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

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
• 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
• 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
• 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
• 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
• 6 Hauptmann J, Markwardt F. Studies on the anticoagulant and antithrombotic action of an irreversible thrombin inhibitor. Thromb Res 1980; 20: 347-351
• 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
• 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
• 9 Molnar J, Lorand L. Studies on apyrases. Arch Biochem Biophys 1961; 93: 353-363
• 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
• 12 Ardlie NG, Packham MA, Mustard JF. Adenosine diphosphateinduced platelet aggregation in suspensions of washed rabbit platelets. Br J Haematol 1970; 19: 7-17
• 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
• 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
• 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
• 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
• 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