Thromb Haemost 1999; 82(03): 1078-1087
DOI: 10.1055/s-0037-1614332
Letters to the Editor
Schattauer GmbH

Interspecies Loop Grafting in the Protease Domain of Human Protein C Yielding Enhanced Catalytic and Anticoagulant Activity

Lei Shen
1   From the Lund University, The Wallenberg Laboratory, Dept. of Clinical Chemistry, University Hospital, Malmö, Malmö, Sweden
,
Bruno O. Villoutreix
1   From the Lund University, The Wallenberg Laboratory, Dept. of Clinical Chemistry, University Hospital, Malmö, Malmö, Sweden
,
Bjorn Dahlbäck
1   From the Lund University, The Wallenberg Laboratory, Dept. of Clinical Chemistry, University Hospital, Malmö, Malmö, Sweden
› Institutsangaben
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Publikationsverlauf

Received 16. Februar 1999

Accepted after revision . Mai 1999

Publikationsdatum:
09. Dezember 2017 (online)

Summary

Human anticoagulant activated protein C (hAPC) is less potent than the bovine APC (bAPC) molecule and our aims were to elucidate the molecular background for this difference and to create an APC with enhanced anticoagulant activity. In the protease domain of human protein C (hPC), the loop 148 (GWGYHSSREKEAKRN) is four residues longer than the corresponding loop in bovine APC (GWGY RDETKRN). To investigate whether this caused the species difference, the loop in hPC was replaced by the shorter bovine loop, whereas the longer human loop was introduced in bovine protein C. The mutation in hAPC yielded enhanced catalytic activity against chromogenic (4-fold) as well as natural (factors Va and VIIIa) substrates and 2-3-fold increased anticoagulant activity. The opposite effects were obtained with the bovine mutant. As compared to wild-type hAPC, the mutant hAPC was inhibited slightly faster by the protein C inhibitor, whereas the inhibition by α 1-antitrypsin was unaffected by the mutation. A computer model of bAPC was developed in order to analyse further our data. Collectively, our results demonstrate enhanced catalytic efficiency to result from mutagenesis in the loop 148 and show that APC mutant with increased anticoagulant activity can be created.

Abbreviations: PC, protein C; APC, Activated protein C. SDS-PAGE, polyacrylamide gel electrophoresis run in the presence of SDS, The chymotrypsinogen nomenclature for APC and thrombin is used throughout the text while the PC numbering is indicated between brackets whenever appropriate. P2, P1 and P1’, P2’.. designate inhibitor residues amino- and carboxy-terminal to the scissile peptide bond, respectively, and S2, S1 and S1’, S2’.. the corresponding subsites of the protease. APTT, activated partial thromboplastin time; rmsd, root mean square deviation. The coordinates of the bAPC model are freely available.

 
  • References

  • 1 Esmon CT, Ding W, Yasuhiro K, Gu JM, Ferrell G, Regan LM, Stearns-Kurosawa DJ, Kurosawa S, Mather T, Laszik Z, Esmon NL. The protein C pathway: new insights. Thromb Haemost 1997; 78: 70-4.
  • 2 Sadler JE. Thrombomodulin structure and function. Thromb Haemost 1997; 77: 392-5.
  • 3 Dahlbäck B. The protein C anticoagulant system: inherited defects as basis for venous thrombosis. Thromb Res 1995; 78: 1-43.
  • 4 Rosing J, Tans G. Coagulation factor V: an old star shines again. Thromb Haemost 1997; 78: 427-33.
  • 5 Aiach M, Borgel D, Gaussem P, Emmerich J, Alhenc-Gelas M, Gandrille S. Protein C and protein S deficiencies. Semin Hematol 1997; 34: 205-16.
  • 6 Koeleman BP, Reitsma PH, Bertina RM. Familial thrombophilia: a complex genetic disorder. Semin Hematol 1997; 34: 256-64.
  • 7 Dahlbäck B. Resistance to activated protein C as risk factor for thrombosis: molecular mechanisms, laboratory investigation, and clinical management. Semin Hematol 1997; 34: 217-34.
  • 8 Smith OP, White B, Vaughan D, Rafferty M, Claffey L, Lyons B, Casey W. Use of protein-C concentrate, heparin, and haemodiafiltration in meningococcus-induced purpura fulminans. Lancet 1997; 350: 1590-3.
  • 9 Dreyfus M, Maghey JF, Bridey F, Schultz HP, Planche C, Dehan M, Tchernia G. Treatment of homozygous protein C deficiency and neonatal purpura fulminans with a purified protein C concentrate. N Engl J Med 1991; 325: 1565-8.
  • 10 Gruber A, Griffin JH, Harker L, Hanson SR. Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model. Blood 1989; 73: 639-42.
  • 11 Gresele P, Momi S, Berrettini M, Nenci GG, Schwarz HP, Semeraro N, Colucci M. Activated human protein C prevents thrombin-induced thromboembolism in mice. Evidence that activated protein C reduces intravascular fibrin accumulation through the inhibition of additional thrombin generation. J Clin Invest 1998; 101: 667-76.
  • 12 Okajima K, Koga S, Kaji M, Inoue M, Nakagaki T, Funatsu A, Okabe H, Takatsuki K, Aoki N. Effect of protein C and activated protein C on coagulation and fibrinolysis in normal human subjects. Thromb Haemost 1990; 63: 48-53.
  • 13 Taylor Jr FB, Chang A, Esmon CT, D’Angelo A, Vigano-D’Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79: 918-25.
  • 14 Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem 1994; 269: 31869-80.
  • 15 Fay PJ, Smudzin TM, Walker FJ. Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity. J Biol Chem 1991; 266: 20139-45.
  • 16 Rosing J, Hoekema L, Nicolaes GAF, Thomassen MCLGD, Hemker HC, Varadi K, Schwarz HP, Tans G. Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 27852-8.
  • 17 Regan LM, Lamphear BJ, Huggins CF, Walker FJ, Fay PJ. Factor IXa protects factor VIIIa from activated protein C. Factor IXa inhibits activated protein C-catalyzed cleavage of factor VIIIa at Arg562. J Biol Chem 1994; 269: 9445-52.
  • 18 Shen L, Dahlbäck B. Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa. J Biol Chem 1994; 269: 18735-8.
  • 19 Lu D, Kalafatis M, Mann KG, Long GL. Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V. Blood 1996; 87: 4708-17.
  • 20 Varadi K, Rosing J, Tans G, Pabinger I, Keil B, Schwarz HP. Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: influence of the factor VR506Q mutation. Thromb Haemost 1996; 76: 208-14.
  • 21 Shen L, He X, Dahlbäck B. Synergistic cofactor function of factor V and protein S to activated protein C in the inactivation of the factor VIIIa –factor IXa complex – species specific interactions of components of the protein C anticoagulant system. Thromb Haemost 1997; 78: 1030-6.
  • 22 Walker F J. Regulation of bovine activated protein C by protein S: the role of the cofactor protein in species specificity. Thromb Res 1981; 22: 321-7.
  • 23 He X, Shen L, Dahlbäck B. Expression and functional characterization of chimeras between human and bovine vitamin-K-dependent protein-S-defining modules important for the species specificity of the activated protein C cofactor activity. Eur J Biochem 1995; 227: 433-40.
  • 24 He X, Shen L, Villoutreix BO, Dahlbäck B. Amino acid residues in thrombin-sensitive region and first epidermal growth factor domain of vitamin K-dependent protein S determining specificity of the activated protein C cofactor function. J Biol Chem 1998; 273: 27357-66.
  • 25 España F, Gruber AG, Heeb MJ, Hanson SR, Harker LA, Griffin JH. In vivo and in vitro complexes of activated protein C with two inhibitors in baboons. Blood 1991; 77: 1754-60.
  • 26 Heeb M J, Griffin JH. Physiologic inhibition of human activated protein C by alpha 1-antitrypsin. J Biol Chem 1988; 263: 11613-6.
  • 27 Suzuki K, Deyashiki Y, Nishioka J, Toma K. Protein C inhibitor: structure and function. Thromb Haemost 1989; 61: 337-42.
  • 28 Hermans JM, Stone SR. Interaction of activated protein C with serpins. Biochem J 1993; 295: 239-45.
  • 29 Holly RD, Foster DC. Resistance to inhibition by alpha-1-anti-trypsin and species specificity of a chimeric human/bovine protein C. Biochemistry 1994; 33: 1876-80.
  • 30 Mather T, Oganessyan V, Hof P, Huber R, Foundling S, Esmon CT, Bode W. The 2.8 Å crystal structure of Gla-domainless activated protein C. EMBO J 1996; 15: 6822-31.
  • 31 Dang QD, Sabetta M, Di Cera E. Selective loss of fibrinogen clotting in a loop-less thrombin. J Biol Chem 1997; 272: 19649-51.
  • 32 Hopfner KP, Brandstetter H, Karcher A, Kopetzki E, Huber R, Engh RA, Bode W. Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants. EMBO J 1997; 16: 6626-35.
  • 33 Sun Z, Jiang Y, Ma Z, Wu H, Liu BF, Xue Y, Tang W, Chen Y, Li C, Zhu D, Gurewich V, Liu JN. Identification of a flexible loop region (297-313) of urokinase-type plasminogen activator, which helps determine its catalytic activity. J Biol Chem 1997; 272: 23818-23.
  • 34 Smith JW, Tachias K, Madison EL. Protein loop grafting to construct a variant of tissue-type plasminogen activator that binds platelet integrin alpha IIb beta 3. J Biol Chem 1995; 270: 30486-90.
  • 35 Laurell CB, Dahlqvist I, Persson U. The use of thiol-disulphide exchange chromotography for the automated isolation of alpha 1-antitrypsin and other plasma proteins with reactive thiol groups. J Chromatogr 1983; 278: 53-61.
  • 36 Laurell M, Carlson TH, Stenflo J. Monoclonal antibodies against the heparin-dependent protein C inhibitor suitable for inhibitor purification and assay of inhibitor complexes. Thromb Haemost 1988; 60: 334-9.
  • 37 Elisen MGLM, Maseland MHH, Church FC, Bouma BN, Meijers JCM. Role of the A+ helix in heparin binding to protein C inhibitor. Thromb Haemost 1996; 75: 760-6.
  • 38 Dahlbäck B, Hildebrand B, Malm J. Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies. J Biol Chem 1990; 265: 8127-35.
  • 39 Dahlbäck B, Hildebrand B. Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor V. Proc Natl Acad Sci USA 1994; 91: 1396-400.
  • 40 Pepper DS, Prowse C. Chromatography of human prothrombin complex on dextran sulphate agarose. Thromb Res 1977; 11: 687-92.
  • 41 Dahlbäck B, Stenflo J. Binding of bovine coagulation factor Xa to platelets. Biochemistry 1978; 17: 4938-45.
  • 42 Shen L, Shah AM, Dahlbäck B, Nelsestuen GL. Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: studies related to proline-10. Biochemistry 1997; 36: 16025-31.
  • 43 Mann KG, Williams EB, Krishnaswamy S, Church W, Giles A, Tracy RP. Active site-specific immunoassays. Blood 1990; 76: 755-6.
  • 44 Bernstein FC, Koetzle TF, Williams GJB, Meyer EFJr, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasmui M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol 1977; 112: 535-42.
  • 45 Ponder JW, Richards FM. Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes. J Mol Biol 1987; 193: 775-91.
  • 46 Villoutreix BO, Dahlbäck B. Molecular model for the C-type lectin domain of human thrombomodulin. J Mol Model 1998; 4: 310-22.
  • 47 Knobe KE, Berntsdotter A, Shen L, Morser J, Dahlbäck B, Villoutreix BO. Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site directed mutagenesis and computer modeling. Proteins 1999; 35: 218-34.
  • 48 Miletich JP, Broze Jr GJ. Beta protein C is not glycosylated at asparagine 329. The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites. J Biol Chem 1990; 265: 11397-404.
  • 49 Hedstrom L, Szilagyi L, Rutter W. Converting trypsin to chymotrypsin: the role of surface loops. Science 1992; 255: 1259-3.
  • 50 Priestle JP, Rahuel J, Rink H, Tones M, Grutter MG. Changes in interactions in complexes of hirudin derivatives and human alpha-thrombin due to different crystal forms. Protein Sci 1993; 2: 1630-42.
  • 51 van de Locht A, Bode W, Huber R, Le Bonniec BF, Stone SR, Esmon CT, Stubbs MT. The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin. EMBO J 1997; 16: 2977-84.
  • 52 Bartunik HD, Summers LJ, Bartsch HH. Crystal structure of bovine beta-trypsin at 1.5 Å resolution in a crystal form with low molecular packing density. Active site geometry, ion pairs and solvent structure. J. Mol. Biol 1989; 210: 813-28.
  • 53 Perona JJ, Craik CS. Structural basis of substrate specificity in the serine proteases. Protein Sci 1995; 4: 337-60.
  • 54 McDonald JF, Shah AM, Schwalbe RA, Kisiel W, Dahlbäck B, Nelsestuen GL. Comparison of naturally occurring vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site. Biochemistry 1997; 36: 5120-7.