Structural and physiological relationships in blood clotting proteins: thrombin and prothrombin

Structural aspects of the behaviour of prothrombin and its fragments have been examined by circulae dichroism spectroscopy. It has been noted that a correlation exists between the ellipticity of the aromatic bands and the physiological activity of partially denatured and abnormal prothrombins. The origin of these bands appears to be predominantly based in the region of one or more tyrosine residues. It is shown that whereas complexation of calcium with prothrombin causes little change in the dromatic c.d. spectrum, the effect on prothrombin fragment 1 is quite dramatic. It is concluded that the binding of calcium to the dicarboxyglutamate residues in fragment 1 causes a concomitant ionization of one or more tyrosine residues. The behaviour of fragment 1 is indicative of an intact protein with a tertiary structure which supports our previous trimodular model of prothrombin, which is activated in part by the unlocking of an ‘ionic’ lock. This lock consists of the highly negatively charged dicarboxyglutamyl patch at or near the N terminus of prothrombin and a positively charged basic patch near the C terminus.     

Kinetic intermediates in prothrombin activation. Bovine prethrombin 1 conversion to thrombin by factor X

Two pathways are possible during the proteolytic formation of alpha-thrombin (alpha-IIa) from prothrombin (II) or prethrombin 1 (P1). One of the pathways, with prethrombin 2 or prethrombin 2 associated with fragment 2 (P2F2) as intermediates, has long been known to exist when activation is catalyzed by Factor Xa (Xa) alone. The second pathway, with meizothrombin or meizothrombin (des fragment 1) (MzIIa(-F1)) as intermediate, has been shown to exist when Factor Va and phospholipids are present with Xa. Until now, MzIIa(-F1) has not been detected in reactions catalyzed by Xa alone. In this study, we demonstrate that P1 activation by Xa alone occurs via both pathways, and we provide rate constants and kinetic equations for calculating the relative contributions of each of the pathways to the formation of alpha-IIa by Xa. Investigation of the initial rates of proteolytic cleavage of P2F2 and P1 by Xa alone indicated first-order dependence on substrate concentration with no evidence of saturation of Xa with either substrate at concentrations as high as 200 microM. Apparent second-order rate constants (kc/Km) of 113 +/- 9 M-1 s-1 for the formation of thrombin from P2F2 and 1,410 +/- 19 M-1 s-1 for the disappearance of P1 were determined at pH 7.5, 25 degrees C, 10 mM CaCl2, 0.15 M ionic strength. A two-step sequential first-order pathway employing these rate constants for thrombin activity production from P1 via P2F2 could not, however, account for the quantity of thrombin that was produced during the early stages of P1 activation. Addition of a parallel first-order reaction to produce thrombin activity from P1 independently of P2F2, tentatively identified as the formation of MzIIa(-F1), yielded progress curves in quantitative agreement with the experimental data. kc/Km for the parallel reaction was estimated to be 98 +/- 10 M-1 s-1. Independent determination of the second-order rate constant for the cleavage of isolated MzIIa (-F1), 15,000 +/- 420 M-1 s-1, indicated that MzIIa(-F1) could meet the kinetic requirements for an intermediate in the parallel activation pathway. The transient formation of MzIIa (-F1), as well as the generation of alpha-IIa, was directly demonstrated during activation of P1 by active site-affinity labeling of the reaction products with a biotin derivative of D-Phe-Pro-Arg chloromethyl ketone and visualization by semiquantitative Western blotting.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of thrombin exosites I and II in the activation of human coagulation factor V

Human blood coagulation Factor V (FV) is a plasma protein with little procoagulant activity. Limited proteolysis at Arg(709), Arg(1018), and Arg(1545) by thrombin or Factor Xa (FXa) results in the generation of activated FV, which serves as a cofactor of FXa in prothrombin activation. Both thrombin exosites I and II have been reported to be involved in FV activation, but the relative importance of these regions in the individual cleavages remains unclear. To investigate the role of each exosite in FV activation, we have used recombinant FV molecules with only one of the three activation cleavage sites available, in combination with exosite I- or II-specific aptamers. In addition, structural requirements for exosite interactions located in the B-domain of FV were probed using FV B-domain deletion mutants and comparison with FV activating enzymes from the venom of Russell's viper (RVV-V) and of Levant's viper (LVV-V) known to activate FV by specific cleavage at Arg(1545). Our results indicate that thrombin exosite II is not involved in cleavage at Arg(709) and that both thrombin exosites are important for recognition and cleavage at Arg(1545). Efficient thrombin-catalyzed FV activation requires both the N- and C-terminal regions of the B-domain, whereas only the latter is required by RVV-V and LVV-V. This indicates that proteolysis of FV by thrombin at Arg(709), Arg(1018), and Arg(1545) show different cleavage requirements with respect to interactions mediated by thrombin exosites and areas that surround the respective cleavage sites. In addition, interactions between exosite I of thrombin and FV are primarily responsible for the different cleavage site specificity as compared with activation by RVV-V or LVV-V.     

Model of a specific interaction: Salt-bridges form between prothrombin and its activating enzyme blood clotting factor Xa

In order for intricate biochemical pathways to function properly in the heterogeneous environment of a cell or organism, high specificity of enzymesubstrate reactions is essential. The molecular nature of this specificity is examined for the case of the specific activation of prothrombin by factor X_a of the blood coagulation system using model-building methods.The structure of blood clotting factor X_a was modeled by homology to the known structures of the mammalian serine proteases. The sequence about the cleaved peptide bond in prothrombin was placed into the active site of factor X_a in a conformation similar to that of bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor, when bound to trypsin, and to that of the tripeptide chloromethyl ketones, when bound to γ-chymotrypsin and Streptomyces griseus protease B.The model of the complex between prothrombin and factor X_a shows the expected salt-bridge between the Arg preceding the cleaved peptide bond and the primary specificity residue Asp189 of factor X_a. Two other salt-bridges appear to be formed: between a Glu three residues before the cleaved bond in prothrombin and Arg143 of factor X_a, and between a second Glu three residues after the cleaved bond and Lys62 in factor X_a. Several hydrogen bonds and hydrophobic interactions also occur in the complex. A stereogeometric requirement for a Gly two residues before the cleaved bond is also imposed by the factor X_a structure.Examination of the known serine protease sequences and model building suggest that the two new salt-bridges are unique to the prothrombin-factor X_a complex. Therefore, these charge interactions, and the requirement for a Gly, are likely to be at least partially responsible for the high specificity of the activation of prothrombin by factor X_a.     

Activation of duck prothrombin by factor Xa and thrombin

Prothrombin isolated from duck sodium citrate plasma was activated in a system containing duck factor Xa and calcium ions. Polyacrylamide gel electrophoresis showed that intermediates and the final product, thrombin, of Mr in the range 21 500-52 000 were present in the incubation mixture Serine and isoleucine were found to be the N-terminal amino acids of the intermediate form 1 and thrombin, respectively.     

Electron microscopy and hydrodynamic properties of blood clotting factor V and activation fragments of factor V with phospholipid vesicles

The electron microscopic and hydrodynamic properties of factor V and factor Va-vesicle complexes were determined. Images of negatively stained factor V bound to vesicles showed the protein as a relatively large globular domain (9.5 nm diameter) connected to the membrane through a narrow protein region 0.5-3 nm in length. This connecting region was not always visible and was measured as the distance between the globular region and the apparent vesicle edge. Factor V protein alone usually appeared as two connected globular regions of 10.2 and 6.5 nm diameter. The two-domain protein structure appeared consistent with both the image of factor V alone and bound to the membrane. Factor V had no biological activity in a phospholipid-free prothrombinase assay system used. The proteolytically activated form of factor V generated by digestion with thrombin (factor Va) was at least 30,000 times more active. The electron microscopic images of factor Va-vesicle complexes showed a smaller protein that was more closely associated with the vesicle surface than was factor V. The light chain (Mr about 80,000) component of factor Va also bound to the surface of the vesicles and appeared to be largely external to the membrane. Protein-induced hydrodynamic radius changes for the factor V-vesicle and factor Va-vesicle complexes were 12.8 and 6.3 nm, respectively. The images observed in the electron microscope were used to calculate protein-induced radius changes. Comparison of these values with the experimentally determined hydrodynamic radius changes showed approximate agreement for factor Va-membrane complexes. However, the images of factor V-vesicle complexes suggested smaller hydrodynamic radius changes than were actually observed.     

Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.     

Role of prothrombin fragment 1 in the pathway of regulatory exosite I formation during conversion of human prothrombin to thrombin

Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin(54-65) labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir(54-65)(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir(54-65)(SO3-)). [NBD]Hir(54-65)(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir(54-65)(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir(54-65)(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin.F1.2 complex bound [5F]Hir(54-65)(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.     

Mathematical model of the activation of prothrombin by factor X a and factor V t

A mathematical model of prothrombin activation is being proposed which includes the feedback mechanism of thrombin and the alteration of factor V by thrombin. This model is in good agreement with experimental data for the dependence of the rate of thrombin formation on the concentrations of factors V and X _a . In particular, it correctly predicts the existence and location of a maximum in both of these cases.