The Effect of Lysed Platelets on Neutralization of Heparin In Vitro with Protamine as Measured by the Activated Coagulation Time (ACT)

Summary

The effect of lysed platelets on the activated coagulation time (ACT) was studied in heparinized whole blood during titration with protamine. Frozen-thawed washed platelet suspension, or a chromatography fraction thereof, or autologous frozen-thawed platelet-rich plasma was added in various dilutions to freshly drawn blood anticoagulated with 3,000 USP units/1 heparin. After a 10 min incubation, the amount of protamine needed to restore the ACT to baseline ("protamine titration dose") was determined. We found that the protamine titration dose decreased in proportion to the amount of lysed platelet material added; expressed as a percentage of the total number of platelets present, each unit increase in lysed platelets produced a 1.7% ±0.8 (SD) reduction in the protamine dose needed to normalize the ACT. A heparin activity assay showed that this effect was not due to antiheparin activity of lysed platelets such as platelet factor 4 (PF4). Our data indicate that the procoagulant activity of platelet membranes reduced the sensitivity of the ACT to heparin. These findings suggest that membranous platelet microparticles may cause an inaccurate calculation, based on the ACT, of a protamine dose to reverse heparin anticoagulation in cardiopulmonary bypass procedures.

• References

• 1 Bull BS, Korpman RA, Huse WM, Briggs BD. Heparin therapy during extracorporeal circulation. J Thorac Cardiovasc Surg 1975; 69: 674-84
• 2 Hattersley PG. Heparin anticoagulation. In: Laboratory Hematology.. Koepke JA. (ed) Churchill Livingstone, New York: 1984. pp 789-818
• 3 Ellison N, Horrow J. Chapters 1, 2. In: Effective Hemostasis in Cardiac Surgery.. Ellison N, Jobes D. (eds) W. B. Saunders Company, New York: 1988. pp 1-39
• 4 Bode AP, Eick L. Lysed platelets shorten the activated coagulation time (ACT) of heparinized blood. Am J Clin Pathol 1989; 9 1(4) 430-4
• 5 George JN, Pickett EB, Heinz R. Platelet membrane microparticles in blood bank fresh frozen plasma and cryoprecipitate. Blood 1986; 6 8(1) 307-9
• 6 Howard MA, Coghlan M, David R, Pfueller SL. Coagulation activities of plasma microparticles. Thromb Res 1988; 50: 145-56
• 7 McGill M, Fugman DA, Vittorio N, Darrow C. Platelet membrane vesicles reduced microvascular bleeding times in thrombocytopenic rabbits. J Lab Clin Med 1987; 109: 127-33
• 8 George JN, Pickett EB, Saucerman S. et al. Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest 1986; 78: 340-8
• 9 Abrams CS, Ellison N, Budzynski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 1990; 7 5(1) 128-38
• 10 Pifarre R, Babka R, Sullivan HJ. et al. Management of postoperative heparin rebound following cardiopulmonary bypass. J Thorac Cardiovasc Surg 1981; 81: 378-81
• 11 Kaul TK, Crow MJ, Rajah SM. et al. Heparin administration during extracorporeal circulation. J Thorac Cardiovasc Surg 1979; 7 8(1) 95-102
• 12 Bode AP, Miller DT. Analysis of platelet factor 3 in platelet concentrates stored for transfusion. Vox Sang 1986; 51: 299-305
• 13 Manufacturer's brochure
• 14 Bode AP, Sandberg H, Dombrose FA, Lentz BR. Association of factor V activity with membranous vesicles released from human platelets: Requirement for platelet stimulation. Thromb Res 1985; 39: 49-61
• 15 Culliford AT, Gitel SN, Starr N, Thomas ST. et al. Lack of correlation between activated clotting time and plasma heparin during cardiopulmonary bypass. Ann Surg 1981; 193: 105-11
• 16 Gravlee GP, Case LD, Angert KC, Roger AT, Miller GS. Variability of the activated coagulation time. Anesth Analog 1988; 67: 469-72
• 17 Stenbjerg S, Berg E, Albrechtsen OK. Heparin levels and activated clotting time (ACT) during open heart surgery. Scand J Haematol 1981; 26: 281-4
• 18 White II GC. Heparin-neutralizing proteins. In: Chemistry and Biology of Heparin.. Lundblad RL, Brown WV, Mann KG, Roberts HR. (eds) Elsevier North-Holland, Inc., Amsterdam: 1981 377
• 19 Harada K, Zucker MB. Simultaneous development of platelet factor 4 activity and release of ¹⁴C-serotonin. Thromb Diath Haemorrh 1971; 25: 41-6
• 20 Oosta GM, Favreau LV, Beeler DL, Rosenberg RD. Purification and properties of human platelet heparitinase. J Biol Chem 1982; 2 57(19) 11249-55
• 21 Taylor C, Baugh R. The influence of platelet activation on the recalcified activated clotting time of citrated, heparinized whole blood.. Thromb Haemostas 1985; 54 (1) 28 (Abstr 167)
• 22 Zwaal RFA. Membrane and lipid involvement in blood coagulation. Biochim Biophys Acta 1978; 515: 163-205
• 23 Dombrose FA, Bode AP, Lentz BR. Differentiation of factor V-like coagulant activity from catalytic phospholipid-like surface activity in membrane fractions derived from human platelets. Thromb Res 1981; 22: 603-21
• 24 Mann KG, Tracy PB, Krishnaswamy S. et al. Platelets and coagulation. In: Thrombosis and Haemostasis. Vermylen J, Lijnen HR, Arnout J. (eds) International Society on Thrombosis and Haemostasis and Leuven University Press, Leuven: 1987. pp 505-23
• 25 Chenoweth DE, Cooper SW, Hugli TE. et al. Complement activation during cardiopulmonary bypass. N Engl J Med 1981; 3 04(9) 497-503
• 26 Wiedmer T, Esmon CT, Sims PJ. Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase. Blood 1986; 6 8(4) 875-80