원저 J Korean Neurol Assoc / Volume 24 / June, 2006 급성뇌경색환자에서항혈소판제투여후 PAC-1 과 P-selectin 발현의변화 순천향대학교의과대학신경과학교실, 내과학교실 a 박선아김은영 a 하성래박정호이태경성기범박춘식 a The Change of PAC-1 and P-selectin Expression after Initiation of Antiplatelets in Patients with Acute Ischemic Stroke Sun Ah Park, M.D., Eun Young Kim, B.S. a, Sung Rye Ha, M.D., Jeong Ho Park, M.D., Tae Kyeong Lee, M.D., Ki Bum Sung, M.D., Choon Sik Park, M.D. a Departments of Neurology and Internal Medicine a, Soonchunhyang University College of Medicine, Bucheon, Korea Background: The purpose of this study was to evaluate the time course of PAC-1 and the P-selectin expression after the initiation of antiplatelets in acute ischemic stroke. Methods: We serially measured PAC-1 and the P-selectin expression level using flow cytometry immediately before and at: one day, seven days, and one month after starting antiplatelets in 25 patients with acute cerebral infarctions. Results: The P-selectin expression increased initially (p<0.05) and then from the seventh day, it began to decrease continually (post-hoc analysis, p<0.05). However, the PAC-1 expression did not reveal significant alterations during the follow-up period of one month. Conclusions: Although the P-selectin expression rapidly declined, the PAC-1 expression remained elevated for one month. Our results suggest that the P-selectin may be useful in monitoring platelet inhibition during the early period after cerebral infarction. J Korean Neurol Assoc 24(3):210-214, 2006 Key Words: Antiplatelets, Cerebral infarction, Flowcytometry, Platelets 서 론 항혈소판제는뇌경색재발방지를위한중요한치료제이다. 1 그러나그효능은제한적이어서이차예방을위해항혈소판제를지속적으로복용하더라도 7-8% 의환자가뇌경색이재발한 Received December 21, 2005 Accepted January 16, 2006 *Sun Ah Park, M.D. Department of Neurology, Soonchunhyang University College of Medicine 1174 Jung-dong, Wonmi-gu, Bucheon, Gyeonggi-do, 420-767, Korea Tel: +82-32-621-5221 Fax: +82-32-621-5018 E-mail: sapark@schbc.ac.kr * 이연구는 2005 년도순천향대학교학술연구비지원에의해수행되었습니다. 다. 2-5 항혈소판제의주된작용기전이혈소판활성화억제인점을고려해볼때이에대한주기적인평가를통해약물효과를감시하는것은재발방지를위한효과적인치료전략개발에도움이되리라생각한다. 가장보편적으로이용하는혈소판활성화검사법인응집측정기 (aggregometer) 는전혈이아닌혈소판풍부혈장을이용하기때문에검사과정에서인위적으로혈소판을활성화시키는것이심각한단점이다. 6 따라서이를보완한검사법의개발과임상적활용의필요성이지속적으로대두되었다. 혈소판활성표지인자의발현을측정하는 flowcytometry 는전혈을빠른시간내에고정시킨후활성도를측정하여혈소판의인위적활성화를최소화하기에, 응집측정기를대체할검사법으로그가능성이제시되었다. 6,7 그러나, 지금까지뇌경색환자를대상으로한 210
급성뇌경색환자에서항혈소판제투여후 PAC-1 과 P-selectin 발현의변화 연구는충분히이루어지지않아, 항혈소판제투여시작후혈소판활성표지인자의연속적반응에대한연구조차드물다. 8-11 저자들은항혈소판제를새로이투여받게된급성뇌경색환자를대상으로뇌경색의재발이가장흔한시기인발병후한달동안 12,13 대표적인혈소판활성표지인자인 PAC-1 및 P- selectin 을이용한 flowcytometry 를실시하여이들의발현변화를연속으로추적하였고, 향후이검사법의임상적활용을위한기초자료를얻고자하였다. 대상과방법 1. 대상 2005 년 4월부터 2005 년 7월까지급성뇌경색으로진단받고새로이항혈소판제를투여한환자중한달간뇌경색재발없이정해진계획에따라혈소판의 PAC-1 과 P-selectin 의발현을측정한 25명을대상으로하였다. 고혈압은 19명, 당뇨병은 4명, 그리고고지혈증은 6명에게있었다. Trial of Org 10172 in Acute Stroke Treatment (TOAST) 분류 14 에따라대혈관동맥경화성 (large artery atherosclerosis) 뇌경색 8명, 소혈관폐색성 (small vessel occlusion) 뇌경색 11명, 그리고기타및분류되지않는뇌경색이 6명이었다. 심인성색전이원인으로생각되어항응고제를투여한환자는대상에서제외하였다. 아스피린과클로피도그렐병용투여는 12명, 아스피린은 8명, 클로피도그렐은 2명, 그리고아스피린과티클로피딘병용투여는 3명 이었다. 대조군은신경과에입원한환자중뇌혈관질환병력이없으며항혈소판제를투여하지않은 10명이었고, 편두통 4명, 양성체위성현훈 3명, 간질 2명, 파킨슨병 1명이었다. 2. 방법대상환자는치료시작전, 치료후 1일, 7일, 그리고한달의혈소판활성도를측정하였다. 혈액은 3.8% sodium citrate tube 에 vacutaine 을이용하여채취하였다. 측정하고자하는혈소판활성인자각각에대해채취한전혈 50 µl에 Tris buffer 450 µl 를넣어희석한검체를준비하여별도로실험을진행하였다. 그후형광물질인 FITC 와결합된 PAC-1 (Pharmingen, USA) 이나 phycoerythrin (PE) 과결합된항 P-selectin 항체 (Pharmingen, USA) 와 20분간반응시켰고, 400 µl의 2% paraformaldehyde 를이용하여고정한후 flowcytometry (Becton Dickinson FACScan flowcytometer, USA) 를이용하여전혈중혈소판표면에 PAC-1 또는 P-selectin 이양성인부분을측정하였다. 대조군으로 PE와결합된 CD41a 를이용하였다. PAC-1 에대해서는 mean fluorescence intensity 값을얻었고, P-selectin 에대해서는정상혈소판의 98-99% 를넘는 fluorescence intensity를가지는 percent positivity 값을얻어분석하였다 (Fig. 1). Figure 1. Flowcytometry analysis for the surface expression of PAC-1 and P-selectin on platelets. One-dimensional histograms show the fluorescence intensity of FITC-conjugated PAC-1 (A) or phycoerythrin-conjugated P-selectin (B) on the x-axis, and number of cells on the y-axis. Mean fluorescence intensity or percent positivity above 98% was used to express the extent of PAC-1 or P-selectin expression, respectively. 211
박선아김은영하성래박정호이태경성기범박춘식 Table 1. The expression of PAC-1 and P-selectin in controls and in the patients with acute cerebral infarction Normal Baseline 1 day 7 day 1 month PAC-1 (MFI) P-selectin (%) 1.8±1.2 31±8 2.3±2.7 2.1±1.9 39±13 a 33±14 1.7±0.9 30±13 2.9±2.2 a 26±7 a Values are mean±sd. MFI; mean fluorescence intensity, a p<0.05 vs. control by t-test Table 2. The expression of PAC-1 and P-selectin depending on TOAST classification Baseline 1 day 7 day 1 month PAC-1 (MFI) LAA SVO UC 3.4±4.2 1.6±1.6 2.3±2.7 2.6±2.6 1.7±1.5 2.1±1.4 1.9±1.3 1.5±0.7 1.5±0.5 2.6±1.4 3.4±2.9 2.4±1.2 P-selectin (%) LAA SVO UC 46±14 37±15 34±8 41±16 27±12 34±8 27±14 28±14 37±5 25±8 25±5 29±10 Values are mean±sd. TOAST; Trial of Org 10172 in Acute Stroke Treatment, MFI; mean fluorescence intensity, LAA; large artery atherosclerosis, SVO; small vessel occlusion, UC; stroke caused by other or undetermined etiology, ; not significant by Kruskal-Wallis test 3. 통계 두연속변수에대한비교는 t-test 를이용하였고, 3개이상의연속변수에대한비교는 ANOVA 와 Kruskal-Wallis test 를이용하였다. 사후검정으로 Tukey-B test 를이용하였다. 결 과 1. PAC-1 의발현 1) 대조군과의비교 (Table 1) 25명대상환자의 PAC-1 값은약물치료전, 약물치료후 1일, 7일까지대조군에비해유의한차이가없었고 (p>0.05), 한달후에는대조군에비해증가하였다 (p<0.05). 2) 항혈소판제투여후 PAC-1 발현의연속적변화 (Fig. 2) Figure 2. Expression of PAC-1 on circulation platelets in patients immediately before and at various days after starting antiplatelets. There is no significant change over time by ANOVA. 치료시작전, 치료후 1 일, 7 일, 그리고한달을측정시점으 로잡았을때 PAC-1 의발현은시간경과에따라 7일째까지는감소하였고, 한달후에는오히려증가하였으나, 이같은변화는통계적으로의미가없었다 (p>0.05). 3) 뇌경색아형에따른발현 (Table 2) 측정한모든시점에서 TOAST 분류에따라구분된세군사이에 PAC-1 발현에는차이가없었다. 2. P-selectin 의발현 1) 대조군과의비교 (Table 1) P-selectin 값은치료전에는환자군이유의하게높았다가 (p<0.05), 항혈소판제를투여하면서점차감소하여투여한달후에는정상인에비해서오히려낮았다 (p<0.05). 2) 항혈소판제투여후 P-selectin 발현의연속적변화 (Fig. 3) 212
급성뇌경색환자에서항혈소판제투여후 PAC-1 과 P-selectin 발현의변화 Figure 3. Expression of P-selectin on circulating platelets in patients immediately before and at various days after starting antiplatelets. The significant changes are noted over time (p<0.05 by ANOVA). Post-Hoc analysis revealed that the significant difference was present from the seventh day ( a p<0.05 by Tukey-B test). P-selectin 발현은투약후시간경과에따라의미있게감소하였는데 (p<0.05), 사후검정을해보면투약전과투약후 1 일사이는의미있는차이가없었고, 투약 1주일후부터이전에비해유의한차이를보이면서감소하였다 (p<0.05). 3) 뇌경색아형에따른발현 (Table 2) 측정한모든시점에서 TOAST 분류에따라구분된세군사이에 P-selectin 발현에는차이가없었다. 고 찰 Glycoprotein IIb/IIIa는자극이없는비활성화상태의혈소판에서는피브리노겐에대해약한친화성을보이지만활성화시키는여러자극이오면구조가변하면서피브리노겐에대한친화성이커져혈소판을강하게부착시키고주위의혈소판들과의응집을크게증가시키는등혈소판응집에있어서핵심적인역할을한다. 15 본연구에서이용한 PAC-1 은혈소판세포막에존재하는활성화형태의 glycoprotein IIb/IIIa를인식하는특이항체이다. P-selectin 은비활성화상태에서는혈소판내부의 alpha 과립에모여있다가혈소판이활성화되면혈소판표면으로이동하여염증세포표면에위치한 P-selectin glycoprotein-1 과결합, interleukin-8 과같은강력한백혈구동원인자들을분비시켜혈관내염증반응을악화시킨다. 15 이두인자는기존연구에서 flowcytometry 를이용한혈소판의활성화 정도를평가함에있어그지표로서이용되어왔다. 8-10,16,17 저자들은급성뇌경색환자에게항혈소판제투여시작후 PAC-1 과 P-selectin 발현의연속적변화를한달동안관찰하였다. 항혈소판제투여직전의 PAC-1 발현은정상군에비해서증가되었으나통계적으로유의한수준에는이르지않았고, P-selectin 발현은통계적으로유의한수준으로증가하였다 (p <0.05). 이는뇌경색급성기에혈소판의활성화가정상인에비해서증가한다는기존의보고와일치한다. 9,10 항혈소판제복용후시간경과에따라두표지인자는서로다른반응을보여, PAC-1 발현은투여한달후까지지속적으로증가한반면, P-selectin 발현은 1주일경과시점부터의미있게감소하였다. 새로이항혈소판제를투여한급성뇌경색환자에서혈소판의활성도를 1일, 14일, 그리고 90일에비교분석한기존연구에서 P-selectin 발현은이번연구결과와유사하게 2주후부터의미있게감소하였다. 10 그러나, 수개월간이미항혈소판제를복용하였던뇌경색환자를대상으로한달간추적관찰한연구에서 P-selectin 발현은수개월이후더이상의미있는변화가없었다. 8,18 따라서 P-selectin 발현은항혈소판제투여시작후 1-2 주내에감소하고, 그후에는감소된상태를유지하는것으로생각하며, 혈소판활성화억제효과의초기반응을보는데적합한표지인자로생각한다. 그러나이번연구결과는대혈관동맥경화성뇌경색환자만을대상으로 P- selectin 발현을 90일까지추적관찰하여이의지속적인증가를보고한연구와다르다. 9 그이유로이번연구에는대혈관동맥경화성외에도소혈관폐색이나명확히분류할수없는원인에의한뇌경색환자를포함했던점을생각할수있다. 즉, P- selectin 발현은소혈관폐색이나심인성색전에의한뇌경색에서는항혈소판제에의해감소하나대혈관동맥경화성뇌경색에서는지속적으로증가하였다는횡단면분석 (cross-sectional analysis) 보고 17 를고려하면, 이번연구에서대혈관동맥경화성뇌경색이 8명으로적어 P-selectin 의감소효과가현저하게나타났을가능성이있다. 대상환자를 TOAST 분류에따라세군으로나누어 P-selectin 발현을모든측정시점에서비교해보면세군사이에의미있는차이는없었다. 그러나대상환자수가각군에따라충분하지않았기에항혈소판제투여후 P-selectin 발현의빠른감소가뇌경색아형에따라차이가있을지여부는더많은환자를포함한추가연구로규명해야하겠다. PAC-1 의경우는급성뇌경색환자를대상으로한추적연구가없기에이번연구결과와직접비교는할수없으나, 수개월간뇌경색재발방지를위해이미항혈소판제를복용한환자를대상으로한달동안추적관찰한연구에서 PAC-1 발현은수개 213
박선아김은영하성래박정호이태경성기범박춘식 월후에도의미있게감소한경우가있다. 8 따라서급성뇌경색후한달이내에 PAC-1 이의미있게감소하지않았던이번연구결과와기존의연구결과를종합해볼때 PAC-1 발현은항혈소판제에대해수개월에걸쳐천천히반응한것으로생각한다. 그러나, 관상동맥스텐트삽입으로인한항혈소판제투여시작후한달동안추적검사한연구에서, PAC-1 발현은관상동맥질환에서약물시작 24시간후부터의미있게감소하였다. 16 그러므로항혈소판제투여에대한 PAC-1 발현의느린감소가급성뇌경색환자에서지속적으로관찰되는현상인지, 그렇다면그의미가무엇인지를알기위해서는보다많은환자를대상으로한추가연구가필요할것으로생각한다. 결론적으로, P-selectin 발현은한달동안의미있게감소하여뇌경색재발률이가장높은이시기에투여중인항혈소판제의혈소판활성화억제효과를감시하는데유용한도구가될것으로생각한다. 반면 PAC-1 은한달동안의미있는감소가없었는데이의증가지속시점및그의미를알기위해서는더많은수의환자를대상으로한달이상의장기관찰을하는추가연구가필요할것으로생각한다. REFERENCES 1. Antiplatelet trialists collaboration. Secondary prevention of vascular disease by prolonged antiplatelet treatment. Br Med J 1988;296:320-331. 2. CAPRIE Steering Committee. A randomized, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). Lancet 1996;348:1329-1339. 3. Jung B, Yoon OY, Park KH, Lee KY, Lee YJ, Kim HT, et al. Analysis of risk factors for recurrent ischemic stroke: Based on data of outpatient clinic in an university hospital. J Korean Neurol Assoc 2004;22:598-603. 4. Diener HC, Bogousslavsky J, Brass LM, Cimminiello C, Csiba L, Kaste M, et al. Aspirin and clopidogrel compared with clopidogrel alone after recent ischaemic stroke or transient ischaemic attack in high-risk patients (MATCH): randomised, double-blind, placebocontrolled trial. Lancet 2004;364;331-337 5. Shin DH, Bang OY. Mechanisms of recurrence in subtypes of ischemic stroke: A hospital-based follow-up study. J Korean Neurol Assoc 2005;23:158-164. 6. Michelson AD. Flow cytometry: a clinical test of platelet function. Blood 1996;87:4925-4936. 7. Harrison P. Progress in the assessment of platelet function. Br J Haematol 2000;111:733-744. 8. Serebruany VL, Malinin AI, Ziai W, Pokov AN, Bhatt DL, Alberts MJ, et al. Effects of clopidogrel and aspirin in combination versus aspirin alone on platelet activation and major receptor expression in patients after recent ischemic stroke. For the Plavix use for treatment of stroke (PLUTO-Stroke) trial. Stroke 2005;36; 2289-2292. 9. Cha JK, Jo WS, Shin HC, Bae HR, Ho JM, Kim JW. Increased platelet CD63 and P-selectin expression persist in atherosclerotic ischemic stroke. Platelets 2004;15:3-7. 10. Marquardt L, Ruf A, Mansmann U, Winter R, Schuler M, Buggle F, et al. Course of platelet activation markers after ischemic stroke. Stroke 2002;33:2570-2574. 11. Park MJ, Oh YM, Park KW, Cha JK, Kim SH, Kim JW. The serial change of platelet activation for 90 days in patients with atherosclerotic ischemic stroke. J Korean Neurol Assoc 2003;21:334-338. 12. Lovett JK, Coull AJ, Rothwell PM. Early risk of recurrence by subtype of ischemic stroke in population-based incidence studies. Neurology 2004;62:569-573. 13. Coull BM, Williams LS, Goldstein LB, Meschia JF, Heitzman D, Chaturvedi S, et al., Anticoagulants and antiplatelet agents in acute ischemic stroke: report of the Joint Stroke Guideline Development Committee of the American Academy of Neurology and the American Stroke Association (a division of the American Heart Association). Stroke 2002;33:1934-1942. 14. Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL,et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in acute stroke treatment. Stroke 1993;24: 35-41. 15. Parise LV, Smyth SS, Coller BS. Platelet morphology, biochemistry, and function. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U. Williams Hematology. 6th ed. New York: McGraw- Hill. 2001;1357-1408. 16. Grubel PA, Biliden KP, Hiatt BL, O Connor CM. Clopidogrel for coronary stenting. Response variability, drug resistance and the effect of pretreatment platelet reactivity. Circulation 2003;107: 2908-2913. 17. Yamazaki M, Uchiyama S, Iwata M. Measurement of platelet fibrinogen binding and p-selectin expression by flow cytometry in patients with cerebral infarction. Thromb Res 2001;104:197-205. 18. Grau AJ, Reiners S, Lichy C, Buggle F, Ruf A. Platelet function under aspirin, clopidogrel, and both after ischemic stroke. A case-crossover study. Stroke 2003;34:849-855. 214