High-dose erythropoietin alters platelet reactivity and bleeding time in rodents in contrast to the neuroprotective variant carbamyl-erythropoietin (CEPO)
Agnete Kirkeby
1
H. Lundbeck A/S, Valby, Denmark
,
Lars Torup
1
H. Lundbeck A/S, Valby, Denmark
,
Louise Bochsen
2
Department of Clinical Immunology, University Hospital of Copenhagen, Copenhagen, Denmark
,
Marianne Kjalke
3
Novo Nordisk A/S, Måløv, Denmark
,
Kristin Abel
3
Novo Nordisk A/S, Måløv, Denmark
,
Kim Theilgaard-Monch
4
Department of Haematology, University Hospital of Copenhagen, Copenhagen, Denmark
,
Pär I. Johansson
2
Department of Clinical Immunology, University Hospital of Copenhagen, Copenhagen, Denmark
The haematopoietic hormone erythropoietin (EPO) has neuroprotective properties and is currently being explored for treatment of stroke and other neurological disorders. Short-term, high-dose treatment with EPO seems to improve neurological function of stroke patients but may be associated with increased thrombotic risk, whereas alternative non-erythropoietic neuroprotective derivatives of EPO, such as carbamylated EPO (CEPO), may be devoid of such side-effects. We investigated the effects of short-term, high-dose treatment with EPO and CEPO on platelet function and haemostasis in healthy mice and rats. Animals received three daily doses of EPO or CEPO (50 μg/kg), and blood was compared with respect to alterations in haematology and platelet reactivity. In rats, treatment with EPO increased the haematocrit to >50% and the mean platelet volume by 37%,while CEPO had no effect on these parameters. Platelets from EPO-treated rats showed an increased sensitivity to thrombin receptor agonist peptides and elevated plasma levels of soluble P-selectin (sP-selectin) were found in treated mice. Further indicators of platelet hyperreactivity in EPO, but not CEPO-treated animals, were significantly increased aggregatory responses to collagen in whole blood and platelet-rich plasma (PRP).The increased platelet reactivity was paralleled by a decreased bleeding time after tail transection in rats. Samples from EPO-treated rats showed an attenuated response to ADP in whole blood aggregometry and thrombelastography (TEG) platelet mapping but not in apyrase-treated PRP, suggesting involvement of ADP receptor desensitization. These findings suggest that while EPO affects various aspects of platelet function, CEPO is devoid of such effects.
Keywords
ADP -
collagen -
P-selectin -
MPV -
stroke
* These authors have contributed equally to this work.
References
1
Fisher J W.
Erythropoietin: physiologic and pharmacologic aspects. Proc Soc Exp Biol Med 1997; 216: 358-369.
2
Hasselblatt M,
Ehrenreich H,
Siren AL.
The brain erythropoietin system and its potential for therapeutic exploitation in brain disease. J Neurosurg Anesthesiol 2006; 18: 132-138.
4
Ehrenreich H,
Hinze-Selch D,
Stawicki S.
et al. Improvement of cognitive functions in chronic schizophrenic patients by recombinant human erythropoietin. Mol Psychiatry 2007; 12: 206-220.
7
Cases A,
Escolar G,
Reverter JC.
et al. Recombinant human erythropoietin treatment improves platelet function in uremic patients. Kidney Int 1992; 42: 668-672.
8
Wolf RF,
Gilmore LS,
Friese P.
et al. Erythropoietin potentiates thrombus development in a canine arteriovenous shunt model. Thromb Haemost 1997; 77: 1020-1024.
9
Wolf RF,
Peng J,
Friese P.
et al. Erythropoietin administration increases production and reactivity of platelets in dogs. Thromb Haemost 1997; 78: 1505-1509.
10
Stohlawetz PJ,
Dzirlo L,
Hergovich N.
et al. Effects of erythropoietin on platelet reactivity and thrombopoiesis in humans. Blood 2000; 95: 2983-2989.
11
Lip GY,
Blann AD,
Farooqi IS.
et al. Sequential alterations in haemorheology, endothelial dysfunction, platelet activation and thrombogenesis in relation to prognosis following acute stroke: The West Birmingham Stroke Project. Blood Coagul Fibrinolysis 2002; 13: 339-347.
14
Villa P,
Van Beek J,
Larsen AK.
et al. Reduced functional deficits, neuroinflammation, and secondary tissue damage after treatment of stroke by nonerythropoietic erythropoietin derivatives. J Cereb Blood Flow Metab 2006; 27: 552-563.
15
Mennini T,
De Paola M,
Bigini P.
et al. Nonhematopoietic erythropoietin derivatives prevent motoneuron degeneration in vitro and in vivo. Mol Med 2006; 12: 153-160.
17
Cauwenberghs S,
Feijge MA,
Hageman G.
et al. Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation. Transfusion 2006; 46: 1018-1028.
19
Zhong X,
Kriz R,
Seehra J.
et al. N-linked glycosylation of platelet P2Y12 ADP receptor is essential for signal transduction but not for ligand binding or cell surface expression. FEBS Lett 2004; 562: 111-117.
21
Herbert JM,
Bernat A,
Samama M.
et al. The antiaggregating and antithrombotic activity of ticlopidine is potentiated by aspirin in the rat. Thromb Haemost 1996; 76: 94-98.
22
Momi S,
Pitchford SC,
Alberti PF.
et al. Nitroaspirin plus clopidogrel versus aspirin plus clopidogrel against platelet thromboembolism and intimal thickening in mice. Thromb Haemost 2005; 93: 535-543.
23
Wang Y,
Zhang ZG,
Rhodes K.
et al. Post-ischemic treatment with erythropoietin or carbamylated erythropoietin reduces infarction and improves neurological outcome in a rat model of focal cerebral ischemia. Br J Pharmacol 2007; 151: 1377-1384.
26
Macdougall IC,
Roberts DE,
Coles GA.
et al. Clinical pharmacokinetics of epoetin (recombinant human erythropoietin). Clin Pharmacokinet 1991; 20: 99-113.
29
Bath P,
Algert C,
Chapman N.
et al. Association of mean platelet volume with risk of stroke among 3134 individuals with history of cerebrovascular disease. Stroke 2004; 35: 622-626.
30
Greisenegger S,
Endler G,
Hsieh K.
et al. Is elevated mean platelet volume associated with a worse outcome in patients with acute ischemic cerebrovascular events?. Stroke 2004; 35: 1688-1691.
31
Andre P,
Hartwell D,
Hrachovinova I.
et al. Pro-coagulant state resulting from high levels of soluble P-selectin in blood. Proc Natl Acad Sci USA 2000; 97: 13835-13840.
32
Blann AD,
Faragher EB,
McCollum CN.
Increased soluble P-selectin following myocardial infarction: a new marker for the progression of atherosclerosis. Blood Coagul Fibrinolysis 1997; 8: 383-390.
33
Li-Saw-Hee FL,
Blann AD,
Lip GY.
A cross-sectional and diurnal study of thrombogenesis among patients with chronic atrial fibrillation. J Am Coll Cardiol 2000; 35: 1926-1931.
34
Toth O,
Calatzis A,
Penz S.
et al. Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood. Thromb Haemost 2006; 96: 781-788.
35
Baurand A,
Eckly A,
Hechler B.
et al. Differential regulation and relocalization of the platelet P2Y receptors after activation: a way to avoid loss of hemostatic properties?. Mol Pharmacol 2005; 67: 721-733.
36
Wang L,
Olivecrona G,
Gotberg M.
et al. ADP acting on P2Y13 receptors is a negative feedback pathway for ATP release from human red blood cells. Circ Res 2005; 96: 189-196.
37
Shankar H,
Garcia A,
Prabhakar J.
et al. P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation. J Thromb Haemost 2006; 4: 638-647.
38
Vassilev AO,
Lorenz DR,
Tibbles HE.
et al. Role of the leukemia-associated transcription factor STAT3 in platelet physiology. Leuk Lymphoma 2002; 43: 1461-1467.
39
Barry FA,
Graham GJ,
Fry MJ.
et al. Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?. FEBS Lett 2003; 553: 173-178.
41
Martinichen-Herrero JC,
Carbonero ER,
Sassaki GL.
et al. Anticoagulant and antithrombotic activities of a chemically sulfated galactoglucomannan obtained from the lichen Cladonia ibitipocae
. Int J Biol Macromol 2005; 35: 97-102.
42
Berridge MV,
Fraser JK,
Carter JM.
et al. Effects of recombinant human erythropoietin on megakaryocytes and on platelet production in the rat. Blood 1988; 72: 970-977.
43
Fraser JK,
Tan AS,
Lin FK.
et al. Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes. Exp Hematol 1989; 17: 10-16.
45
Haiden N,
Cardona F,
Schwindt J.
et al. Changes in thrombopoiesis and platelet reactivity in extremely low birth weight infants undergoing erythropoietin therapy for treatment of anaemia of prematurity. Thromb Haemost 2005; 93: 118-123.
46
Tassies D,
Reverter JC,
Cases A.
et al. Effect of recombinant human erythropoietin treatment on circulating reticulated platelets in uremic patients: association with early improvement in platelet function. Am J Hematol 1998; 59: 105-109.
47
Peng J,
Friese P,
Heilmann E.
et al. Aged platelets have an impaired response to thrombin as quantitated by P-selectin expression. Blood 1994; 83: 161-166.
48
Coleman TR,
Westenfelder C,
Togel FE.
et al. Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities. Proc Natl Acad Sci USA 2006; 103: 5965-5970.
49
Grasso G,
Buemi M,
Alafaci C.
et al. Beneficial effects of systemic administration of recombinant human erythropoietin in rabbits subjected to subarachnoid hemorrhage. Proc Natl Acad Sci USA 2002; 99: 5627-5631.
50
Lee ST,
Chu K,
Sinn DI.
et al. Erythropoietin reduces perihematomal inflammation and cell death with eNOS and STAT3 activations in experimental intracerebral hemorrhage. J Neurochem 2006; 96: 1728-1739.
