enhancement of water proton relaxation by the PKC alpha.Gd3+ complex, consistent with displacement of Gd3+ by Ca2+. Titrations of the PKC alpha.Gd3+ complex with Co(NH3)4ATP, a substitution-inert analogue of ATP, caused a substantial decrease in the observed water proton relaxation enhancement, consistent with formation of a ternary enzyme.metal.substrate complex with a KPKC alpha.Gd.[CoATP] of 30-100 nM. Titration of the metal enzyme complex with a model peptide substrate derived from the pseudosubstrate sequence of PKC alpha caused a similar decrease in enhancement at stoichiometric concentrations consistent with the formation of a PKC alpha.Gd3+.peptide complex with a KPKC alpha.Gd.[peptide] of less than or equal to 13 nM. Titrations of the fully formed PKC alpha.Gd3+.peptide complex with Co(NH3)4ATP caused a further decrease in enhancement consistent with formation of a quaternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of porphyrins with purified DNA and more highly organized structures.

Studies of the solution properties of gold(III)tetrakis(4-N-methylpyridyl) porphine and its DNA binding characteristics have been conducted utilizing uv/vis absorption spectroscopy, circular dichroism (CD), Mossbauer spectroscopy, and temperature-jump relaxation techniques. These studies indicate that over the concentration range considered this water soluble gold(III) porphyrin does not aggregate, binds axial ligands only weakly with a preference for soft Lewis bases, and is capable of intercalation into nucleic acids of appropriate base pair content. The interaction of this and several other porphyrins with the synthetic polynucleotide poly(dA-dC).poly(dT-dG) has been studied. Spectroscopic signatures for intercalation were found for those derivatives not having axial ligands. Intercalation into chromatin in vitro can also occur with those porphyrins and metalloporphyrins which do not have axial ligands. Finally, studies utilizing microinjection techniques indicate that once within the cell, tetrakis(4-N-methylpyridyl)porphine tends to localize in the nucleus.