A Natural Prothrombin Mutant Reveals an Unexpected Influence of A-chain Structure on the Activity of Human α-Thrombin
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m312430200
ISSN1083-351X
AutoresRaimondo De Cristofaro, Sepideh Akhavan, Cosimo Altomare, Andrea Carotti, Flora Peyvandi, Pier Mannuccio Mannucci,
Tópico(s)Cancer-related gene regulation
ResumoWe have recently identified in two unrelated patients with bleeding tendency a homozygous mutation causing a deletion of one of the two contiguous Lys9/Lys10 residues in the A-chain of α-thrombin (ΔK9). We used in vitro expression analysis to clarify the role of the deletion of Lys9 or Lys10 in the thrombin function. The kcat/Km value of the hydrolysis by ΔK9 of the synthetic substrate Phe-Pip-Arg-p-nitroanilide (where Pip represents l-pipecolyl) and fibrinopeptide A was 18- and 60-fold lower, respectively, compared with wild type (WT). Interaction with antithrombin was also reduced in the mutant, the association rate being about 20-fold lower than in the WT thrombin. The sensitivity to sodium ion of ΔK9 was found significantly attenuated compared with the WT form. ΔK9 has a very weak platelet-activating capacity, attributed to a severely defective PAR1 interaction, whereas the binding to the platelet glycoprotein Ibα was unaffected. Likewise, the interaction with protein C was severely impaired, whereas interaction with thrombomodulin had a normal Kd value. At variance with these findings, both low affinity (basic pancreatic trypsin inhibitor) and high affinity (N-α-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) thrombin inhibitors displayed a better binding to ΔK9 than to the WT form, indicating a better accommodation of these inhibitors into the catalytic pocket of ΔK9. A molecular dynamics simulation of the ΔK9 thrombin in full explicit water solvent provided support to the role of the A-chain in affecting conformation and catalytic properties of the B-chain, especially in some insertion loops of the enzyme, such as the 60-loop, as well as in the geometry of the catalytic triad residues. We have recently identified in two unrelated patients with bleeding tendency a homozygous mutation causing a deletion of one of the two contiguous Lys9/Lys10 residues in the A-chain of α-thrombin (ΔK9). We used in vitro expression analysis to clarify the role of the deletion of Lys9 or Lys10 in the thrombin function. The kcat/Km value of the hydrolysis by ΔK9 of the synthetic substrate Phe-Pip-Arg-p-nitroanilide (where Pip represents l-pipecolyl) and fibrinopeptide A was 18- and 60-fold lower, respectively, compared with wild type (WT). Interaction with antithrombin was also reduced in the mutant, the association rate being about 20-fold lower than in the WT thrombin. The sensitivity to sodium ion of ΔK9 was found significantly attenuated compared with the WT form. ΔK9 has a very weak platelet-activating capacity, attributed to a severely defective PAR1 interaction, whereas the binding to the platelet glycoprotein Ibα was unaffected. Likewise, the interaction with protein C was severely impaired, whereas interaction with thrombomodulin had a normal Kd value. At variance with these findings, both low affinity (basic pancreatic trypsin inhibitor) and high affinity (N-α-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) thrombin inhibitors displayed a better binding to ΔK9 than to the WT form, indicating a better accommodation of these inhibitors into the catalytic pocket of ΔK9. A molecular dynamics simulation of the ΔK9 thrombin in full explicit water solvent provided support to the role of the A-chain in affecting conformation and catalytic properties of the B-chain, especially in some insertion loops of the enzyme, such as the 60-loop, as well as in the geometry of the catalytic triad residues. Recently, a homozygous deletion mutation of one of the two contiguous Lys9/Lys10 residues 1The thrombin amino acid residues are numbered by the chymotrypsin(ogen) numbering system. 1The thrombin amino acid residues are numbered by the chymotrypsin(ogen) numbering system. in the A-chain of α-thrombin was identified in two unrelated Iranian patients with severe prothrombin deficiency and hemorrhagic diathesis (1Akhavan S. Mannucci P.M. Lak M. Mancuso G. Mazzucconi M.G. Rocino A. Jenkins P.V. Perkins S.J. Thromb. Haemost. 2000; 84: 989-997Crossref PubMed Scopus (60) Google Scholar). The level of prothrombin antigen measured in plasma was 15%, whereas the coagulant activity ranged from less than 1% to about 2.5% (1Akhavan S. Mannucci P.M. Lak M. Mancuso G. Mazzucconi M.G. Rocino A. Jenkins P.V. Perkins S.J. Thromb. Haemost. 2000; 84: 989-997Crossref PubMed Scopus (60) Google Scholar). Prothrombin deficiency is an autosomal recessive bleeding disorder characterized by two phenotypes: hypoprothrombinemia, with concomitantly low levels of coagulant activity and antigen (type I), and dysprothrombinemia, with very low activity but subnormal or normal antigen levels (type II). These disorders are rare, and there is always residual prothrombin procoagulant activity measurable in patients, the phenotype found in prothrombin-deficient mice indicating that complete prothrombin deficiency may be lethal in humans (2Sun W.Y. Witte D.P. Degen J.L. Colbert M.C. Burkart M.C. Holmback K. Xiao Q. Bugge T.H. Degen S.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7597-7602Crossref PubMed Scopus (205) Google Scholar, 3Xue J. Wu Q. Westfield L.A. Tuley E.A. Lu D. Zhang Q. Shim K. Zheng X. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7603-7607Crossref PubMed Scopus (136) Google Scholar). To date, 38 defects in the prothrombin gene have been identified in patients with dysprothrombinemia or hypoprothrombinemia (1Akhavan S. Mannucci P.M. Lak M. Mancuso G. Mazzucconi M.G. Rocino A. Jenkins P.V. Perkins S.J. Thromb. Haemost. 2000; 84: 989-997Crossref PubMed Scopus (60) Google Scholar, 4Lefkowitz J.B. Haver T. Clarke S. Jacobson L. Weller A. Nuss R. Manco-Johnson M. Hathaway W.E. Br. J. Haematol. 2000; 108: 182-187Crossref PubMed Scopus (30) Google Scholar, 5Sun W.Y. Smirnow D. Jenkins M.L. Degen S.J. Thromb. Haemost. 2001; 85: 651-654Crossref PubMed Scopus (40) Google Scholar, 6Akhavan S. Luciani M. Lavoretano S. Mannucci P.M. Br. J. Haematol. 2003; 120: 142-144Crossref PubMed Scopus (12) Google Scholar, 7Ortiz I. Lefkowitz J. Weller A. Santiago-Borrero P. Haemophilia. 2002; 8 (abstr.): 836Crossref Google Scholar). Among these natural dysprothrombins, amino acid residues of the thrombin A-chain were found to be rarely involved (1Akhavan S. Mannucci P.M. Lak M. Mancuso G. Mazzucconi M.G. Rocino A. Jenkins P.V. Perkins S.J. Thromb. Haemost. 2000; 84: 989-997Crossref PubMed Scopus (60) Google Scholar, 4Lefkowitz J.B. Haver T. Clarke S. Jacobson L. Weller A. Nuss R. Manco-Johnson M. Hathaway W.E. Br. J. Haematol. 2000; 108: 182-187Crossref PubMed Scopus (30) Google Scholar, 5Sun W.Y. Smirnow D. Jenkins M.L. Degen S.J. Thromb. Haemost. 2001; 85: 651-654Crossref PubMed Scopus (40) Google Scholar, 6Akhavan S. Luciani M. Lavoretano S. Mannucci P.M. Br. J. Haematol. 2003; 120: 142-144Crossref PubMed Scopus (12) Google Scholar, 7Ortiz I. Lefkowitz J. Weller A. Santiago-Borrero P. Haemophilia. 2002; 8 (abstr.): 836Crossref Google Scholar). The A-chain in human thrombin is composed of 36 amino acid residues, which are connected to the catalytic B-chain by a single disulfide bond (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). This chain has no analog in the prototypic serine protease trypsin, although chymotrypsin and other trypsin-like blood coagulation enzymes, such as factor IX and factor XI, contain A-chain analogs (9Hartley B.S. Phil. Trans. R. Soc. London Ser. B. 1970; 257: 77-87Crossref PubMed Scopus (195) Google Scholar, 10Fujikawa K. Legaz M.E. Kato H. Davie E.W. Biochemistry. 1974; 13: 4508-4516Crossref PubMed Scopus (128) Google Scholar). Some structural analogies between the A-chains present in different serine proteases, as in the case of thrombin and chymotrypsin, were identified, although the function of the A-chain has not yet been well established. The thrombin A-chain is organized mainly in a multiple-turn and partly helical conformation and a boomerang-like shape, making a smooth contour of the B-chain part, opposite to the active site pocket (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). The A-chain is topologically similar to the activation peptide of chymotrypsin(ogen) and connected in a similar manner through the Cys1-Cys122 disulfide bridge to the B-chain. Most of the A-B chain interactions involve charged side chains; six are buried salt bridges, and 10 are involved in interchain hydrogen bonds (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). Stabilization within the A-chain also occurs, mostly via polar and salt bridge interactions. The N-terminal segment of the A-chain up to E1c is characterized by relatively weak electron density in the d-Phe-Pro-Arg-methyl ketone-thrombin crystal (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar), and seems to have a high degree of conformational flexibility (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). The C-terminal segment up to Y14j is an amphiphilic α-helix forming one and one-half turns and showing a high degree of flexibility as well (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). The central region is the most rigid segment of the A-chain and contains strong salt bridges, such as that involving the Asp1a-Lys9 side chains (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar), which is characterized by a high electrostatic energy (-1.8 kcal/mol), significantly contributing to confer the boomerang-like shape to the chain (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar). Not all of the coagulation serine proteases bear an A-chain, hence its general role has not been precisely established. In a previous study, carried out on bovine thrombin, it has been proposed that the A-chain, although strongly linked to the B-chain through strong electrostatic and apolar bonds, does not play a major role in the specificity of the enzyme catalytic activity (11Hageman T.C. Endres G.F. Scheraga H.A. Arch. Biochem. Biophys. 1975; 171: 327-336Crossref PubMed Scopus (36) Google Scholar). In contrast, subsequent studies carried out on human thrombin showed that isolated B-chain has a markedly reduced proteolytic and amidase activity compared with the native enzyme (12Sereiskaia A.A. Osadchuk T.V. Korneliuk A.I. Pekhnik I.V. Serebrianyi S.B. Biokhimiia. 1989; 54: 542-548PubMed Google Scholar). With the aim of gaining insights into the role of the A-chain in human thrombin, we used in vitro expression analysis of the natural mutant bearing a deletion of Lys9 (ΔK9). The wild-type (WT) 2The abbreviations used are: WT, wild type; AT, antithrombin; BPTI, basic pancreatic trypsin inhibitor; α-NAPAP, N-α-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide; MD, molecular dynamics; HPLC, high pressure liquid chromatography; pNA, p-nitroanilide; Pip, l-pipecolyl. 2The abbreviations used are: WT, wild type; AT, antithrombin; BPTI, basic pancreatic trypsin inhibitor; α-NAPAP, N-α-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide; MD, molecular dynamics; HPLC, high pressure liquid chromatography; pNA, p-nitroanilide; Pip, l-pipecolyl. prothrombin and mutant cDNAs were stably transfected in Chinese hamster ovary cell lines expressing high levels of prothrombin for experiments probing their functional properties. In order to understand their different functional properties, we performed a molecular dynamics (MD) simulation of the ΔK9 thrombin mutant in full explicit water solvent. Our simulation, in conjunction with a comparative analysis of available crystal structures of free or ligand-bound WT thrombins, provided insightful support to the role of the A-chain in affecting conformation and catalytic properties of the B-chain. Patients—We investigated the molecular defects in two Iranian patients with prothrombin deficiency and a severe hemorrhagic diathesis. Clinical and laboratory findings pertaining to these patients were previously reported in detail (1Akhavan S. Mannucci P.M. Lak M. Mancuso G. Mazzucconi M.G. Rocino A. Jenkins P.V. Perkins S.J. Thromb. Haemost. 2000; 84: 989-997Crossref PubMed Scopus (60) Google Scholar). DNA Analysis—Following DNA extraction from leukocytes, the coding region, intron/exon boundaries, and 5′- and 3′-untranslated regions of the prothrombin gene were amplified by PCR screened for mutations by single strand conformation polymorphism analysis and sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Kit (ABI 373; PerkinElmer Life Sciences). The primers used in the PCR and sequencing are identical to those used in a previous study (1Akhavan S. Mannucci P.M. Lak M. Mancuso G. Mazzucconi M.G. Rocino A. Jenkins P.V. Perkins S.J. Thromb. Haemost. 2000; 84: 989-997Crossref PubMed Scopus (60) Google Scholar). Site-directed Mutagenesis and Construction of Expression Vectors—Full-length cDNA of human prothrombin (including 38 bp of 5′-untranslated region and 97 bp of 3′-untranslated region) was obtained by PCR amplification of M13mp18 (kindly provided by Dr. Barbara C. Furie). The construction of plasmid PT7SalI FII-WTEcoRI was previously described (13Akhavan S. De Cristofaro R. Peyvandi F. Lavoretano S. Landolfi R. Mannucci P.M. Blood. 2002; 100: 1347-1353Crossref PubMed Scopus (28) Google Scholar). To investigate the influence of the Lys9 deletion (ΔK9) on prothrombin activity, mutant FII-ΔK9 was obtained by site-directed mutagenesis of PT7-SalIFII-WTEcoRI using a commercially available kit (Clontech, Palo Alto, CA). Oligonucleotide (5′-CTGTTCGA---GAAGTCGCTGGAGGACAAAACCGAAAGAGAGCTTCTAGAATCCTACATC-3′) spanning nucleotides 1021-1080 of the human FII cDNA were used to introduce an in-frame deletion of 3 bp, nucleotides 1029-1031 (GAA deletion). This primer also introduced a XbaI restriction site (underlined), arising from two silent CCT to TCT and GGA to AGA mutations at nucleotides 1065 and 1068, respectively, to facilitate screening for clones carrying the mutation. The cloned insert was sequenced, and sequencing confirmed that the mutation had been introduced. To obtain stable cell lines expressing recombinant FII-WT and FII-ΔK9, we used dihydrofolate reductase-deficient Chinese ovary cells (CHO-DUKX-B11). These cells were grown in α-modified essential medium supplemented with 10% fetal bovine serum, 2 mmol/liter l-glutamine, 10 mmol/liter HEPES, pH 7.2, 100 units/ml penicillin G, 100 μg/ml streptomycin, and 8 μg/ml vitamin K1 (Phytonadione; Abbott), 10 μg/ml adenosine, 10 μg/ml deoxyadenosine, and 10 μg/ml thymidine in a 5% CO2 atmosphere at 37 °C. The DNA of pED-FII-WT or pED-FII-ΔK301 (30 ng) were transfected by electroporation into 5 × 106 CHO cells according to the manufacturer's instructions. Two days after transfection, cells were selected for dihydrofolate reductase expression using medium deficient in ribonucleosides and deoxyribonucleotides. A single clone stably transfected with each construct and expressing high levels of FII was selected for further experiments. Purification of Wild Type and ΔK9 Prothrombin—Activation of ΔK9 prothrombin was obtained using the Taipan snake venom, whereas ecarin slowly activated the mutant zymogen (data not shown). It is known that the Taipan snake prothrombin activator acts as a Factor Xa-Factor Va complex, since it directly activates prothrombin through cleavages of the Arg271-Thr272 peptide bond at the junction between A-chain and fragment 2 besides the additional cleavage of the Arg320-Ile321 peptide bond (14Speijer H. Govers-Riemslag J.W. Zwaal R.F. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Prothrombin activation was obtained by incubating for 2 h at 37 °C 1 μm ΔK9 prothrombin (or the WT form) with 0.2 mg/ml Taipan snake venom in 50 mm Tris-HCl, 150 mm NaCl, 2 mm CaCl2, synthetic phospholipid reagent containing a colloidal silica activator and used at a 1:5 dilution of the stock solution (SynthAsil APTT reagent, Instrumentation Laboratory, Milan, Italy), pH 8.00. The generated active ΔK9 thrombin was successfully purified by cation exchange HPLC, being eluted ∼5 min before WT thrombin. This finding is in agreement with the lack of one positive charge related to the missing lysine residue in the A-chain of the mutant thrombin. SDS-PAGE of pooled chromatographic peaks was carried out on 4-20% gradient gels under both reducing (5% β-mercaptoethanol) and nonreducing conditions in a Bio-Rad mini-PROTEAN II apparatus. The pooled peak contained a single band of roughly 36 kDa, whose identity was further checked by N-terminal sequencing. To obtain purified A-chain from both WT and ΔK9 thrombin, the peak obtained in the ion exchange chromatography was reduced by 5% β-mercaptoethanol and analyzed through RP-HPLC using a C4 resin (High Pore RP-304 250 × 4.6 mm; Bio-Rad). The applied gradient was 10-40% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1.0 ml/min. The elution was followed at 224 nm. The peak relative to the A-chain was eluted after 17.8 min for the WT species, whereas it appeared after 17 min in the case of ΔK9 thrombin. These peaks were pooled, dried, and solubilized in acetonitrile/water/trifluoroacetic acid (49:49:2 by volume) before sequencing was performed. An automatic liquid phase sequencer (ABI-PerkinElmer model 477A) connected to an HPLC apparatus (ABI-PerkinElmer model 120A, PTH-analyzer), and a Bio-Rad reverse-phase C18 column were used to determine the N-terminal sequencing. Identification of the cleavage products was based on the amino acid sequences of the human prothrombin (15Degen S.J. Davie E.W. Biochemistry. 1987; 26: 6165-6177Crossref PubMed Scopus (223) Google Scholar). The concentration of recombinant thrombins was measured spectrophotometrically at 280 nm, using an extinction coefficient (0.1%) equal to 1.83. Active site titration was carried out spectrophotometrically, as previously reported (16De Cristofaro R. Rocca B. Bizzi B. Landolfi R. Biochem. J. 1993; 289: 475-480Crossref PubMed Scopus (32) Google Scholar). The purified enzyme was immediately aliquoted and frozen at -80 °C until use. Effect of Na+ and Viscosity on the Michaelis-Menten Parameters Pertaining to Hydrolysis of the Synthetic Substrates d-Phe-Pip-Arg-pNA—Michaelis-Menten parameters, kcat and Km, were calculated as previously detailed (16De Cristofaro R. Rocca B. Bizzi B. Landolfi R. Biochem. J. 1993; 289: 475-480Crossref PubMed Scopus (32) Google Scholar), in 10 mm Tris-HCl, 0.15 m NaCl, 0.1% polyethylene glycol 6000, pH 8.00, at 25 °C (buffer A). The enzyme concentration was typically 0.5 nm for WT and 5-10 nm for the ΔK9 thrombin form. In the experiments without sodium, NaCl was substituted with a cation not interacting with thrombin, tetramethylammonium chloride, which was used at the same concentration to keep constant the ionic strength of the solution (17Stone S.R. Betz A. Hofsteenge J. Biochemistry. 1991; 30: 9841-9848Crossref PubMed Scopus (61) Google Scholar). The effect of the viscogenic agent sucrose, used over a 0-0.8 m concentration range in buffer A, was investigated according to a previously detailed kinetic and analytical scheme (16De Cristofaro R. Rocca B. Bizzi B. Landolfi R. Biochem. J. 1993; 289: 475-480Crossref PubMed Scopus (32) Google Scholar, 17Stone S.R. Betz A. Hofsteenge J. Biochemistry. 1991; 30: 9841-9848Crossref PubMed Scopus (61) Google Scholar, 18Wells C.M. Di Cera E. Biochemistry. 1992; 31: 11721-11730Crossref PubMed Scopus (225) Google Scholar, 19De Cristofaro R. Landolfi R. J. Mol. Biol. 1994; 239: 569-577Crossref PubMed Scopus (21) Google Scholar). Accordingly, the canonical scheme for serine proteases amidase activity was applied, E+S⇌k-1k+1ES→k2EP*→k3E+PScheme 1 where k+1, k-1, k2, and k3 are the kinetic rate constant of the substrate association and dissociation, acylation, and deacylation of thrombin, respectively, and EP* is the acyl-enzyme intermediate, and P is the final product. The equilibrium dissociation constant, Kd, can be calculated from the k-1/k+1 ratio. Experimental data sets were simultaneously fitted to the appropriate equations (16De Cristofaro R. Rocca B. Bizzi B. Landolfi R. Biochem. J. 1993; 289: 475-480Crossref PubMed Scopus (32) Google Scholar, 17Stone S.R. Betz A. Hofsteenge J. Biochemistry. 1991; 30: 9841-9848Crossref PubMed Scopus (61) Google Scholar), using the GRAFIT software (Erithacus Software Ltd., Staines, UK). Fibrinopeptide A Release—Fibrinopeptide A release by both WT and ΔK9 thrombin in buffer A was monitored by reverse transcriptase-HPLC, using a previously reported method (20Picozzi M. Landolfi R. De Cristofaro R. Eur. J. Biochem. 1994; 219: 1013-1021Crossref PubMed Scopus (14) Google Scholar). Purified plasmin- and fibronectin-free fibrinogen from American Diagnostica (Instrumentation Laboratory, Milan, Italy) was used as substrate. The enzyme concentration was 0.2 and 2 nm, for the WT and the mutated form, respectively. The Michaelis parameters were computed as indicated above for the synthetic substrate. Measurement of kcat/Km for Protein C Activation and Interaction with Thrombomodulin—Hydrolysis by WT and ΔK9 thrombin of zymogen protein C, purchased from American Diagnostica (Instrumentation Laboratory, Milan, Italy), was performed in buffer A, as previously reported in detail (21De Cristofaro R. Landolfi R. Eur. J. Biochem. 1999; 260: 97-102Crossref PubMed Scopus (14) Google Scholar). Hydrolysis of protein C (2 μm) was followed in the presence of 100 nm human thrombomodulin (American Diagnostica), since in the absence of the cofactor, the reaction was too slow to be correctly analyzed. Likewise, binding of the thrombin forms to immobilized human thrombomodulin (nonlipidated form, from American Diagnostica) was investigated according to a previously reported method (21De Cristofaro R. Landolfi R. Eur. J. Biochem. 1999; 260: 97-102Crossref PubMed Scopus (14) Google Scholar). Inhibition of WT and ΔK9 Thrombin Forms by Antithrombin, Bovine Pancreatic Trypsin Inhibitor (BPTI), and N-α-[2-Naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide (α-NAPAP)—Pseudo-first order kinetics formalism was used to investigate the interaction of WT and ΔK9 thrombin forms with antithrombin (AT) (Enzyme Research, Indianapolis, IN) in the absence and presence of heparin in buffer A. In the absence of heparin, AT was used in concentrations ranging between 10 and 15 μm in buffer A at 25 °C. The reaction with thrombin was started by adding 0.1 nm WT or 5-10 nm ΔK9, and the release of p-nitroaniline was measured as a function of time at 405 nm. The measurements were performed in duplicate. The association second order rate constant, kon, for thrombin-AT interaction was calculated as reported (22De Cristofaro R. De Candia E. Rutella S. Weitz J.I. J. Biol. Chem. 2000; 275: 3887-3895Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Progress curve kinetics was also used to derive the kon value of AT interaction with WT and ΔK9 mutant thrombin in the presence of high molecular weight heparin from porcine intestinal mucosa (Sigma; sodium salt Grade I-A, 170 United States Pharmacopeia/mg, average Mr = 16,500), as described (22De Cristofaro R. De Candia E. Rutella S. Weitz J.I. J. Biol. Chem. 2000; 275: 3887-3895Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Heparin concentration was equal to 75 nm in functional experiments using 0.15 μm AT, 0.15 nm WT form, or 10 nm ΔK9 and 100 μm Phe-Pip-Arg-pNA as the substrate. WT and ΔK9 mutant thrombin interaction with BPTI (Sigma) was performed in buffer A, according to a previously described "matrix" method (21De Cristofaro R. Landolfi R. Eur. J. Biochem. 1999; 260: 97-102Crossref PubMed Scopus (14) Google Scholar). Competitive inhibition of the synthetic substrate Phe-Pip-Arg-pNA hydrolysis was used to monitor the inhibitor interaction with both the WT and ΔK9 thrombins. BPTI effect on the substrate's hydrolysis was investigated using the inhibitor over a concentration range from 0 to 1 mm, whereas α-NAPAP (Sigma) was used between 0 and 100 nm concentration. All of the experimental points taken at different inhibitor concentrations (BPTI or α-NAPAP) as a function of the substrate concentration were simultaneously fitted to a competitive inhibition of the Michaelis equation, as previously reported (19De Cristofaro R. Landolfi R. J. Mol. Biol. 1994; 239: 569-577Crossref PubMed Scopus (21) Google Scholar), using the GRAFIT software (Erithacus Software). This method allowed us to calculate the equilibrium dissociation constant of the inhibitor's binding to thrombin. Measurement of the PAR1 Peptide Hydrolysis—Hydrolysis of the PAR1 peptide (PAR1P, NH2-LDPRSFLLRNPNDKYEPFWEDEE-COOH, synthesized by Primm s.r.l., Milan, Italy) by the different thrombin forms was followed by measuring the release of the peptide LDPR, resulting from the cleavage of the NH2 terminus of PAR1, according to a previously described method (22De Cristofaro R. De Candia E. Rutella S. Weitz J.I. J. Biol. Chem. 2000; 275: 3887-3895Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, 0.5 μm PAR1P peptide was incubated with 50-100 pm WT or 10 nm ΔK9 thrombins in 10 mm HEPES, 0.15 m NaCl, 0.1% polyethylene glycol 6000, pH 7.5, at 25 °C. At time intervals (1, 2, 3, 4, 8, 12, and 15 min), the reaction was stopped with 0.3 m HClO4, and the cleaved peptide was measured by reversed-phase HPLC, using a 250 × 4.6-mm RP-304 column (Bio-Rad), as previously reported (22De Cristofaro R. De Candia E. Rutella S. Weitz J.I. J. Biol. Chem. 2000; 275: 3887-3895Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Interactions with Platelets—We compared the effects of WT and ΔK9 thrombins on the enzyme capacity to activate gel-filtered platelets, which were prepared in 10 mm Hepes, 0.15 m NaCl, 5.5 mm glucose, 0.2% bovine serum albumin, pH 7.50, at 25 °C, according to a previously described method (22De Cristofaro R. De Candia E. Rutella S. Weitz J.I. J. Biol. Chem. 2000; 275: 3887-3895Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In these experiments, thrombin concentrations ranged from 0.5 to 128 nm. Aggregation of gel-filtered platelets (3 × 105/μl) was studied in a PACKS 4 aggregometer (Helena Laboratories, Milan, Italy), using a final volume of 500 μl. Aggregation response was evaluated by taking into account the maximum velocity of transmittance increase per minute (expressed as a percentage of transmittance, set by using plain buffer). Molecular Dynamics Calculations—The x-ray structure of human thrombin covalently bound to d-Phe-Pro-Arg-methyl ketone, determined at 1.9-Å resolution (Protein Data Bank entry 1ppb) (8Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar), was used as the starting structure. The covalently bound inhibitor was removed, and the structure of the free enzyme was used as a template for generating the ΔK9 mutant protein (Swiss-Prot; available on the World Wide Web at www.expasy.ch). The protein structure was solvated with water in a periodic truncated octahedron, to produce a 12-Å water shell. All solvent molecules within 1.5 Å of any protein atom were then removed. The GROMACS 3.1.4-1 software package, running on a Linux PC cluster, was used for the MD simulations and analysis of the trajectories. The total charge of the ΔK9 protein was +2, and no counterions was added. The entire system consisted of 3060 protein atoms and 12,759 water molecules. The initial structure ΔK9 mutant thrombin was energy-minimized by using 200 steps of steepest descent with the protein constrained and then 1000 steps of conjugate gradient, with the entire system free, in order to remove bad contacts. During the simulations, the system was coupled to an external temperature bath with a coupling constants of 0.1 ps (23Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Di Nola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23441) Google Scholar). The Gromos-96 force field (24van Gunsteren W.F. Daura X. Mark A.E. Encycl. Comput. Chem. 1998; 2: 1211-1216Google Scholar) was used for minimization procedures, with a time step of 2 fs. Water molecules were modeled according to the "simple point charge" model (25Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Hermans J. Pullman B. Intermolecular Forces. Reidel, Dordrecht, The Netherlands1981: 331-342Google Scholar). The LINCS algorithm (26Hess B. Bekker H. Fraaije J. Berendsen H.J.C. J. Comput. Chem. 1997; 18: 1463-1472Crossref Scopus (11564) Google Scholar) was used to constrain all bond lengths. The nonbonded interactions were cut off at 8 Å (short range cut-off radius) and 12 Å (long range cut-off radius) for both Coulombic and Lennard-Jones interactions. The short range nonbonded interactions were updated every time step, whereas the interactions within the long range radius were updated every five time steps. Initial velocities were assigned according to a Maxwellian distribution at the desired temperature. The density of the system was adjusted by performing the first equilibration runs under number pressure temperature conditions by weak coupling to a bath of constant pressure (p = 1 bar, coupling time of 0.5 ps). The simulation, starting from the initial structure, was equilibrated by 50 ps of MD run with position restraints on the protein to allow the solvent molecules to be relaxed, followed by another 50-ps run without restraints. Production dynamics were performed under number volume temperature conditions for 18 ns, and the coordinates saved every 10 ps. To investigate the influence of the Lys9 deletion on prothrombin biosynthes
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