Prothrombin Molecule Research Articles

Prothrombin “Carmel”: Identification of a Novel Homozygous Mutation in the Prothrombin Gene Causing Coagulation Factor II Deficiency and Inherited Bleeding Disorder in an Israeli Arab Family

Congenital prothrombin deficiency, also known as hypoprothrombinemia is a rare autosomal recessive disorder characterized by severe bleeding manifestations and decreased prothrombin antigen levels below 10% activity of normal. Dysprothrombinemia, which is characterized by normal antigen levels but a dysfunctional prothrombin molecule, shows a more variable level in bleeding tendency, and there is often a good correlation between the levels of prothrombin activity and clinical severity. We have studied a large consanguineous family of thirteen members contains two patients with a prolonged protrombine time (PT) and activated partial thromboplastin time (aPTT) with prothrombin activity of 11%. DNA was isolated from leukocytes and the thrombin exons were amplified by polymerase chain reaction and sequenced. Exon 14 revealed a point mutation in the codon of Tyrosine 560 which is substituted by Histidine (Tyr560His). Mutation at this residue most probably distorts the structure of the peptidase catalytic domain. The variation segregated as expected in the family, it did not appear in dbSNP, EVS or the 1000 genome project. Further investigation is needed to determine the antigen level and to elucidate the potential effect of the mutation on the structure and the activity of the enzyme. This prothrombin is designated as Prothrombin “Carmel”. DisclosuresNo relevant conflicts of interest to declare.

The appearance of molecules of prothrombin origin in blood upon development of atherothrombotic and cardioembolic ischemic stroke

Increase of molecules of prothrombin origin in patients with atherothrombotic and cardioembolic stroke subtypes had been shown. It was found the presence of protein fractions in the range of molecular weights corresponding to both prothrombin molecules and its degraded forms.

Crystal Structure of Prothrombin Reveals Conformational Flexibility and Mechanism of Activation

The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36° angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.

Conformational Dynamics of Prothrombin Dictate Its Ordered Cleavage by Prothrombinase

Abstract 3359The prothrombinase complex (membranes/Xa/Va) binds prothrombin through exosite interactions which orient and present the substrate to the protease active site for cleavage. Although both cleavage sites within the substrate are accessible, prothrombin is preferentially activated by prothrombinase through sequential cleavage at R320 followed by cleavage at R271. We investigated the exosite-dependent interaction between prothrombin and prothrombinase using fluorescence resonance energy transfer (FRET). An acceptor fluorophore was placed onto the prothrombin cleavage site by introducing the mutation R271C and fluorescently modifying the introduced thiol (IIC271*). The active site of Xa was covalently modified with a chloromethyl ketone conjugated to a donor fluorescent compound. Titrations of the fluorescently labeled prothrombinase complex with IIC271* revealed a maximum energy transfer efficiency of 70–80 % observed with two different donor probes. Resonance energy transfer arising from the substrate docking interaction was confirmed by equivalent changes in the excited state lifetime of the donors. The calculated distance between the probe at C271 and the probe tethered at the active site of Xa ranged between 34 and 40 Å. Variation in the measured distance reflects the different lengths of the tethers used to connect the donor probes. The FRET measurements, validated with two donor-acceptor pairs, indicate that the 271 site within prothrombin docked to prothrombinase is positioned distant from the active site of Xa within the enzyme complex. This distant constraint prevents R271 from effectively engaging the active site of Xa within prothrombinase, thereby providing a physical explanation for the ordered action of prothrombinase on prothrombin. It follows that initial cleavage at R320 must reorient the substrate to allow R271 access to the Xa active site. We pursued an intramolecular FRET approach in which both donor and acceptor probes were placed on the prothrombin molecule to investigate the putative conformational change of prothrombin as a result of initial cleavage. Orthogonal labeling of prothrombin was accomplished using a prothrombin variant containing C271 and a LPETG extension at its C terminus. The optimized C271 label was used for the first modification. For the second, the sequence-specific transpeptidation activity of bacterial Sortase A was used to incorporate a fluorophore at the C-terminus (IICterm*). To assess the new fluorescent modification, FRET studies of prothrombin binding to prothrombinase were repeated with the new IICterm* construct. Titrations revealed a maximal energy transfer of 16 %, confirmed by corresponding changes in excited state lifetime and yielded a Kd = 39 nM indistinguishable from the Kd= 41 nM seen for IIC271* binding. Thus, two different approaches illustrate that the affinity of prothrombin for prothrombinase is independent of active site docking by the substrate. To examine the intramolecular dynamics of prothrombin during activation, the C271 site was labeled with BADAN (donor) and the Sortase labeling method placed the Alexa532 (acceptor) at the C terminus. The orthogonal placement of BADAN and Alexa532 on the prothrombin molecule resulted in extensive energy transfer between the two probes. The cleavage of doubly-labeled prothrombin to produce meizothrombin was associated with a change in intramolecular energy transfer. The variation of the energy transfers observed between the zymogen and protease forms can be interpreted as global rearrangement of the prothrombin molecule renders the second cleavage site accessible of active site docking. The FRET studies provide a comprehensive physical explanation for the ordered cleavage of prothrombin by prothrombinase. Initial cleavage occurs at the spatially accessible cleavage site R320. The prothrombin molecule then significantly re-orients itself making the second R271 cleavage site accessible to prothrombinase in order for activation to proceed and produce thrombin. Disclosures:No relevant conflicts of interest to declare.

Thomas Addis, MD (1881-1949): Scottish-American clinical laboratory researcher, social activist and pioneer of renal medicine

Addis was born and educated in Edinburgh, from the University of which he graduated MB in 1905, and MD in 1908, in which year he also gained membership of Edinburgh's Royal College of Physicians. After researching disordered haemostasis associated with various clinical conditions, he spent over a year in Germany: in Berlin with Dr. E.L. Salkowski learning urinalysis and at Heidelberg under Ludolph von Krehl studying haemophilics. Back in Edinburgh he concluded that the ultimate cause of haemophilia was an 'anatomical defect in the molecule of prothrombin'. He was the first to monitor the effects on plasma clotting times of transfusion of anticoagulated blood into a haemophilic. In 1911 he was recruited by Ray Lyman Wilbur, the first dean of the Stanford University School of Medicine, to investigate metabolic disorders including jaundice, diabetes and ultimately chronic renal disease. In 1917 he described the 'urea ratio'--the mathematical and conceptual forerunner of clearance formulae--and over the next 30 years developed a combined clinical and laboratory service for patients with inexorably failing kidneys. He devised an effective, rational and individually based dietary treatment--some patients such as Linus Pauling, who presented in 1941 with marked nephrosis, responded completely. Addis' Calvinist upbringing gave him a strong sense of 'mission' which during the American Depression developed into support for poverty-stricken workers in America, and against the fascists in Spain. He died before the full development of the 'McCarthy Witch Hunts' of the 1950s, although many associates, including Robert Oppenheimer, were interrogated.

The Role of Amino Acids 659-663 of Factor Va Heavy Chain During Prothrombin Activation by Prothrombinase.

Abstract 2131Poster Board II-108The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), and the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of Ca2+. FXa alone can activate prothrombin following sequential cleavages at Arg271 and Arg320 yielding the transient inactive intermediate prethrombin 2. However, the interaction of fVa with fXa on a membrane/cell surface in the presence of divalent metal ions and formation of the prothrombinase complex results in the reversal of the order of cleavages and a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. A first cleavage of prothrombin by prothrombinase at Arg320 produces the active intermediate meizothrombin, while the second cleavage at Arg271 produces thrombin. Thrombin and prothrombin contain two positively charged binding regions (anion binding exosite I, ABE-I and anion binding exosite II, ABE II), that are crucial for protein function. Initial cleavage of prothrombin at Arg320 by prothrombinase which is absolutely factor Va dependent, entirely exposes (pro)exosite I. FVa is required for the specific recognition of prothrombinase by (pro)exosite I of prothrombin. The COOH-terminal region of the heavy chain of fVa contains acidic amino acid clusters that are important for cofactor activity. We have investigated the role of amino acid region 659-663 that contains five consecutive acidic amino acid residues. To ascertain the function of this region, site-directed mutagenesis was performed. We have constructed a mutant molecule with this region deleted (fVD659-663) and a mutant molecule in which all five residues were mutated to lysine (fV5K, charge reversal). The recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to fVaWT. Kinetic analyses studying prothrombinase assembled with the mutant molecules demonstrated diminished kcat values, while the affinity of all mutant molecules for factor plasma-derived fXa was similar to fVaWT. Gel electrophoresis analyzing plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg320 and Arg271 by prothrombinase assembled with the mutant molecules. Using recombinant prothrombin molecules we determined that cleavage at Arg271 by prothrombinase assembled with either fVaD659-663 or fVa5K was severely impaired compared to cleavage at Arg320 by prothrombinase assembled with the same recombinant cofactor molecules, resulting in lingering of meizothrombin throughout the activation process. To ascertain the effect of the mutations of the fVa heavy chain on the cleavage at Arg271 alone following the transition that occurs after cleavage at Arg320, we compared the rate of cleavage of active-site blocked meizothrombin (FPR-meizothrombin) by prothrombinase assembled with either fVaWT or fVaD659-663. The data demonstrate a delay for cleavage of FPR-meizothrombin at Arg271 by prothrombinase assembled with fVaD659-663 as compared to the same reaction catalyzed by prothrombinase assembled with fVaWT. Quantitative scanning densitometry of fragment 1•2-A demonstrated a ∼4-fold delay in cleavage of FPR-meizothrombin at Arg271 by prothrombinase assembled with fVaD659-663, compared to cleavage at Arg271 by prothrombinase assembled with fVaWT. Direct comparison between the rates of cleavage of FPR-meizothrombin by membrane-bound fXa alone or by prothrombinase assembled with fVaRVVD659-663 do not show any significant differences. Thus, deletion of amino acid region 659-663 virtually eliminates the acceleration in the rate of cleavage at Arg271 of meizothrombin attributed to the interaction of fVa with fXa. These data demonstrate that amino acid sequence 659DDDED663 from the factor Va heavy chain, regulates meizothrombin concentration during activation of prothrombin by prothrombinase. Disclosures:No relevant conflicts of interest to declare.

A Control Switch for Prothrombinase: CHARACTERIZATION OF A HIRUDIN-LIKE PENTAPEPTIDE FROM THE COOH TERMINUS OF FACTOR Va HEAVY CHAIN THAT REGULATES THE RATE AND PATHWAY FOR PROTHROMBIN ACTIVATION

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg(271) and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg(320) (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695-699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg(320). These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg(320) and Arg(271), respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg(271) of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in prothrombinase.

Controlling the Pathway for Prothrombin Activation by Prothrombinase.

Prothrombinase is the enzymatic complex responsible for timely thrombin formation. Activation of human prothrombin is the consequence of two cleavages at Arg271 and Arg320 in prothrombin by factor Xa. Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have previously shown that a pentapeptide encompassing amino acid sequence 695–699 from the COOH-terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) interacts with anion binding exosite I (ABE-I) of thrombin and inhibits prothrombin activation by prothrombinase. The peptide was found to be a competitive inhibitor of prothrombinase with respect to substrate. According to the mode of inhibition, we postulated that the peptide binds prothrombin in competition with the binding of the substrate to the enzyme, and inhibits prothrombinase activity by substrate depletion. This mode of DYDYQ inhibition of prothrombin activation by the factor Va-factor Xa complex is similar to that previously demonstrated for sulfated hirugen. To understand the mechanism of inhibition of thrombin formation by DYDYQ we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by fully assembled prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. Higher peptide concentrations were required to impair thrombin formation through the latter pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg320. These findings were corroborated by studying the kinetics of activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271 respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ while high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with thrombin formation by membrane-bound factor Xa alone in the absence of factor Va. Nonetheless, while the rate for cleavage at Arg271 of plasma-derived prothrombin or rP2-II by membrane-bound factor Xa alone was significantly accelerated in the presence of DYDYQ, resulting in accumulation of prethrombin 2, the rate for cleavage at Arg320 of plasma-derived prothrombin or rMZ-II by membrane-bound factor Xa alone was only moderately affected by the pentapeptide. Our data demonstrate that a pentapeptide mimicking amino acids 695–699 of the heavy chain of factor Va has opposing effects on membrane-bound factor Xa for prothrombin activation, depending on the incorporation of factor Va in prothrombinase. In the presence of the cofactor the peptide inhibits the rate of thrombin generation by specifically interfering with initial cleavage of prothrombin at Arg320, while in the absence of factor Va the pentapeptide accelerates cleavage of prothrombin by factor Xa at Arg271. Thus, the amino acid region spatially surrounding proexosite I in prothrombin most likely has two interactive sites for the components of prothrombinase, a factor Va interactive site and a factor Xa binding site.

The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII

Alpha-thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. Alpha-thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by alpha-thrombin. We have used plasma-derived alpha-thrombin, beta-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg(320) (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO(3)(-))DY(SO(3)(-))Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with beta-thrombin was increased by approximately 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than alpha-thrombin under similar experimental conditions. Alpha-thrombin readily activated factor V following cleavages at Arg(709), Arg(1018), and Arg(1545) and factor VIII following proteolysis at Arg(372), Arg(740), and Arg(1689). Cleavage of both procofactors by alpha-thrombin was significantly inhibited by D5Q1,2. In contrast, beta-thrombin was unable to cleave factor V at Arg(1545) and factor VIII at both Arg(372) and Arg(1689). The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. Beta-thrombin was found to cleave factor V at Arg(709) and factor VIII at Arg(740), albeit less efficiently than alpha-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by beta-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of alpha-thrombin can account for the interaction of both procofactors with alpha-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of alpha-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.

Decreased inhibitory activity of prothrombin to calcium oxalate crystallization by specific chemical modification of its gamma-carboxyglutamic acid residues

Objectives To investigate the inhibitory role of gamma (γ)-carboxyglutamic acid (Gla) in the Gla domain of urinary prothrombin in the formation of calcium oxalate crystals. Methods Morpholine and formaldehyde at different concentrations were added to the solution of prothrombin to cause the conversion of Gla to γ-methyleneglutamic acid (MGlu). The extent of the modification was controlled by the relative amount of modification reagents to prothrombin. The 100-fold molar excess of modification reagents to prothrombin produced the prothrombin molecule containing 2γ-MGlu residues, and the 10,000-fold molar excess produced the molecule containing 8γ-MGlu residues. The inhibitory activities of prothrombin and modified prothrombin to calcium oxalate crystallization were evaluated by the seeded crystallization technique. Results The inhibitory index of Gla prothrombin to calcium oxalate crystallization was 14.2%, that of 2γ-MGlu prothrombin was 12.8%, decreased by about 10%, and that of 8γ-MGlu prothrombin was 5.0%, decreased by about 65%. Conclusions The Gla in the Gla domain of urinary prothrombin may play an important role in inhibiting the formation of renal calcium oxalate calculus.

Human Prothrombin Fragment 1 Conformational Changes Following Binding to Phospholipid Vesicles: A Computational Study.

Our long term goal is to create a model of the prothrombinase complex. We have recently proposed a complete model in solution of human coagulation factor Va (pdb entry 1y61). In the present study we create the framework needed to accomplish our final goal. We first created a lipid bilayer that mimics the cell membrane surface. The lipid bilayer mimetic was set up as a random mixture of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) using a 4:1 ratio. Human prothrombin fragment one is a highly homologous region among species, except zebrafish and hagfish ( Jagadeeswaran et al., Blood Cells Mol. Dis., 2000). In the absence of a crystal structure of the entire human prothrombin molecule, we created a homology model of human prothrombin fragment 1 (amino acid residues 1–155). In the homology modeling procedure we used three X-ray structures available for the bovine prothrombin fragment 1 molecule (Soriano-Garcia et al., Biochemistry, 1992, Seshadri et al., J. Mol. Biol., 1991, and Huang et al., Nat. Struct. Biol., 2003). One extra Ca2+ was added as proposed in an earlier human prothrombin fragment 1 homology model (Leping et al., Protein Sci., 1995). The modeling was performed using the Modeller software package. Prothrombin fragment 1 was then inserted into the lipid bilayer so the region composed of amino acid residues 1–10 were positioned in the hydrophobic core, whereas the γ-carboxyglutamic acid rich region was on the surface of the lipid bilayer as proposed (Falls et al., J. Biol. Chem., 2001). Molecular dynamics simulations on the 10 ns time scale of the prothrombin fragment 1 bound to the POPC/POPS lipid bilayer showed a conformational change in prothrombin fragment 1. This conformational change can be described as a rearrangement of three regions: region A (composed of amino acid residues 1–30), region B (composed of amino acid residues 31–69), and region C (composed of amino acid residues 70–155). Root mean square deviation (RMSD) of region A compared to the starting model was found to be 1.61 Å whereas for region C was found to be 1.91 Å. The RMSD of region B was found to be 1.99 Å. Because region B contains two α- helix motifs the deviation is due to the amino acids found at both ends (i.e., the ones that connect region A and region C with region B). During the simulations the initial Ca2+ ions positions were found to be preserved in the vicinity of the γ-carboxyglutamic amino acid residues. The secondary structure elements were preserved during simulations. Following a 10 ns simulation three γ-carboxyglutamic residues (7, 14 and 25) of prothrombin fragment 1 were found to form polar contacts with POPS molecules. Due to the conformational change, Asp55 was also found to interact with one POPS molecule. This amino acid residue was previously shown to be located near the active site of meizothrombin. Overall, our studies present for the first time a POPC/POPS lipid bilayer model, and show that in the presence of Ca2+ ions a conformational change occurs in prothrombin fragment 1 following its interaction with the lipid bilayer.

Exposure of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII.

The intrinsic tenase complex and the prothrombinase complex are composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. Incorporation of the protein cofactor in both complexes results in a substantial increase in the catalytic efficiency of both enzymes, factor IXa and factor Xa, for cleavage and activation of factor X and prothrombin respectively, resulting in normal hemostasis. The procofactors, factor V (FV) and factor VIII (fVIII), do not interact with the components of prothrombinase and intrinsic tenase respectively and must be activated. α-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate binding necessary for efficient catalysis. α-Thrombin catalyzes the activation of fV and fVIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by α-thrombin. We have used plasma-derived α-thrombin, β-thrombin (a thrombin molecule that has only ABE-II available) and a recombinant prothrombin molecule rMz-II (R155A/R284A/R271A) that can only be cleaved at Arg320 (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a sulfated pentapeptide (DY(SO3−)DY(SO3−)Q, named D5Q1,2) as exosite-directed inhibitor of thrombin. D5Q1,2 was found to increase thrombin time in a dose dependent manner yielding an eight-fold increase in thrombin time at 250 μM in the presence of 10 nM α-thrombin. This clotting time was equivalent to the thrombin time obtained with 10 nM β-thrombin alone. The clotting time of rMZ-IIa was increased four-fold compared to the clotting time of α-thrombin under similar experimental conditions. α-Thrombin readily activated fV following cleavages at Arg709, Arg1018, and Arg1545 and fVIII following proteolysis at Arg372, Arg740, and Arg1689. Cleavage of both procofactors by α-thrombin was significantly inhibited by D5Q1,2. In contrast, β-thrombin was unable to cleave fV at Arg1545 and fVIII at both Arg372 and Arg1689. The former is required for expression of factor Va (fVa) cofactor activity while the latter two cleavages are a prerequisite for expression of factor VIIIa (fVIIIa) cofactor activity. β-Thrombin was found to cleave fV at Arg709 and fVIII at Arg740, albeit less efficiently than α-thrombin. D5Q1,2 inhibited moderately both cleavages by β-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules with a rate similar to that observed for the activation of fV and fVIII by α-thrombin. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. These data demonstrate that ABE-I alone of α-thrombin can account for the interaction of both procofactors with α-thrombin resulting in their timely and efficient activation. Our data also show that a sulfated pentapeptide inhibits several procoagulant ABE-I-related functions of α-thrombin and provide a target as well as the scaffold for the synthesis of an exosite-directed anticoagulant molecule that could inhibit and/or attenuate therapeutically thrombin function in individuals with thrombotic tendencies.

Distribution of the heterogeneity of des‐γ‐carboxyprothrombin in patients with hepatocellular carcinoma

Our aim was to evaluate the heterogeneity of des-gamma-carboxyprothrombin (DCP) in the plasma of patients with hepatocellular carcinoma (HCC), benign liver diseases, and normal controls and compare the DCP values by enzyme-linked immunosorbent assay (ELISA) for two types monoclonal antibodies (MU-3 and 19B7). We purified DCP from the plasma of 17 patients with HCC, three patients with metastatic liver tumors (MTLT), 12 with acute hepatitis (AH), five with chronic hepatitis (CH), nine with liver cirrhosis (LC), and 10 normal controls (NC). The DCP was analyzed by using immunoaffinity chromatography, reversed-phase high-performance liquid chromatography and measured by using an ELISA. In HCC, the synthesis of the 1-, 3-, and 4-Gla (gamma-carboxyglutamic acid) DCP variants markedly increased, and those levels accounted for more than 50% of the DCP. The synthesis of the 3-, and 4-Gla DCP gradually increased in order of AH, CH, and LC patients. The MU-3 antibody reacted with the 1-, 3- and 4-Gla DCP variants, whereas the 19B7 antibody reacted with the 6-, 7- and 8-Gla variants. The DCP was measured by ELISA, markedly increased in order of NC, AH, CH, LC, and HCC cases. The correlation of the ratios (1 + 3 + 4)-Gla/(6 + 7 + 8)-Gla DCP and MU-3/19B7 was positive and statistically significant (r = 0.786, n = 56). According to the severity of liver damages, the synthesis of the 1-, 3- and 4-Gla DCP variants that lost the Ca binding from the outside of the Gla-domain of the prothrombin molecule increases and the MU-3/19B7 ratio is believed to reflect this.

Incorporation of Factor Va into Prothrombinase Is Required for Coordinated Cleavage of Prothrombin by Factor Xa

Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg271 followed by cleavage at Arg320. Factor Va, along with phospholipid and Ca2+, enhances the rate of the process by 300,000-fold, reverses the order of cleavages, and directs the process through the meizothrombin pathway, characterized by initial cleavage at Arg320. Previous work indicated reduced rates of prothrombin activation with recombinant mutant factor Va defective in factor Xa binding (E323F/Y324F and E330M/V331I, designated factor VaFF/MI). The present studies were undertaken to determine whether loss of activity can be attributed to selective loss of efficiency at one or both of the two prothrombin-activating cleavage sites. Kinetic constants for the overall activation of prothrombin by prothrombinase assembled with saturating concentrations of recombinant mutant factor Va were calculated, prothrombin activation was assessed by SDS-PAGE, and rate constants for both cleavages were analyzed from the time course of the concentration of meizothrombin. Prothrombinase assembled with factor VaFF/MI had decreased k(cat) for prothrombin activation with Km remaining unaffected. Prothrombinase assembled with saturating concentrations of factor VaFF/MI showed significantly lower rate for cleavage of plasma-derived prothrombin at Arg320 than prothrombinase assembled with saturating concentrations of wild type factor Va. These results were corroborated by analysis of cleavage of recombinant prothrombin mutants rMz-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A), which can be cleaved only at Arg320 or Arg271, respectively. Time courses of these mutants indicated that mutations in the factor Xa binding site of factor Va reduce rates for both bonds. These data indicate that the interaction of factor Xa with the heavy chain of factor Va strongly influences the catalytic activity of the enzyme resulting in increased rates for both prothrombin-activating cleavages.

Relationship between clinical features and binding domains of anti-prothrombin autoantibodies in patients with systemic lupus erythematosus and antiphospholipid syndrome.

Autoantibodies against prothrombin, including lupus anticoagulant antibodies (LAC), have been identified in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). To identify the epitopes of LAC in patients with SLE and APS, we analyzed B cell epitopes of anti-prothrombin Abs. Prothrombin was purified from fresh plasma samples from healthy subjects, and fragmented by thrombin. Two fragments (prethrombin-1, 50 kDa, and fragment-1, 22 kDa) were separated and used for further experiments. The two fragments were coated on irradiated plate and the binding activities of sera from 13 patients with anti-prothrombin Abs (SLE, 7; APS, 4; SLE+APS, 2) were determined by using ELISA. The assay was conducted under the following conditions: use of irradiated plates, and TBS containing Tween-20. We detected two types of anti-prothrombin Abs. The first was anti-prethrombin-1 (n=5) while the other was Ab against fragment-1 (n=8). There were no patients with Abs that showed binding activities to both fragments. A higher proportion of patients with thrombosis were positive for anti-prethrombin-1 Abs (80%) than for anti-fragment-1 Abs (25%). Two patients with anti-prethrombin-1 Ab were positive for LAC and negative for anti-cardiolipin-beta2 glycoprotein I antibody (aCL-beta2GPI). Our results strongly support the notion that both prethrombin-1 and fragment-1 on prothrombin molecule are B cell epitopes.

Domains of human prothrombin embedded into vesicles: relation with biological activity

In our previous works, ac polarography experiments gave evidence of penetration of prothrombin into phosphatidylserine (PS) containing monolayers and labeling of prothrombin by the highly apolar azide 5[ 125 I ]-iodonaphthalene-1-azide (INA) could be used to monitor penetration of the zymogen into phospholipid vesicles. In the present study, the photochemical labeling method was used to detect which domains in the whole prothrombin molecule are embedded in 75% PS–25% PC containing vesicles; the results are compared to those obtained on monolayers by ac polarography with isolated purified fragments of the protein. The kinetics of cleavage of prothrombin following INA labeling, in the presence of phospholipids, by factor Xa or thrombin, as well as the nature of the products formed, are reported here. The cleavages of radiolabeled INA-II and non-radiolabeled II could be independently followed by the formation of radiolabeled and non-radiolabeled products. It was found that prethrombin 2 is the main penetrating domain, as compared with prethombin 1 and fragment 1, while fragment 2 does not penetrate. Both INA-II and II were activated by factor Xa to give the expected products but INA-II was cleaved faster at Arg 271. This finding suggests that the interaction with vesicles which leads to INA-labeling of prothrombin, results in a conformational change of the protein that enhances its susceptibility to activation. In gel electrophoresis under reducing conditions, subsequent cleavage at Arg 320 resulted in the formation of radiolabeled thrombin B-chain (TB). Since thrombin is formed from radiolabeled prothrombin, this shows that the prothrombin molecules which are embedded in the membrane are biologically active.

Fluorescence resonance energy transfer study of shape changes in membrane-bound bovine prothrombin and meizothrombin.

Prothrombin activation to thrombin is a key control reaction in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg323-Ile and Arg274-Thr) by factor X(a) to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-containing membranes from platelets are believed to facilitate this two-step process. Using fluorescence energy transfer (FRET), we determined the distances of closest approach between a specifically located C-terminal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C)-FM) or meizothrombin (M(S528A, G581C)-FM) and phosphatidylethanolamine-N-rhodamine B (PE-Rh; 0-8.7 mol %) contained in membranes composed of PS (25 mol %) and phosphatidylcholine (66.3-75 mol %). Plots of the energy transfer efficiency as a function of membrane concentration, at six PE-Rh surface densities, were analyzed globally to obtain dissociation constants and binding stoichiometries as global parameters and saturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were estimated to be 0.32 +/- 0.10 and 0.28 +/- 0.12 microM with stoichiometries of 42 +/- 12 and 44 +/- 9 lipid/protein for prothrombin and meizothrombin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the acceptor (PE-Rh) surface density. With the assumptions of kappa2 = 2/3 and n = 1.4, the distances were 94 +/- 3 A for prothrombin and 114 +/- 2 A for meizothrombin. Since both prothrombin and meizothrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 A, our FRET measurements suggest that binding to PS-containing membranes induced tighter folding of the prothrombin molecule but not of the meizothrombin intermediate. This observation is consistent with our hypothesis that membrane binding plays an essential role in the sequential alignment of the bond Arg323-Ile in prothrombin and Arg274-Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating process.

Coagulation protein function. III. Effect of acetyldehyde upon the activation of prothrombin

When prothrombin is mixed with acetaldehyde (AcH), the primary metabolite of ethanol, at room temperature for 30 min, dialyzed to remove excess AcH, and then activated with Echis carinatus venom (ECV), a prolonged clotting time is observed. Similarly treated prothrombin, however, readily hydrolyzes the synthetic substrate, benzoyl- dl-arginine-β-naphthylamide (BANA). These results sugegst that AcH does not react with the catalytic site of thrombin, which is protected in the prothrombin molecule. However, it does react with susceptible sites of the prothrombin surface which remain alkylated during extensive dialysis to remove excess AcH and subsequent activation of the molecule by ECV. These alkylated sites on the newly formed thrombin molecule may inhibit or prevent the effective/efficient binding of fibrinogen at its binding sites, causing prolonged clotting times. The binding sites for accommodating fibrinogen on the thrombin molecule are apparently quite different from those that bind BANA. These data further suggest that prolonged clotting times that are reported in chronic alcoholics may be due, in part, to ready interaction of acetaldehyde with circulating prothrombin. Fibrinogen, which has been exposed to AcH and subsequently dialyzed to remove excess AcH, also responds with a prolonged clotting time in the presence of added thrombin, signifying that sites on fibrinogen that are essential for its function/conformation are susceptible to AcH.

Inhibition of blood coagulation activation and oral anticoagulants in patients with mechanical heart valve prostheses

Oral anticoagulants are widely used for preventing thromboembolic events in many pathological conditions, such as mechanical or biological heart valve prosthesis, atrial fibrillation, deep vein thrombosis and post-myocardial infarction (1). Particularly, the intensity of anticoagulation to be induced in patients with mechanical heart prosthesis is not well established. In fact, the Dutch Thrombosis Policy recommends deep anticoagulation (3.6-4.8 INR) irrespective of whether the prosthesis is aortal or mitral (2). In contrast the Consensus Committee for antithrombotic therapy in patients with mechanical prosthetic valves, with North American thromboplastins, suggests a therapeutic range between 2.2 and 3.3 INR for both ball and tilt disk prosthesis (3). Again, the guidelines on oral anticoagulation published on behalf of the British Society for Hematology recommend a target of between 3 and 4.5 for mechanical heart valves (1). But is deeper anticoagulation more effective in the biochemical inhibition of the coagulation cascade ? Is there any difference between aortal and mitral prosthesis considering that a higher thromboembolic risk may be present in the latter due to hypercoagulability secondary to atrial fibrillation and blood stagnation ? For this purpose we chose to measure prothrombin F 1+2 peptide that is cleaved by factor Xa from the prothrombin molecule, since it has recently been shown that F 1+2 is reduced during oral anticoagulants (4).

Homozygosity for a Novel Missense Mutation in the Prothrombin Gene Causing a Severe Bleeding Disorder

A patient with a severe bleeding tendency and hypoprothrombinemia (Factor II activity 2%, Factor II antigen 5%) was screened for the presence of alterations in his prothrombin gene. Direct sequencing of PCR fragments derived from the coding and flanking regions of the prothrombin gene, revealed that the patient was homozygous for an A-->G substitution in exon 3. This substitution predicts the replacement of Tyr44 (TAC) by Cys (TGC) in the prothrombin molecule. Both parents were found to be heterozygous for the same mutation. Further family studies revealed complete cosegregation of the mutation with the prothrombin deficiency. Only the five homozygous brothers and sisters of the propositus were clinically affected (severe hemorrhages including epistaxis, soft tissue, muscle and joint bleedings in all, and severe hemorrhages in the two women). The bleeding tendency therefore seems to inherit as an autosomal recessive trait.