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Platelet dysfunction, platelet function tests and cardiopulmonary bypass with heparin anticoagulation
Thrombosis Research 107 (2002) 81 – 82
Letter to the Editors-in-Chief Platelet dysfunction, platelet function tests and cardiopulmonary bypass with heparin anticoagulation Greilich et al. [1] report a series of studies in which they measured changes in platelet contractile force, platelet macroaggregation, platelet counts and platelet membrane expression of the receptors CD42b (GP Ib) and CD61 (GP IIb/IIIa) during cardiopulmonary bypass. Although their findings are interesting, their interpretation of them may be flawed because they omitted several important considerations. Regarding the markers measured, it is important to consider what phases of platelet activation they detect. The binding of a weak agonist to a platelet membrane receptor triggers a tightly regulated series of events that culminate in platelet secretory release and aggregation, but strong platelet agonists can trigger a full aggregatory response without requiring platelet secretory release [2]. The events that follow stimulation with a weak agonist can be divided into three phases: (1) shape change, (2) microaggregation (primary aggregation) and (3) macroaggregation (secondary aggregation) [3]. During and immediately after cardiopulmonary bypass, when bleeding times are increased, platelet macroaggregation in response to stimulation by weak agonists is impaired [4– 6] but the initial responses of platelets to stimulation up to and including microaggregation are well preserved [4– 6]. Macroaggregation is essential for primary haemostasis, as this process gives strength to a platelet plug and so paves the way for clot retraction. The decrease in platelet contractility observed by these workers might well be a direct or indirect consequence of impaired platelet macroaggregation. Platelet macroaggregation in whole blood may be measured by impedance aggregometry as it correlates well with optical aggregometry [7] and both methods are insensitive to microaggregation but reliably detect the coalescence of microaggregates into macroaggregates [8]. Although Dr. Greilich’s aggregometry results also demonstrate an impairment of platelet macroaggregation, the collagen concentration (2 Ag/ml) used for aggregometry was excessive and subtle changes in platelet aggregability may have been masked. Collagen in low concentrations acts as a weak platelet agonist, requiring platelet secretion of thromboxane A2, and platelet release of ADP and serotonin to stimulate macroaggregation [9,10]. Higher concentrations of collagen can induce a full platelet aggregatory response independently of platelet autocrine positive feedback. Using collagen (Hormon Chemie, Munich, Germany), we showed that 1 Ag/
ml for platelet studies in platelet-rich plasma and 0.6 Ag/ml for studies in whole blood gave near-maximal responses [4,6], and in our studies we observed greater reductions in platelet macroaggregation than Greilich et al. [4,6,11]. The agonist concentration is an important consideration when investigating changes in platelet function during cardiopulmonary bypass with heparin anticoagulation, as other workers have shown that haemorrhagic tendencies induced by heparin, and other glycosaminoglycans were associated with a platelet defect that was not detected when high concentrations of agonists were used during aggregation studies [12]. Greilich et al. [1] also studied changes in platelet expression of membrane receptors. Changes in these markers do not necessarily reflect platelet damage; they may indicate a normal platelet response to stimulation. These changes start to occur in the early stages of platelet activation during the shape change response. Therefore, while these markers are sensitive indicators of platelet activation they give little information on the ability of platelets to proceed through the full spectrum of responses and to form microaggregates, macroaggregates or both. Storey et al. [13] demonstrated that macroaggregation is inhibited more effectively than microaggregation by GP IIb/IIIa receptor antagonists, which suggests that the early and late roles of these receptors during platelet aggregation are differentially triggered. The multiple roles of these membrane receptors in the platelet activation response hamper the interpretation of levels of their expression, in terms of the platelet activation response. Furthermore, changes in the percentage of platelets expressing a receptor may reflect a preferential removal (or return) of activated platelets from (or to) circulation, as occurs during extracorporeal circulation [14,15]. Regarding the factors that generate platelet dysfunction during cardiopulmonary bypass, Greilich et al. [1] incompletely address the role of heparin. While they refer to the direct effects of heparin on platelets, they neglect to consider that there are some effects of heparin on platelets in vivo that are not reproduced when whole blood is heparinised in vitro [6,11,16,17]. In particular, we and others have shown that intravenous heparin administration markedly impairs platelet macroaggregation in a manner that is not reproduced by heparinisation of whole blood in vitro [6,11,16]. Our later studies suggest that the breakdown of plasma lipoproteins by lipoprotein lipase and hepatic lipase, which are released from endothelium by heparin, causes this platelet dysfunction [18]. The mediation by released endothelial enzymes would explain why we observed that ex vivo neutralisation of
0049-3848/02/$ - see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 9 - 3 8 4 8 ( 0 2 ) 0 0 2 0 9 - 8
82
Letter to the Editors-in-Chief
heparin with heparinase did not restore platelet macroaggregation after heparinisation in vivo [6]. In the studies by Greilich et al. [1], although both the direct and the antithrombin III-mediated effects of heparin may have been neutralised by the addition of heparinase ex vivo, indirect effects of heparin caused by released endothelial proteins may have caused the changes in platelet contractility. Greilich et al. [1], after observing that impaired platelet responses correlated with the duration of extracorporeal circulation, concluded that platelet dysfunction was caused by contact with the membranes of the oxygenator. However, we have observed that the inhibition of platelet macroaggregation after intravenous heparin administration [11] or after induction of lipolysis [18] in vitro is a gradual process. We therefore suggest that the correlation between duration of extracorporeal circulation and platelet dysfunction was observed because the platelet dysfunction induced by heparin is time-dependent and not because additional (intrinsic) platelet dysfunction occurred, as Greilich et al. [1] have concluded. Heparin is usually administered just before starting extracorporeal circulation, therefore the period of extracorporeal circulation closely approximates to the duration of heparinisation.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
References [1] Greilich PE, Brouse CF, Beckham J, Jessen ME, Martin EJ, Carr ME. Reductions in platelet contractile force correlate with duration of cardiopulmonary bypass and blood loss in patients undergoing cardiac surgery. Thromb Res 2002;105:523 – 9. [2] Brass LF. Molecular basis of platelet activation. In: Hoffmann R, Benz Jr EJ, Shattill SJ, Furie B, Cohen HJ, Silberstein LE, editors. Haematology: basic principles and practice. 2nd ed. London: Churchill Livingstone; 1995. p. 1536 – 52. [3] Pedvis LG, Wong T, Frojmovic MM. Differential inhibition of the platelet activation sequence: shape change, micro- and macro-aggregation, by a stable prostacyclin analogue (Iloprost). Thromb Haemost 1988;59:323 – 8. [4] Menys VC, Belcher PR, Noble M, Evans RD, Drossos GE, Pillai R, et al. Macroaggregation of platelets in plasma, as distinct from microaggregation in whole blood (and plasma), as determined using optical aggregometry and platelet counting respectively, is specifically impaired following cardiopulmonary bypass in man. Thromb Haemost 1994;72:511 – 8. [5] Kawahito K, Kobayashi E, Iwasa H, Misawa Y, Fuse K. Platelet aggregation during cardiopulmonary bypass evaluated by a laser light-scattering method. Ann Thorac Surg 1999;67:79 – 84. [6] Belcher P, Muriithi EW, Milne EM, Wanikiat P, Wheatley DJ, Armstrong RA. Heparin, platelet aggregation, neutrophils and cardiopulmonary bypass. Thromb Res 2000;98:249 – 56. [7] Podczasy JJ, Lee J, Vucenik I. Evaluation of whole-blood lumiaggregation. Clin Appl Thromb/Hemost 1997;3:190 – 5. [8] Born GV, Hume M. Effects of the numbers and sizes of platelet
[17]
[18]
aggregates on the optical density of plasma. Nature 1967;215: 1027 – 9. Kinlough-Rathbone RL, Packham MA, Reimers HJ, Cazenave JP, Mustard JF. Mechanisms of platelet shape change, aggregation, and release induced by collagen, thrombin, or A23,187. J Lab Clin Med 1977;90:707 – 19. Menys VC. Collagen induced human platelet aggregation: serotonin receptor antagonism retards aggregate growth in vitro. Cardiovasc Res 1993;27:1916 – 9. Muriithi EW, Belcher PR, Day SP, Menys VC, Wheatley DJ. Heparininduced platelet dysfunction and cardiopulmonary bypass. Ann Thorac Surg 2000;69:1827 – 32. Fernandez F, N’guyen P, Van Ryn J, Ofosu FA, Hirsh J, Buchanan MR. Hemorrhagic doses of heparin and other glycosaminoglycans induce a platelet defect. Thromb Res 1986;43:491 – 5. Storey RF, Wilcox RG, Heptinstall S. Differential effects of glycoprotein IIb/IIIa antagonists on platelet microaggregate and macroaggregate formation and effect of anticoagulant on antagonist potency. Implications for assay methodology and comparison of different antagonists. Circulation 1998;98:1616 – 21. Wahba A, Black G, Koksch M, Rothe G, Preuner J, Schmitz G, et al. Cardiopulmonary bypass leads to a preferential loss of activated platelets. A flow cytometric assay of platelet surface antigens. Eur J Cardiothorac Surg 1996;10:768 – 73. Muriithi EW, Belcher PR, Rao JN, Chaudhry MA, Nicol D, Wheatley DJ. The effects of heparin and extracorporeal circulation on platelet counts and platelet microaggregation during cardiopulmonary bypass. J Thorac Cardiovasc Surg 2000;120:538 – 43. Besterman EM, Gillett MP. Heparin effects on plasma lysolecithin formation and platelet aggregation. Atherosclerosis 1973;17:503 – 13. Heiden D, Mielke CHJ, Rodvien R. Impairment by heparin of primary haemostasis and platelet [14C]5-hydroxytryptamine release. Br J Haematol 1977;36:427 – 36. Muriithi EW, Belcher PR, Day SP, Chaudhry MA, Caslake MJ, Wheatley DJ. Lipolysis generates platelet dysfunction after in vivo heparin administration. Clin Sci 2002 [in press].
Elijah W. Muriithi * Department of Cardiac Surgery, Royal Sussex County Hospital, Eastern Road, Brighton BN2 5BE, UK E-mail address: [email protected] Philip R. Belcher Department of Cardiac Surgery, University of Glasgow, Royal Infirmary, 16 Alexandra Parade, Glasgow G31 2ER, UK Valentine C. Menys Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK 19 June 2002
* Corresponding author. Tel.: +44-1273-696955; fax: +44-1273664464.
Letter to the Editors-in-Chief Platelet dysfunction, platelet function tests and cardiopulmonary bypass with heparin anticoagulation Greilich et al. [1] report a series of studies in which they measured changes in platelet contractile force, platelet macroaggregation, platelet counts and platelet membrane expression of the receptors CD42b (GP Ib) and CD61 (GP IIb/IIIa) during cardiopulmonary bypass. Although their findings are interesting, their interpretation of them may be flawed because they omitted several important considerations. Regarding the markers measured, it is important to consider what phases of platelet activation they detect. The binding of a weak agonist to a platelet membrane receptor triggers a tightly regulated series of events that culminate in platelet secretory release and aggregation, but strong platelet agonists can trigger a full aggregatory response without requiring platelet secretory release [2]. The events that follow stimulation with a weak agonist can be divided into three phases: (1) shape change, (2) microaggregation (primary aggregation) and (3) macroaggregation (secondary aggregation) [3]. During and immediately after cardiopulmonary bypass, when bleeding times are increased, platelet macroaggregation in response to stimulation by weak agonists is impaired [4– 6] but the initial responses of platelets to stimulation up to and including microaggregation are well preserved [4– 6]. Macroaggregation is essential for primary haemostasis, as this process gives strength to a platelet plug and so paves the way for clot retraction. The decrease in platelet contractility observed by these workers might well be a direct or indirect consequence of impaired platelet macroaggregation. Platelet macroaggregation in whole blood may be measured by impedance aggregometry as it correlates well with optical aggregometry [7] and both methods are insensitive to microaggregation but reliably detect the coalescence of microaggregates into macroaggregates [8]. Although Dr. Greilich’s aggregometry results also demonstrate an impairment of platelet macroaggregation, the collagen concentration (2 Ag/ml) used for aggregometry was excessive and subtle changes in platelet aggregability may have been masked. Collagen in low concentrations acts as a weak platelet agonist, requiring platelet secretion of thromboxane A2, and platelet release of ADP and serotonin to stimulate macroaggregation [9,10]. Higher concentrations of collagen can induce a full platelet aggregatory response independently of platelet autocrine positive feedback. Using collagen (Hormon Chemie, Munich, Germany), we showed that 1 Ag/
ml for platelet studies in platelet-rich plasma and 0.6 Ag/ml for studies in whole blood gave near-maximal responses [4,6], and in our studies we observed greater reductions in platelet macroaggregation than Greilich et al. [4,6,11]. The agonist concentration is an important consideration when investigating changes in platelet function during cardiopulmonary bypass with heparin anticoagulation, as other workers have shown that haemorrhagic tendencies induced by heparin, and other glycosaminoglycans were associated with a platelet defect that was not detected when high concentrations of agonists were used during aggregation studies [12]. Greilich et al. [1] also studied changes in platelet expression of membrane receptors. Changes in these markers do not necessarily reflect platelet damage; they may indicate a normal platelet response to stimulation. These changes start to occur in the early stages of platelet activation during the shape change response. Therefore, while these markers are sensitive indicators of platelet activation they give little information on the ability of platelets to proceed through the full spectrum of responses and to form microaggregates, macroaggregates or both. Storey et al. [13] demonstrated that macroaggregation is inhibited more effectively than microaggregation by GP IIb/IIIa receptor antagonists, which suggests that the early and late roles of these receptors during platelet aggregation are differentially triggered. The multiple roles of these membrane receptors in the platelet activation response hamper the interpretation of levels of their expression, in terms of the platelet activation response. Furthermore, changes in the percentage of platelets expressing a receptor may reflect a preferential removal (or return) of activated platelets from (or to) circulation, as occurs during extracorporeal circulation [14,15]. Regarding the factors that generate platelet dysfunction during cardiopulmonary bypass, Greilich et al. [1] incompletely address the role of heparin. While they refer to the direct effects of heparin on platelets, they neglect to consider that there are some effects of heparin on platelets in vivo that are not reproduced when whole blood is heparinised in vitro [6,11,16,17]. In particular, we and others have shown that intravenous heparin administration markedly impairs platelet macroaggregation in a manner that is not reproduced by heparinisation of whole blood in vitro [6,11,16]. Our later studies suggest that the breakdown of plasma lipoproteins by lipoprotein lipase and hepatic lipase, which are released from endothelium by heparin, causes this platelet dysfunction [18]. The mediation by released endothelial enzymes would explain why we observed that ex vivo neutralisation of
0049-3848/02/$ - see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 9 - 3 8 4 8 ( 0 2 ) 0 0 2 0 9 - 8
82
Letter to the Editors-in-Chief
heparin with heparinase did not restore platelet macroaggregation after heparinisation in vivo [6]. In the studies by Greilich et al. [1], although both the direct and the antithrombin III-mediated effects of heparin may have been neutralised by the addition of heparinase ex vivo, indirect effects of heparin caused by released endothelial proteins may have caused the changes in platelet contractility. Greilich et al. [1], after observing that impaired platelet responses correlated with the duration of extracorporeal circulation, concluded that platelet dysfunction was caused by contact with the membranes of the oxygenator. However, we have observed that the inhibition of platelet macroaggregation after intravenous heparin administration [11] or after induction of lipolysis [18] in vitro is a gradual process. We therefore suggest that the correlation between duration of extracorporeal circulation and platelet dysfunction was observed because the platelet dysfunction induced by heparin is time-dependent and not because additional (intrinsic) platelet dysfunction occurred, as Greilich et al. [1] have concluded. Heparin is usually administered just before starting extracorporeal circulation, therefore the period of extracorporeal circulation closely approximates to the duration of heparinisation.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
References [1] Greilich PE, Brouse CF, Beckham J, Jessen ME, Martin EJ, Carr ME. Reductions in platelet contractile force correlate with duration of cardiopulmonary bypass and blood loss in patients undergoing cardiac surgery. Thromb Res 2002;105:523 – 9. [2] Brass LF. Molecular basis of platelet activation. In: Hoffmann R, Benz Jr EJ, Shattill SJ, Furie B, Cohen HJ, Silberstein LE, editors. Haematology: basic principles and practice. 2nd ed. London: Churchill Livingstone; 1995. p. 1536 – 52. [3] Pedvis LG, Wong T, Frojmovic MM. Differential inhibition of the platelet activation sequence: shape change, micro- and macro-aggregation, by a stable prostacyclin analogue (Iloprost). Thromb Haemost 1988;59:323 – 8. [4] Menys VC, Belcher PR, Noble M, Evans RD, Drossos GE, Pillai R, et al. Macroaggregation of platelets in plasma, as distinct from microaggregation in whole blood (and plasma), as determined using optical aggregometry and platelet counting respectively, is specifically impaired following cardiopulmonary bypass in man. Thromb Haemost 1994;72:511 – 8. [5] Kawahito K, Kobayashi E, Iwasa H, Misawa Y, Fuse K. Platelet aggregation during cardiopulmonary bypass evaluated by a laser light-scattering method. Ann Thorac Surg 1999;67:79 – 84. [6] Belcher P, Muriithi EW, Milne EM, Wanikiat P, Wheatley DJ, Armstrong RA. Heparin, platelet aggregation, neutrophils and cardiopulmonary bypass. Thromb Res 2000;98:249 – 56. [7] Podczasy JJ, Lee J, Vucenik I. Evaluation of whole-blood lumiaggregation. Clin Appl Thromb/Hemost 1997;3:190 – 5. [8] Born GV, Hume M. Effects of the numbers and sizes of platelet
[17]
[18]
aggregates on the optical density of plasma. Nature 1967;215: 1027 – 9. Kinlough-Rathbone RL, Packham MA, Reimers HJ, Cazenave JP, Mustard JF. Mechanisms of platelet shape change, aggregation, and release induced by collagen, thrombin, or A23,187. J Lab Clin Med 1977;90:707 – 19. Menys VC. Collagen induced human platelet aggregation: serotonin receptor antagonism retards aggregate growth in vitro. Cardiovasc Res 1993;27:1916 – 9. Muriithi EW, Belcher PR, Day SP, Menys VC, Wheatley DJ. Heparininduced platelet dysfunction and cardiopulmonary bypass. Ann Thorac Surg 2000;69:1827 – 32. Fernandez F, N’guyen P, Van Ryn J, Ofosu FA, Hirsh J, Buchanan MR. Hemorrhagic doses of heparin and other glycosaminoglycans induce a platelet defect. Thromb Res 1986;43:491 – 5. Storey RF, Wilcox RG, Heptinstall S. Differential effects of glycoprotein IIb/IIIa antagonists on platelet microaggregate and macroaggregate formation and effect of anticoagulant on antagonist potency. Implications for assay methodology and comparison of different antagonists. Circulation 1998;98:1616 – 21. Wahba A, Black G, Koksch M, Rothe G, Preuner J, Schmitz G, et al. Cardiopulmonary bypass leads to a preferential loss of activated platelets. A flow cytometric assay of platelet surface antigens. Eur J Cardiothorac Surg 1996;10:768 – 73. Muriithi EW, Belcher PR, Rao JN, Chaudhry MA, Nicol D, Wheatley DJ. The effects of heparin and extracorporeal circulation on platelet counts and platelet microaggregation during cardiopulmonary bypass. J Thorac Cardiovasc Surg 2000;120:538 – 43. Besterman EM, Gillett MP. Heparin effects on plasma lysolecithin formation and platelet aggregation. Atherosclerosis 1973;17:503 – 13. Heiden D, Mielke CHJ, Rodvien R. Impairment by heparin of primary haemostasis and platelet [14C]5-hydroxytryptamine release. Br J Haematol 1977;36:427 – 36. Muriithi EW, Belcher PR, Day SP, Chaudhry MA, Caslake MJ, Wheatley DJ. Lipolysis generates platelet dysfunction after in vivo heparin administration. Clin Sci 2002 [in press].
Elijah W. Muriithi * Department of Cardiac Surgery, Royal Sussex County Hospital, Eastern Road, Brighton BN2 5BE, UK E-mail address: [email protected] Philip R. Belcher Department of Cardiac Surgery, University of Glasgow, Royal Infirmary, 16 Alexandra Parade, Glasgow G31 2ER, UK Valentine C. Menys Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK 19 June 2002
* Corresponding author. Tel.: +44-1273-696955; fax: +44-1273664464.