A Control Switch for Prothrombinase
2006; Elsevier BV; Volume: 281; Issue: 51 Linguagem: Inglês
10.1074/jbc.m604482200
ISSN1083-351X
AutoresMichael A. Bukys, Paul Y. Kim, Michael E. Nesheim, Michael Kalafatis,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoMembrane-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 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 Arg320. 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 Arg320 and Arg271, 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 Arg271 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. 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 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 Arg320. 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 Arg320 and Arg271, 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 Arg271 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. Prothrombinase is the enzymatic complex responsible for timely thrombin formation in response to vascular injury (1Kalafatis M. Egan J.O. van't Veer C. Cawthern K.M. Mann K.G. Crit. Rev. Eukaryotic Gene Expression. 1997; 7: 241-280Crossref PubMed Scopus (114) Google Scholar, 2Mann K.G. Kalafatis M. Blood. 2003; 101: 20-30Crossref PubMed Scopus (177) Google Scholar). Activation of human prothrombin is the consequence of two cleavages at Arg271 and Arg320 in prothrombin by factor Xa. Depending on the order of peptide bond cleavage, different intermediates are formed (Fig. 1). Cleavage first at Arg271 produces fragment 1·2 and prethrombin-2, whereas initial cleavage at Arg320 results in the formation of meizothrombin, which has enzymatic activity (3Mann K.G. Bajaj S.P. Heldebrant C.M. Butkowski R.J. Fass D.N. Semin. Hematol. 1973; 6: 479-493Google Scholar, 4Heldebrant C.M. Butkowski R.J. Bajaj S.P. Mann K.G. J. Biol. 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The prothrombinase complex, which is formed following the interaction of factor Va with membrane-bound factor Xa in the presence of divalent metal ions, catalyzes the activation of prothrombin following the opposite pathway (Arg320 followed by Arg271; see Fig. 1, pathway II, meizo pathway), resulting in a substantial increase in the catalytic efficiency of factor Xa required for normal hemostasis (15Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). Although both cleavages are phospholipid-dependent, only initial cleavage of prothrombin at Arg320 is strictly dependent on factor Va. The increase in the rate of the overall enzymatic reaction is attributed to an increase in the kcat, which in turn is solely credited to the interaction of the cofactor molecule with both the membrane-bound enzyme and the membrane-bound substrate (11Rosing J. Tans G. Govers-Riemslang J.W. Zwaal R.F. Hemker H.C. J. Biol. 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J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We have recently identified a pentapeptide from the COOH terminus of the heavy chain of the cofactor (spanning amino acid residues 695–699, DYDYQ) as potent inhibitor of prothrombinase function (40Beck D.O. Bukys M.A. Singh L.S. Szabo K.A. Kalafatis M. J. Biol. Chem. 2004; 279: 3084-3095Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 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 demonstrated previously for sulfated hirugen (28Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. 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Drug Metab. Dispos. 2004; 32: 572-580Crossref PubMed Scopus (21) Google Scholar). The present work was undertaken to elucidate the molecular mechanism underlying the inhibition of prothrombin activation by DYDYQ. Materials, Reagents, and Proteins—l-α-Phosphatidylserine (PS) 2The abbreviations used are: PS, l-α-phosphatidylserine; PC, l-α-phosphatidylcholine; HPLC, high performance liquid chromatography; rMZ-II, (prothrombin with the substitutions R155A, R284A, and R271A); rP2-II, (prothrombin with the substitution R155A, R284A, and R320A); ABE-I, anion binding exosite I; ABE-II, anion binding exosite II; DYDYQ, pentapeptide mimicking factor Va heavy chain sequence: Asp695–Tyr696–Asp697–Tyr698–Gln699; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amine. and l-α-phosphatidylcholine (PC) were from Avanti Polar Lipids (Alabaster, AL). The chromogenic substrate Spectrozyme-TH was from American Diagnostica, Inc. (Greenwich, CT). Human α-thrombin and human prothrombin were from Hematologic Technologies, Inc. (Essex Junction, VT). The monoclonal antibody αhFV1 coupled to Sepharose was provided by Dr. Kenneth G. Mann (Department of Biochemistry, University of Vermont, Burlington, VT). Human factor Xa was from Enzyme Research Laboratories (South Bend, IN). The pentapeptide DYDYQ that was previously shown to inhibit prothrombinase activity and delay factor V activation (40Beck D.O. Bukys M.A. Singh L.S. Szabo K.A. Kalafatis M. J. Biol. Chem. 2004; 279: 3084-3095Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) was custom-synthesized under the form H2N-DYDYQ-amide and purchased from New England Peptide, Inc. (Gardner, MA) and from American Peptide Co. (Sunnyvale, CA). In each case, the peptide was purified by HPLC to more than 96% homogeneity. Its molecular weight and composition were usually verified by mass spectrometry and amino acid composition analysis, respectively. DYDYQ is a highly negatively charged peptide, and all mass spectrometry analysis was conducted in a negative mode. The peptide was stored lyophilized at –20 °C in a dessicator. It is noteworthy that significant solubility problems were encountered when using the peptide. DYDYQ was insoluble at concentrations higher than 5 mm in water or in the assay buffer. DYDYQ at high concentrations slowly precipitated when incubated in an ice bucket, resulting in a gelatinous insoluble mass. Thus, for all experiments DYDYQ was made fresh in Milli Q (Millipore Corp., Bedford, MA) water at concentrations ranging between 2 and 4 mg/ml and kept at room temperature. DYDYQ was also frozen in small aliquots. All frozen peptide aliquots were thawed and used only once. Peptide solutions were always centrifuged prior to use to remove potential microaggregates and insoluble material. After the peptide was dissolved in water or buffer, the molar concentration of DYDYQ was initially calculated to a theoretical concentration assuming the degree of purity specified by the supplier and 100% peptide content. However, because DYDYQ is a highly negatively charged pentapeptide, theoretically it could adsorb water and counter ions during HPLC purification. Invariably, significant amounts of salt were present in the initial lyophilized, purified peptide preparations obtained from the manufacturers and used in this study. Thus, the exact concentration of each peptide solution used and provided throughout this study was determined following amino acid composition analysis of an aliquot in the laboratory of Dr. Alex Kurosky and Steve Smith (University of Texas Medical Branch, Galveston). Briefly, peptide samples were centrifuged for 5 min at 16,000 rpm to remove any precipitation that might be present in the tube. 10 μl of each supernatant was placed in a Wheaton 1-ml pre-scored vacule and dried (a vacule is a small thin-walled glass vial with a long neck that can be sealed with a torch while a vacuum is being applied). 100 μl of 6 n constant boiling HCl was added to each vacule. The vacules were sealed with a torch under vacuum and hydrolyzed for 20 h at 107 °C. After hydrolysis the vacules were dried, and the contents were resuspended in 40 μl of 0.02 n HCl. 10 μl of each sample was subsequently injected into a Hitachi L-8800 amino acid analyzer. Average concentration (micromoles/ml) was determined by data analysis using the Hitachi L-8800 AAA System Manager. The mole/ml values obtained directly from the analyzer for each amino acid were multiplied by the molecular weight of the peptide (Mr 702) to determine the milligram/ml of peptide. Because of the harsh conditions of hydrolysis, the mole values found for Tyr were not taken into account for the determination of the final peptide concentrations. It is noteworthy that for each analysis, the average mole numbers of Asp divided by 2 was usually very close to the total mole number obtained with Gln, confirming the accuracy of the method. Usually the mole values obtained with Asp and Gln were averaged to obtain the exact concentration of peptide. This method determines the concentration of a peptide solution with an accuracy of 96 ± 6%. Under these conditions, the calculated starting concentration of peptide in each experiment was systematically found to be lower than its initial concentration calculated after the peptide was dissolved in water or buffer. It is important to note that in order to verify peptide bond integrity of DYDYQ during storage, several peptide solutions used in this study were randomly analyzed by NH2-terminal sequence analysis in the same laboratory. Again, significant problems were encountered when attempting to sequence DYDYQ using the traditional method (polyvinylidene difluoride sample support). Under these conditions, the yield of amino acid per cycle was low as most of the peptide was washed off the polyvinylidene difluoride sample support. NH2-terminal sequencing of DYDYQ was thus performed on a Biobrene Plus™-treated glass fiber filter sample support (Applied Biosystems, Foster City, CA.). Biobrene Plus is a cationic polymer used to immobilize peptide and protein samples on the glass fiber filter during Edman degradation. A much higher yield of amino acids per cycle was observed using these conditions. NH2-terminal sequencing demonstrated that the majority of peptide remained intact following extensive incubation periods. Recombinant prothrombin rMZ-II and rP2-II that have only one cleavage site for factor Xa were prepared and purified as described (47Côté H.C.F. Stevens W.K. Bajzar L. Banfield D.K. Nesheim M.E. MacGillivray T.A. J. Biol. Chem. 1994; 269: 11374-11380Abstract Full Text PDF PubMed Google Scholar, 48Côté H.C.F. Bajzar L. Stevens W.K. Samis J.A. Morser J. MacGillivray R.T.A. Nesheim M.E. J. Biol. Chem. 1997; 272: 6194-6200Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 49Stevens W.K. Côté H.C.F. MacGillivray R.T.A. Nesheim M.E. J. Biol. Chem. 1996; 271: 8062-8067Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Human factor V was purified using methodologies previously described employing the monoclonal antibody αhFV1 coupled to Sepharose (50Nesheim M.E. Katzmann J.A. Tracy P.B. Mann K.G. Methods Enzymol. 1980; 80: 243-275Google Scholar) and activated to factor Va with thrombin as described recently (51Bukys M.A. Blum M.A. Kim P.Y. Bruffato N. Nesheim M.E. Kalafatis M. J. Biol. Chem. 2005; 280: 27393-27401Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Phospholipid vesicles composed of 75% PC and 25% PS (referred to as PCPS vesicles throughout the study) were prepared as described previously (52Barenholz Y. Gibbs D. Litmann B.J. Goll J. Thompson T. Carlson D. Biochemistry. 1977; 16: 2806-2910Crossref PubMed Scopus (730) Google Scholar). Assay Measuring Thrombin Formation—Initial experiments performed to identify the best conditions allowing peptide inhibition of prothrombin activation revealed that preincubation of prothrombin with DYDYQ in a small volume is required to observe maximum inhibition of prothrombinase activity by the peptide. Thus, the ability of the peptide to inhibit prothrombin activation by either prothrombinase or factor Xa alone was conducted exactly as follows. In a typical experiment, a constant concentration of prothrombin was preincubated with increasing concentrations of peptide at room temperature in a 15-μl volume. Following a 10-min incubation period, the prothrombin/peptide sample was centrifuged, and the entire mixture was added to different tubes containing a solution composed of PCPS vesicles (1 or 10 μm), DAPA (50 μm), and factor Va (1–5 nm) in the presence of 5 mm Ca2+ in 20 mm HEPES, 0.15 m NaCl, pH 7.4. The reaction was started by the addition of factor Xa (0.5 nm) at room temperature. The final concentration of prothrombin was 1.4 μm or 300 nm, whereas the final concentration of peptide is provided in the text. All final concentrations of reagents provided in the text and relating to the measure of activity of prothrombinase in the presence or absence of peptide were calculated assuming a final reaction volume of 225 μl. At selected time intervals, aliquots of the mixture were diluted 2-fold in a buffer containing EDTA (50 mm) to quench the reaction. The assay verifying thrombin formation was conducted as described by measuring the initial rate of thrombin formation by the change in the absorbance of a chromogenic substrate at 405 nm (Spectrozyme-TH) monitored with a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA) (51Bukys M.A. Blum M.A. Kim P.Y. Bruffato N. Nesheim M.E. Kalafatis M. J. Biol. Chem. 2005; 280: 27393-27401Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Percent inhibition of thrombin generation was calculated by comparing the initial rates of thrombin formation obtained in the presence of peptide with the control reaction in the absence of peptide. Inhibition of Prothrombin Cleavage by DYDYQ and Analysis of Prothrombin Activation by Gel Electrophoresis—Initial preliminary experiments carried out to determine the optimum conditions necessary to observe the effect of DYDYQ on prothrombin activation by prothrombinase by gel electrophoresis established that the best results (maximum inhibition) are obtained when the peptide is preincubated with prothrombin in a small volume. Thus, all samples analyzed by gel electrophoresis examining the effect of DYDYQ on prothrombin activation were prepared precisely as follows. In a typical experiment, plasma-derived prothrombin or recombinant mutant prothrombin at a constant concentration were preincubated with varying (increasing) concentrations of DYDYQ for 10 min in a 50-μl volume. Following centrifugation, the entire supernatant containing the prothrombin/peptide mixture was added to separated solutions containing PCPS vesicles (1 or 10 μm), DAPA (50 μm), and factor Va (1–5 nm) in the presence of 5 mm Ca2+ in 20 mm Tris, 0.15 m NaCl, pH 7.4. The reaction was started by the addition of factor Xa (0.5 nm) at room temperature. The final volume of each reaction mixture was 1,200 μl when the prothrombin concentration was 1.4 μm. The final concentration of DYDYQ reported throughout the study in experiments studying prothrombin activation by gel electrophoresis was always calculated using the final volume of the reaction mixture. The effect of the peptide on prothrombin activation by factor Xa alone (in the absence of factor Va) was studied in a similar manner (preincubation of prothrombin and peptide in a 50-μl volume for 10 min). The reaction was started by the addition of factor Xa. The final concentrations of reagents in this case were as follows: 2.5–5 nm enzyme, 1.4 μm prothrombin, and 50 μm PCPS in a final volume of 1,200 μl. Control experiments studying activation of prothrombin by factor Xa alone in the absence of factor Va and phospholipid were also performed in a similar fashion. At selected time intervals (indicated in the figure legends), aliquots from the reaction mixtures (60 μl) were removed and immediately diluted into 2 volumes of 0.2 m glacial acetic acid and concentrated using a Centrivap concentrator attached to a Centrivap cold trap (Labconco Corp., Kansas City, MO). The dried samples were dissolved in 0.1 m Tris base, pH 6.8, 1% SDS (final concentration), 1% β-mercaptoethanol (final concentration), heated for exactly 65 s at 90 °C, mixed again, and subjected to SDS-PAGE using 9.5% gels according to the method of Laemmli (53Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Usually 6 μg of total protein per lane were applied. Protein bands were visualized following staining by Coomassie Brilliant Blue R-250 and destained in a methanol/acetic acid/water solution. After each experiment using a new peptide solution, an aliquot of the solution was stored at –20 °C. Following amino acid composition analysis, the exact initial concentration of each peptide solution was calculated for every experiment. The final concentration of peptide used was then back-calculated and is reported throughout the study. Finally, it is worth mentioning that despite all the problems encountered when working with DYDYQ, using the precise experimental protocol provided above, highly reproducible results were observed with 18 separate peptide preparations from two different suppliers (New England Peptide, Inc. and American Peptide Co.) representing three different batches of peptide. Scanning Densitometry of SDS-PAGE and Calculation of the Rate of Prothrombin Consumption—Scanning densitometry of the gels was performed as described earlier (54Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar) and recently with several modifications (51Bukys M.A. Blum M.A. Kim P.Y. Bruffato N. Nesheim M.E. Kalafatis M. J. Biol. Chem. 2005; 280: 27393-27401Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The stained gels were scanned with a Lexmark printer/scanner; the final images were imported into Adobe Photoshop, captured as TIFF files, and subsequently imported into the software UN-SCAN-IT gel (Silk Scientific, Orem, UT). Following analysis, the numerical data were saved as a Microsoft Excel file, and the molar concentration of prothrombin as observed on the gels was calculated by normalizing its staining intensity to the initial prothrombin concentration. The data representing prothrombin consumption as a function of time (seconds) were subsequently plotted according to the equation representing a first-order exponential decay using the software Prizm (GraphPad, San Diego). The apparent first-order rate constant, k (s–1), obtained directly from the graph was subsequently divided by the molar concentration of factor Xa used in each experiment (to obtain s–1 × molar factor Xa–1). The number obtained was subsequently multiplied by the starting concentration of prothrombin used (1.4 μm or 300 nm). The final numbers reported throu
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