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Thromb Haemost 1993; 69(05): 430-435
DOI: 10.1055/s-0038-1651628
Original Article
Coagulation
Schattauer GmbH Stuttgart

An International Collaborative Study to Investigate Standardisation of Hirudin Potency

Colin Longstaff
The National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK
,
Man-Yu Wong
The National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK
,
Patrick J Gaffney
The National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK
› Author Affiliations
Further Information

Publication History

Received 29 October 1992

Accepted after revision 14 January 1993

Publication Date:
25 July 2018 (online)

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Summary

An international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.

 

  • References

  • 1 Markwardt F. Hirudin and derivatives as anticoagulant agents. Thromb Haemostas 1991; 66: 141-152
    PubMedSearch in Google Scholar
  • 2 Seemüller U, Dodt J, Fink E, Fritz H. Proteinase inhibitors of the leech Hirudo medicinalis (hirudins, bdellins, eglins). In: Proteinase Inhibitors. Barrett AJ, Salvesen G. (eds). Amsterdam; Elsevier: 1986: 337-359
    Search in Google Scholar
  • 3 Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave J-P, Courtney M, Tolstoshev P, Lecocq J-P. Cloning and expression of a cDNA coding for the anticoagulant hirudin from the blocksucking leech, Hirudo medicinalis . Proc Natl Acad Sci USA 1986; 83: 1084-1088
    CrossrefPubMedSearch in Google Scholar
  • 4 Märki WE, Wallis RB. The anticoagulant and antithrombotic properties of hirudins. Thromb Haemostas 1990; 64: 344-348
    PubMedSearch in Google Scholar
  • 5 Stone SR, Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 1986; 25: 4622-4628
    CrossrefPubMedSearch in Google Scholar
  • 6 Stone SR, Hofsteenge J, Dennis S. Quantitative evaluation of the contribution of ionic interactions to the formation of the thrombin-hirudin complex. Biochemistry 1989; 28: 6857-6863
    CrossrefPubMedSearch in Google Scholar
  • 7 Folkers PJM, Clore GM, Driscoll PC, Doat J, Kohler S, Gronenborn AM. Solution structure of recombinant hirudin and the Lys 47 → Glu mutant: a nuclearmagnetic resonance and hybrid geometry-dynamical simulated annealing study. Biochemistry 1989; 28: 2601-2617
    CrossrefPubMedSearch in Google Scholar
  • 8 Haruyama H, Wuthrick K. Conformation of recombinant desulfate hirudin in aqueous solution determined by nuclear magnetic resonance. Biochemistry 1989; 28: 4301-4312
    CrossrefPubMedSearch in Google Scholar
  • 9 Grütter MG, Priestle JP, Rahuel J, Grossenbacher H, Bode U, Hofsteenge J, Stone SJ. Crystal structure of thrombin-hirudin complex: a novel model of serine protease inhibition. EMBO J 1990; 9: 2361-2365
    PubMedSearch in Google Scholar
  • 10 Rydel TJ, Ravichrandran KG, Tulisky A, Bode W, Huber R, Fenton JW, Roitsch C. The structure of a complex of recombinant hirudin and human-α-thrombin. Science 1990; 249: 277-280
    CrossrefPubMedSearch in Google Scholar
  • 11 Stone SR, Hofsteenge J. Basis for the reduced affinity of βT- and gammaT-thrombin for hirudin. Biochemistry 1991; 30: 3950-3955
    CrossrefPubMedSearch in Google Scholar
  • 12 Hofsteenge J, Stone SR, Donella-Deana A, Pinna LA. The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin. Eur J Biochem 1990; 188: 55-59
    CrossrefPubMedSearch in Google Scholar
  • 13 Gaffney PJ, Heath AB, Fenton JW. A collaborative study to establish an International standard for alpha-thrombin. Thromb Haemostas 1992; 67: 424-427
    PubMedSearch in Google Scholar
  • 14 Finney DJ. In: Statistical Methods in Biological Assay. 3.. London: Griffin and Co; 1978
  • 15 Kirkwood TBL. Geometric means and measures of dispersion. Biometrics 1979; 35: 908-909
    PubMedSearch in Google Scholar
  • 16 Griessbach U, Stürzebecher J, Markwardt F. Assay of hirudin in plasma using a chromogenic thrombin substrate. Thromb Res 1985; 37: 347-350
    CrossrefPubMedSearch in Google Scholar
  • 17 Markwardt F, Stürzebecher J, Walsmann P. The hirudin standard. Thromb Res 1990; 59: 395-400
    CrossrefPubMedSearch in Google Scholar
  • 18 Stürzebecher J. Methods for determination of hirudin. Semin Thromb Hemostas 1991; 17: 99-102
    Thieme ConnectPubMedSearch in Google Scholar
  • 19 Spinner S, Scheffauer F, Maschler R, Stoffler G. A hirudin catching ELISA for quntitating the anticoagulant in biological fluids. Thromb Res 1988; 51: 617-625
    CrossrefPubMedSearch in Google Scholar
  • 20 Walenga JM, Hoppensteadt D, Koza M, Pifarre R, Fareed J. Comparative studies on various assays for the laboratory evaluation of r-hirudin. Semin Thromb Hemostas 1991; 17: 103-112
    Thieme ConnectPubMedSearch in Google Scholar
  • 21 Jameson GW, Roberts DV, Adams RW, Kyle WSA, Elmore DT. Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration. Biochem J 1973; 131: 107-117
    CrossrefPubMedSearch in Google Scholar
  • 22 Degryse E. Determination of the specific activity of recombinant hirudin. Thromb Res 1990; 60: 433-443
    CrossrefPubMedSearch in Google Scholar
  • 23 Degryse E, Ebel C. Influence of storage conditions on the activity of recombinant hirudin. Thromb Res 1991; 61: 87-89
    PubMedSearch in Google Scholar
  • 24 Gray E, Watton J, Cesmeli S, Barrowcliffe TW, Thomas DP. Experimental studies on a recombinant hirudin CGP 39393. Thromb Haemostas 1991; 65: 355-359
    PubMedSearch in Google Scholar