Effects of calcium binding and of EDTA and CaEDTA on the clotting of bovine fibrinogen by thrombin

Studies were carried out at pH 7.0 and gamma/2 0.15 before addition of CaCl2 or EDTA. Clotting time, tau, at 3.03 microM fibrinogen and 0.91 u/ml thrombin was determined for equilibrium systems. With added Ca2+, tau decreases, from tau 0 at 0 added Ca2+ (mean, 29.7 +/- 3 s), by approximately 3 s at 5 mM added Ca2+. With added EDTA, tau increases sigmoidally from tau 0 at 0 EDTA to a maximum (mean tau m = 142 +/- 23 s) at approximately 200 microM EDTA. tau then decreases slightly to a minimum at approximately 1.3 mM and finally increases to infinity at approximately 10 mM EDTA. Between 0 and 1.3 mM EDTA, effects on clotting time are completely reversed by adding Ca2+ and, after equilibration at 400 microM EDTA, tau is independent of EDTA concentration. Thus, up to 400 microM EDTA, effects on clotting time are attributed to decreasing fibrinogen bound Ca2+. Between 5 mM Ca2+ and 200 microM EDTA it is assumed that an equilibrium distribution of fibrinogen species having 3, 2, 1, or 0 bound calcium ions is established and that a clotting time is determined by the sum of products of species fractional abundance and pure species clotting time. Analysis indicates that pure species clotting times increase proportionately with decreasing Ca2+ binding, binding sites are nearly independent, and the microscopic association constant for the first bound Ca2+ is approximately 4.9 X 10(6) M-1. Effects of adding Ca2+ at times t1 after thrombin addition to systems initially equilibrated at 200 microM EDTA were determined. Analysis of the relation between tau and t1 indicates that as Ca2+ binding decreases, rate constants for release of B peptides decrease less than those for release of A peptides. As EDTA concentration is increased above 1.3 mM, inhibitory effects of EDTA and CaEDTA progressively increase.     

Inhibited thrombins. Interactions with fibrinogen and fibrin.

Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins. Also, the elution profile of phenylmethane-sulphonyl fluoride-inhibited thrombin from fibrinogen-Sepharose was identical with that of active thrombin from fibrin-monomer-Sepharose. Thus far the only low-Mr inhibitor that prevents thrombin from binding to fibrin monomer is pyridoxal 5'-phosphate. Preformed hirudin-thrombin complexes do not interact with fibrin. The extent to which the active centre of thrombin associated with fibrin is still accessible to substrates and inhibitors was also studied. Thrombin bound to fibrin hydrolyses a synthetic substrate at the same rate as the free enzyme. Water-soluble low-Mr inhibitors such as D-Phe-Pro-Arg-CH2Cl and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide can readily modify the active centre of the fibrin-associated enzyme, and the active centre is exposed to the degree that displacement of dansylarginine NN-(3-ethylpentane-1,5-diyl)amide by D-Phe-Pro-Arg-CH2Cl is possible without disturbing the binding. Hirudin disrupts the affinity between thrombin and fibrin. These data indicate that the active centre of thrombin associated with fibrin through extended binding is fully exposed and freely accessible. It is possible that extended binding may play a regulatory role in the activation of Factor XIII by thrombin, as well as inactivation of this enzyme by antithrombin III.     

Clotting of bovine fibrinogen. Kinetic analysis of the release of fibrinopeptides by thrombin and of the calcium uptake upon clotting at high fibrinogen concentrations

Kinetic data on the release of fibrinopeptides A and B from bovine fibrinogen by human thrombin were obtained at high fibrinogen concentrations, within the 0.8-8.8% range (0.227 X 10(-4) to 2.60 X 10(-4) M), at 25 degrees C, pH 7.26, 0.30 ionic strength, and 10(-4) M free Ca2+ concentration. Release of fibrinopeptide A followed strictly first-order kinetics at all concentrations, in spite of the fact that the highest concentration was 26 times larger than the value of KM found in the literature. This behavior can be explained by inhibition of thrombin by the reaction products, with KI = KM. The equation describing the course of the reaction under these conditions can be rearranged into a linear relationship between 1/kobsd and substrate concentration. The slope of the line is equal to 1/kcat and the intercept to KM/kcat. The data points fell accurately on a straight line, and with the parameters of the latter, kcat and KM were calculated as (6.3 +/- 0.11) X 10(-10) M s-1 (unit of thrombin)-1 L-1 and (11.0 +/- 3.0) X 10(-6) M, respectively. These values agree well with those found in the literature. Release of fibrinopeptide B follows complex kinetics. Higgins et al. [Higgins, D. L., Lewis, S.D., & Shafer, J.A. (1983) J. Biol. Chem. 258, 9276-9282] suggested that it can be described as the result of two consecutive reactions, the first one being the release of fibrinopeptide A and the second one of fibrinopeptide B from those molecules that have already lost fibrinopeptide A in the previous step.(ABSTRACT TRUNCATED AT 250 WORDS)     

The heparin-accelerated neutralisation of bovine α and β thrombins by antithrombin III

The heparin-accelerated neutralisation of bovine α and β thrombins has been examined using a peptide substrate H-d-phenylalanyl-pipecolyl-arginine-paranitroanilide-HCl to measure thrombin amidase activity. α and β thrombins were both neutralised by antithrombin III and this neutralisation was further accelerated by the presence of small amounts of heparin. Low and high molecular weight heparin and heparins fractionated by their affinity for antithrombin III were all able to accelerate the neutralisation of α and β thrombin. This work is therefore unabel to confirm reports that α and β thrombins have different heparin sensitives.     

Characterization of the amino acids of bovine fibrinogen involved in the fibrinogen-thrombin interaction of the blood clotting process. Comparison with the milk clotting process

Summary Bovine fibrinogen and the A and B chains of bovine fibrinogen have been subjected to chemical modification by a number of reagents and the effects of these procedures on the susceptibility of the proteins to thrombin hydrolysis is described. The reagents used were rose bengal (for photo-oxidation), 2-hydroxy-5-nitrobenzyl bromide, N-acetylimidazole, iodoacetic acid and diethyl pyrocarbonate. Evidence is presented which indicates that the tryptophan and tyrosine residues of fibrinogen are not involved to any great extent in the interaction of this protein with thrombin. Modification with iodoacetic acid suggests that methionine residues play a major role in such interactions, but the fibrinogen chains on which the important residues reside remain uncertain. The use of diethyl pyrocarbonate indicates the participation also of histidine in fibrinogen-thrombin interactions and that, whereas the histidine residues of the B chain are involved to a great extent, it appears that those of the Aa chain are not. The similarities which exist between the fibrinogen-thrombin and the -casein-chymosin systems are discussed.Abbreviations used DEP diethyl pyrocarbonate (ethoxyformic anhydride) - HNBB 2-hydroxy-5-nitrobenzyl bromide - N-Acl N-acetylimidazole - PTC phenylthiocarbamyl - PTH 3-phenyl-2-thiohydantoin.     

Complete sequence of the lamprey fibrinogen alpha chain

The complete amino acid sequence of the lamprey fibrinogen alpha chain has been determined by a combination of peptide sequencing and cDNA and genomic cloning. The chain, which has an apparent molecular weight by dodecyl sulfate-polyacrylamide gel electrophoresis of ca. 100,000, is composed of 961 amino acid residues and has a calculated molecular weight of 96,722. It is distinguished by a large number of 18-residue repeats in a region where mammalian fibrinogens have 13-residue repeats. The data are in accord with our previous finding that the lamprey alpha chain has a distinctive amino acid composition, almost half the residues being glycine, serine, or threonine. The chain differs from mammalian alpha chains in that there are no cysteines in the carboxy-terminal half, and thus no intrachain loop, nor are there any RGD sequences in the lamprey alpha chain. Taken together with previous data on the sequences of the beta and gamma chains, the findings bear significantly on our understanding of fibrin formation. The alpha chain also provides an interesting case of structural convergence during evolution.     

Activation of bovine prothrombin as monitored by the clotting assay

The activation of bovine prothrombin was studied with highly purified clotting factors and using a coagulation assay developed to look at the initial rate of prothrombin conversion as well as the conversion rate over a time course of 75 min. Activation of prothrombin by factor Xa alone was slow. The rate of prothrombin conversion increased markedly with the addition of each of the accessory components Ca2+, phospholipid and bovine factor V, respectively. With the complete prothrombinase complex comprising factor Xa, Ca2+, phospholipid and factor V, the rate increase was about 22,000-fold higher compared to the action of factor Xa and Ca2+ on prothrombin alone. The rates of thrombin formation obtained with activated factor X1 were only about 70% the values obtained with factor X2. The rate of prothrombin activation and the difference between the activities of the activated factors X1 and X2 are discussed.     

Complementary DNA sequence of lamprey fibrinogen beta chain

The cDNA sequence of the beta chain of lamprey fibrinogen has been determined. To that end, an oligonucleotide probe was synthesized that corresponded to an amino acid sequence from the carboxy-terminal region of the lamprey fibrinogen beta chain. The insert actually began with residue 3 of the fibrin beta chain; it ran through to a terminator codon following the carboxy-terminal residue at position 443 and then continued for an additional 606 nucleotides of noncoding sequence to its 3' end. The inferred amino acid sequence was verified by comparison with assorted cyanogen bromide fragments isolated from the beta-chain protein, including two carbohydrate-containing peptides that corresponded to segments containing the carbohydrate-attachment consensus sequence. Overall, the lamprey chain is 49% identical with the beta chain from human fibrinogen. This is the same degree of resemblance as was found for the lamprey and human gamma chains. Moreover, the principal regions of conservation are the same in both the beta and gamma chains. Differences and similarities in the physiological behavior of the two fibrinogens are assessed in terms of the observed amino acid replacements.     

Deciphering the physiological pathway of clotting of fibrinogen in blood plasma

I have been fortunate to have benefited over the years from the friendship and advice of John Ferry in our research to decipher the physiological reactions and regulatory events involved in the clotting of fibrinogen in blood. The article is a tribute to the memory of this creative scientist and remarkable individual.     

High affinity binding of human and bovine thrombins to p-chlorobenzylamido-epsilon-aminocaproyl agarose.

Human thrombin (EC 3.4.21.5) binds tightly to p-chlorobenzylamido-epsilon-aminocaproyl agarose, and is not eluted by 2 M NaCl at pH 8. Its zymogen, human prothrombin, does not bind to the same absorbent. 2 M NaCl partially elutes DFP-treated thrombin. For native human and bovine thrombins, protein and activity are quantitatively eluted by 25% dioxane, and upon rechromatography the active human enzyme exhibits the same binding properties. Equally tight binding of human thrombin occurs with derivatives of the m- and p-chlorobenzylamines. With the o-chloro derivative or benzylamine itself insolubilized to epsilon-aminocaproyl agarose, thrombin is eluted by high ionic strength. Bovine trypsin and bovine factor Xa bind less tightly than thrombin to p-chlorobenzylamido-epsilon-aminocaproyl agarose, being eluted by high ionic strength. It is proposed that the specific thrombin adsorption is related to a secondary binding site of high affinity and with hydrophobic properties. This site is not available in the zymogen. Furthermore, the less specific protease, trypsin, and the more specific protease, factor Xa, lack this binding site.     

Clotting of bovine fibrinogen. Calcium binding to fibrin during clotting and its dependence on release of fibrinopeptide B

Polymerization of bovine fibrinogen acted upon by thrombin is accompanied by binding of Ca2+ and a concomitant decrease of the free Ca2+ concentration. The latter can be recorded by a Ca2+-selective electrode as a shift in the electrode potential. The shift shows marked dependence on the initial free Ca2+ concentration, being maximal at about 10(-4.1) M and decreasing sharply on either side of this. Thus, the effect is limited to the 10(-3)-10(-5) M free Ca2+ concentration range. From the initial and the final value of the electrode potential during a clotting experiment, the amount of Ca2+ bound to fibrinogen and fibrin, respectively, can be calculated. The difference between the two, plotted against free Ca2+ concentration, gives a bell-shaped curve. This indicates that the reason for the Ca2+ binding is a shift of the pK of some groups from a lower to higher value. The recordings can be used for evaluation of the kinetics of the Ca2+ uptake. However, they have to be corrected for the effect of the continuous shift in the free Ca2+ concentration during the experiment. The reaction does not follow simple kinetics, showing a lag period. Therefore, rates were estimated from inverse half-reaction times. Half-times of the corrected curves show that the reaction is first order with respect to thrombin. Moreover, the rate of Ca2+ uptake is identical with that of the conformational change seen in differential scanning calorimetry [Donovan, J.W., & Mihalyi, E. '1985) Biochemistry 24, 3434]. The inverse rate and the final corrected Ca2+ uptake increase linearly with the initial fibrinogen concentration. Concomitant estimates of fibrinopeptide A and B release showed that the Ca2+ uptake runs parallel to the release of fibrinopeptide B. Fibrinopeptide A was released largely during the lag period of the Ca2+ uptake. In agreement with this, clotting with Ancrod, an enzyme that liberates only fibrinopeptide A, was not accompanied by binding of Ca2+. Thus, polymerization is not sufficient for the Ca2+ uptake to occur; liberation of fibrinopeptide B seems to be obligatory. Further support for this was obtained with experiments with the polymerization inhibitor Gly-Pro-Arg-Pro. The tetrapeptide inhibits polymerization and also, proportional to this, release of fibrinopeptide B [Hurlet-Jensen, A., Cummins, H.Z., Nossel, H.L., & Liu, C.Y. (1982) Thromb. Res. 27, 419; Lewis, S.D., Shields, P.P., & Shafer, J.A. (1985) J. Biol. Chem. 260, 10192]. Calcium uptake was also depressed by the tetrapeptide in a way similar to its effect upon fibrinopeptide B release.