Download PDF
Thromb Haemost 1990; 63(02): 282-285
DOI: 10.1055/s-0038-1645210
Original Article
Schattauer GmbH Stuttgart

Agglutination of Rabbit Platelets in Plasma by the Thrombin Inhibitor D-phenylalanyl-L-prolyl-L-arginyl Chloromethyl Ketone

M A Packham
The Department of Biochemistry, University of Toronto, Toronto, Canada
,
N L Bryant
The Department of Biochemistry, University of Toronto, Toronto, Canada
,
M A Guccione
The Department of Biochemistry, University of Toronto, Toronto, Canada
› Author Affiliations
Further Information

Publication History

Received 25 August 1989

Accepted after revision 11 December 1989

Publication Date:
02 July 2018 (online)

Also available at
  PDF Download  Permissions and Reprints

Summary

The specific thrombin inhibitor D-phenylalanyl-L-prolyl-Larginyl chloromethyl ketone (FPRCH2Cl, PPACK) has been used as an anticoagulant with human blood to permit the preparation of platclct-rich plasma in which the physiological concentration of Ca2+ is maintained. When we attempted to use FPRCH2Cl(40 εM) as an anticoagulant for rabbit blood, clotting was prevented for at least 6 h at room temperature, but nearly all the platelets agglutinated. Thromboxane B2 was not formed and indomethacin and prostaglandin E1 were not inhibitory. The presence of citrate delayed this agglutination, but only EDTA (not EGTA) prevented it, indicating a dependence on Mg2+. Addition of FPRCH2Cl to suspensions of rabbit platelets in Tyrode-albumin solution had no effect on responses to ADP or collagen, but completely blocked all responses to human or rabbit thrombin. Thus plasma is required for the agglutination. Intravenous injection of FPRCH2Cl (5 mg/3 kg rabbit) reduced the platelet count to less than 50% by 15 to 25 min. Thus FPRCH2Cl cannot be used as an anticoagulant for rabbit blood when platelets are to be examined, and has a profound effect on the platelet count in vivo in rabbits. These effects should be considered when FPRCH2Cl is used as an antithrombotic agent for in vivo experiments with rabbits.

 

  • References

  • 1 Kettner C, Shaw E. D-Phe-Pro-ArgCH2Cl – a selective affinity label for thrombin. Thromb Res 1979; 14: 969-973
    CrossrefPubMedGoogle Scholar
  • 2 Lalau Keraly C, Kinlough-Rathbone RL, Packham MA, Suzuki H, Mustard JF. Conditions affecting the response of human platelets to epinephrine. Thromb Haemostas 1988; 60: 209-216
    PubMedGoogle Scholar
  • 3 Packham MA, Bryant NL, Guccione MA, Kinlough-Rathbone RL, Mustard JF. Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit plateletes to aggregating agents. Thromb Haemostas 1989; 62: 968-976
    PubMedGoogle Scholar
  • 4 Hanson SR, Harker LA. Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-Larginyl chloromethyl ketone. Proc Natl Acad Sci USA 1988; 85: 3184-3188
    CrossrefPubMedGoogle Scholar
  • 5 Collen D, Matsuo O, Stassen JM, Kettner C, Shaw E. In vivo studies of a synthetic inhibitor of thrombin. J Lab Clin Med 1982; 99: 76-83
    PubMedGoogle Scholar
  • 6 Hauptmann J, Markwardt F. Studies on the anticoagulant and antithrombotic action of an irreversible thrombin inhibitor. Thromb Res 1980; 20: 347-351
    CrossrefPubMedGoogle Scholar
  • 7 Schaeffer Jr RC, Chilton S-M, Hadden TJ, Carlson RW. Pulmonary fibrin microembolism with Echis carinatus venom in dogs: effects of a synthetic thrombin inhibitor. J Appl Physiol: Respirat Environ Exercise Physiol 1984; 57: 1824-1828
    PubMedGoogle Scholar
  • 8 Cazenave J-P, Packham MA, Mustard JF. Adherence of platelets to a collagen-coated surface: development of a quantitative method. J Lab Clin Med 1973; 82: 978-990
    PubMedGoogle Scholar
  • 9 Molnar J, Lorand L. Studies on apyrases. Arch Biochem Biophys 1961; 93: 353-363
    CrossrefPubMedGoogle Scholar
  • 10 Kinlough-Rathbone RL, Packham MA, Mustard JF. Platelet aggregation. In: Methods in Hematology. Measurements of Platelet Function Marker LA, Zimmerman TS. (eds) Churchill Livingstone, New York: 1983: 64-91
  • 11 Aster RH, Jandl JH. Platelet sequestration in man. I. Methods. J Clin Invest 1964; 43: 843-855
    CrossrefPubMedGoogle Scholar
  • 12 Ardlie NG, Packham MA, Mustard JF. Adenosine diphosphateinduced platelet aggregation in suspensions of washed rabbit platelets. Br J Haematol 1970; 19: 7-17
    CrossrefPubMedGoogle Scholar
  • 13 Ardlie NG, Perry DW, Packham MA, Mustard JF. Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc Soc Exp Biol Med 1971; 136: 1021-1023
    CrossrefPubMedGoogle Scholar
  • 14 Greenberg J, Packham MA, Cazenave J-P, Reimers H-J, Mustard JF. Effects on platelet function of removal of platelet sialic acid by neuraminidase. Lab Invest 1975; 32: 476-484
    PubMedGoogle Scholar
  • 15 Buchanan MR, Hirsh J. A comparison of the effects of aspirin and dipyridamole on platelet aggregation in vivo and ex vivo. Thromb Res 1978; 13: 517-529
    CrossrefPubMedGoogle Scholar
  • 16 Honey AC, Lad N, Tuffin DP. Effect of indomethacin and dazoxiben on intravascular platelet aggregation in the anaesthetized rabbit. Thromb Haemostas 1986; 56: 80-85
    PubMedGoogle Scholar
  • 17 Meyers KM. Species differences. In: Platelet Responses and Metabolism Volume 1, Responses Holmsen H. (ed) CRC Press; Boca Raton FL: 1986: 209-234
  • 18 Tsujinaka T, Sakon M, Hawiger J. Contrasting effects of thrombin inhibitors on hemostatic plug formation in vivo. Blood 1985; 66: 345a
    PubMedGoogle Scholar