Kasım ÖZLÜK, İşbil Naciye BÜYÜKCOŞKUN, Engin SAĞDİLEK

Trombositlerin epinefrine yanıtlarının optik agregometre ve PFA-100 ile incelenmesi

Çalışmamızda trombositlerin epinefrine yanıtlarının optik agregometre ve PFA-100 sistemi ile incelenmesi amaçlandı.20-76 yaş arası, sağlıklı ve gönüllü 68 kişiden alınan kan örnekleri PFA-100 sisteminde kollajen/epinefrin ve kollajen/ADP kartuşu ile kapanma zamanı ölçülerek trombosit yanıtları değerlendirildi. Agregasyon testleri için elde edilen trombositten zengin plazma (450µl) üzerine 10 µM ADP, 10 µg/ml kollajen veya 300 µM epinefrin 50 µl ilave edilerek agregasyon süreci 10 dk takip edildi. Optik agregometrede epinefrine karşı gösterdikleri maksimum agregasyon cevaplarına göre cevapsız, yarı cevaplı ve normal olarak üç gruba ayrılan örnekler %14,7 cevapsız, %16,2 yarı cevaplı bulundu. Epinefrine azalan cevap; epinefrinle lag zamanının uzaması, ADP ile maksimum agregasyonunun azalması ve maksimum agregasyona ulaşma süresinin kısalmasıyla ve kollajenle lag zamanının uzamasıyla birlikteydi. Epinefrine cevapsız ve yarıcevaplı grupta PFA-100 sistemi kollajen/epinefrin kartuşu kapanma zamanının uzadığı gözlendi. Bulgularımız, optik agregometre ile elde edilen sonuçlara göre trombositlerin epinefrine yanıtlarının farklı olduğunu ve bu değerlendirmenin daha yeni bir yöntem olan PFA- 100 sistemi ile de yapılabileceğini göstermektedir.

The investigation of platelets response to epinephrine using optical aggregometer and PFA-100

In this study we aimed to examine the response of platelets to epinephrine using optic aggregometer and PFA-100. Blood samples of 68 healthy and volunteered subjects, aged between 20-76, were evaluated for the epinephrine responses of platelets by using collagen/ epinephrine and collagen/ADP cartridges in PFA-100 system for closure time values. The process of aggregation was observed for 10 minutes by adding 10µM ADP, 10 µg/ml collagen or 300 µM epinephrine on platelet rich plasma (450 µl) prepared for aggregation tests. The samples were classified into three groups as nonresponder, semiresponder and normal according to their maximal responses to epinephrine: 14,7 % nonresponder, 16,2 % semiresponder were observed. A decreased response to epinephrine was associated with increased epinephrine lag duration, decreased maximum aggregation of ADP and increased collagen lag duration. The PFA-100 system collagen/ epinephrine cartridge closure time values were observed to be increased in the groups with nonresponder and semiresponder. Our results acquired with optic aggregometer demonstrate that the responses of platelets to epinephrine is various, and the relatively new method of PFA-100 can also be used to evaluate this response.

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1. Colman RW, Hirsh J, Marder VJ, Clowes AW, George JN, (eds). Hemostasis and Thrombosis: Basic Priciples and Clinical Practice. 4th Edition. Philadelphia: J.B.Lippincott Company; 2001.
2. Brass LF. Thrombin and platelet activation. Chest 2003; 124: 18–25.
3. Quinton TM, Kim S, Dangelmaier C, Dorsam RT, et al. Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation. Biochem J 2002; 368: 535–43.
4. Yang J, Wu J, Jiang H, et al. Signaling through Gi family members in platelets. J Biol Chem 2002; 277: 46035–42.
5. Maayani S, Schwarz T, Martinez R, Tagliente TM. Activation of Gi-coupled receptors releases a tonic state of inhibited platelet agregation. Platelets 2001; 12: 94–8.
6. Jin J, Kunapuli SP. Coactivation of two different G proteincoupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998; 95: 8070-74.
7. Paul BZS, Jin J, Kunapuli SP. Molecular mechanism of Thromboxane A2-induced platelet aggregation. J Biol Chem 1999; 274: 29108-14.
8. Woulfe D, Yang J, Brass L. ADP and platelets: the end of the beginning. J Clin Invest 2001; 107: 1591-8.
9. Nieswandt B, Schulte V, Zywietz A, Gratacap MP, Offermanns S. Costimulation of Gi- and G12/13-mediated signaling pathways induces integrin αIIbβ3 activation in platelets. J Biol Chem 2002; 277: 39493-8.
10. Breddin HK. Can platelet aggregometry be standardized? Platelets 2005; 16: 151-8.
11. Keularts IMLW, van Gorp RMA, Feijge MAH, Vuist WMJ, Heemskerk JWM. α2A-adrenergic receptor stimulation potentiates calcium release in platelets by modulating cAMP levels. J Biol Chem 2000; 275: 1763–72.
12. Yang J, Wu J, Kowalska MA, et al. Loss of signalling through te G protein, Gz, results in abnormal platelet activation and altere responses to psychoactive drugs. Proc Natl Acad Sci USA 2000; 97: 9984-89.
13. Swart SS, Pearson D, Wood JK, Barnett DB. Functional significance of the platelet alpha2-adrenoceptor: studies in patients with myeloproliferative disorders. Thromb Res 1984;33: 531-41.
14. Avram S, Lupu A, Angelescu S, Olteanu N, Mut-Popescu D. Abnormalities of platelet aggregation in chronic myeloproliferative disorders. J Cell Mol Med 2001; 5: 79-87.
15. Kaywin P, McDonough M, Insel PA, Shattil SJ. Platelet function in essential thrombocythemia. Decreased epinephrine responsiveness associated with a deficiency of platelet alphaadrenergic receptors. N Engl J Med 1978; 299: 505-9.
16. Yamamoto K, Sekiguchi E, Takatani O. Abnormalities of epinephrine-induced platelet aggregation and adenine nucleotides in myeloproliferative disorders. Thromb Haemost 1984;52: 292-6.
17. Kambayashi J, Shinoki N, Nakamura T, et al. Prevalence of impaired responsiveness to epinephrine in platelets among Japanese. Thromb Res 1996; 81: 85–90.
18. Choi JW. Incidence of nonresponsiveness to epinephrine in platelets from healthy humans. Acta Haematol 2002; 108:106-8.
19. Theodoropoulos I, Christopoulos C, Metcalfe P, Dimitriadou E, Economopoulos P, Loucopoulos D. The effect of human platelet alloantigen polymorphisms on the in vitro responsiveness to adrenaline and collagen. Br J Haematol 2001; 114: 387–93.
20. Pyo MK, Yun-Choi HS, Hong YJ. Apparent heterogeneous responsiveness of human platelet rich plasma to catecholamines. Platelets 2003; 14: 171–8.
21. Nakahashi TK, Kambayashi J, Nakamura T, et al. Platelets in nonresponders to epinephrine stimulation showed reduced response to ADP. Thromb Res 2001; 104: 127-35.
22. Storey RF, Heptinstall S. Laboratory investigation of platelet function. Clin Lab Haem 1999; 21: 317–29.
23. Harrison P, Robinson M, Liesner R, et al. The PFA–100®: a potential rapid screening tool for the assessment of platelet dysfunction. Clin Lab Haem 2002; 24: 225–32.
24. Franchini M. The platelet-function analyzer (PFA–100®) for evaluating primary hemostasis. Hematology 2005; 10: 177–81.
25. DADE BEHRING. To aid in the detection of platelet dysfunction in citrated human whole blood. 2000.
26. Emmanuel JF. Utility of the PFA-100 for assessing bleeding disorders and monitoring therapy: a review of analytical variables, benefits and limitations. Haemophilia 2001: 7; 170–9.
27. Helena Laboratories, PACKS-4, Operator’s Manual. 1991. 28. Kunapuli SP, Dorsam RT, Kim S, Quinton TM. Platelet purinergic receptors. Curr Opin Pharm 2003; 3: 175–80.
29. Cattaneo M, Gachet C. The platelet ADP receptors. Haematologica 2001; 86: 346–8.
30. Dorsam RT, Kunapuli SP. Central role of the P2Y12 receptor in platelet activation. J Clin Invest 2004; 113: 340–5.
31. Sağdilek E. Kardiyovasküler hastalarda antitrombositik tedaviye yanıtın optik agregometre ve PFA–100 ile incelenmesi (Uzmanlık Tezi). Bursa: Uludağ Üniversitesi; 2004.
32. Nakamura T, Ariyoshi H, Kambayashi J, et al. Signal transduction system in epinephrine stimulated platelets; comparison between epinephrine sensitive and insensitive platelets. Thromb Res 1997; 85: 83–93.
33. Kayaalp SO. Rasyonel Tedavi Yönünden Tıbbi Farmakoloji. 9. baskı. Ankara: Hacettepe-Taş Kitapçılık; 2000; 3,45, 581–612.
34. Mammen E, Comp PC, Gosselin R, et al. PFA-100TM System: A new method for assessment of platelet dysfunction. Thromb Haemost 1998: 24: 195–202.
35. Posan E, McBane RD, Grill DE, Motsko CL, Nichols WL. Comparison of PFA–100 testing and bleeding time for detecting platelet hypofunction and von Willebrand disease in clinical practice. Thromb Haemost 2003; 90: 483–90.
36. Buyukasik Y, Karakus S, Goker H, et al. Rational use of the PFA–100 device for screening of platelet function disorders and von Willebrand disease. Blood Coagul Fibrin 2002;13: 1–5.
37. Harrison P, Robinson MSC, Mackie IJ, et al. Performance of the platelet function analyser PFA–100 in testing abnormalities of primary haemostasis. Blood Coagul Fibrin 1999; 10: 25–31.
38. Sourav KK, Eric JH, Reynaldo S, Carmen G, Rosa MD, Roy AO. Description of an in vitro Platelet Function Analyzer - PFA–100. Thromb Haemost 1995; 21: 106–12.
39. Walter AW, Katharina MG, Sacha SZ, Bernhard L. Evaluation of a platelet function analyser (PFA–100) in patients with a bleeding tendency. SwissMed Wkly 2002; 132:443–8.
40. Andersen K, Hurlen M, Arnesen H, Seljeflot I. Aspirin nonresponsiveness as measured by PFA–100 in patients with coronary artery disease. Thromb Res 2003; 108: 37–42.
41. Watala C, Golanski J, Pluta J, et al. Reduced sensitivity of platelets from type 2 diabetic patients to acetylsalicylic acid (aspirin)--- its relation to metabolic control. Thromb Res 2004; 113: 101–13.
42. Meng J, Casey PJ. Activation of Gz attenuates Rap1-mediated differentiation of PC12 cells. J Biol Chem 2002; 277: 43417–24.