( )Kjbt023.hwp

Size: px

Start display at page:

Download "( )Kjbt023.hwp"

지오 갈
6 years ago
Views:

1 대한수혈학회지 : 제 18 권제 3 호, 2007 혈소판제제의냉장보존에있어서마그네슘의유효성 조용곤 1 ㆍ이재현ㆍ김정태ㆍ김달식 1 ㆍ이혜수 1 ㆍ최삼임 1 = Abstract = 전북대학교의학전문대학원진단검사의학교실, 전북대학교임상의학연구소 1 The Effect of Magnesium Sulfate on Platelets Preserved at Cold Temperature Yong Gon Cho 1, Jae Hyeon Lee, Jeong Tae Kim, Dal Sik Kim 1, Hye Soo Lee 1, Sam Im Choi 1 Department of Laboratory Medicine, Chonbuk National University Medical School, Research Institute of Clinical Medicine 1, Chonbuk National University, Jeonju, Korea Background: It is known that magnesium can inhibit platelet function in vitro and ADP-induced platelet activation. We wished to demonstrate if magnesium could inhibit activation of platelets preserved at cold temperature. Methods: We incubated each mixture of platelets and various concentrations of magnesium sulfate (0 10 mm) at room temperature and at a cold temperature (4 o C). On days 1, 3, 5, 7 and 9, we measured the platelet count, ph, LD level, glucose level, HCO 3 level, lactate level, expression of CD62P, expression of annexin V, and ADP-induced aggregation of platelets. Results: The platelets stored at 4 o C showed a similar LD level but a lower platelet count, lactate level, and expression of CD62P and annexin V, and a higher ph and glucose level than platelets stored at room temperature. With an increasing magnesium concentration, expression of CD62P in the platelets stored at 4 o C was slightly decreased, but expression of annexin V was increased. ADP-induced aggregation of the platelets stored at 4 o C was not affected by magnesium. Conclusion: Magnesium did not effectively inhibit activation of platelets during preservation at cold temperature (4 o C). (Korean J Blood Transfus 2007;18: ) Key words: Magnesium, Platelets, Preservation 접수일 :2007년 12월 9일, 승인일 :2007년 12월 17일책임저자 : 조용곤 전북전주시덕진구금암2동 전북대학교의학전문대학원진단검사의학교실 TEL: 063) , FAX: 063) , choyg@chonbuk.ac.kr

2 혈소판제제의냉장보존에있어서마그네슘의유효성 서론 을알아보고자하였다. 농축혈소판은헌혈자로부터채혈된이후 o C의온도에서지속적으로천천히흔들어주는상태 ( 분당 60 80회정도 ) 로 5일동안보존유효하지만, 적혈구제제에비하여매우짧은보존유효기간으로인하여여유분을비축하기어렵고유효기간경과로폐기되는양도상당하여문제가되고있다. 혈소판제제의장기간보관은헌혈혈액의부족이나응급상황과같이혈소판제제의준비가어려운상황에서이를해결하기위한방법중의하나이다. 혈소판을장기간보존하기위해냉동처리하는경우에는형태변화가발생할뿐아니라, 혈관내피세포층 (endothelial cell layer) 에대한부착능이신선한혈소판에비해 40% 까지감소한다고알려져있다. 1) 또한, 혈소판표면수용체의재배치로인해수혈후대식세포에의해쉽게제거된다는것이발견되었다. 2) 이러한제한점을극복하기위해서혈소판표면수용체를당화시키거나, 활성화를차단하는물질을개발하는노력이시도되었다. 3-6) 최근, 마그네슘이혈소판의부착과응집을차단할수있음이보고되었다. 7-12) 조기진통을호소하는산모를대상으로마그네슘을투여한결과, 출혈시간 (bleeding time) 이유의하게연장되는것이관찰되었고, 7) 마그네슘이 ADP에의해유도되는혈소판활성화를억제한다는결과도여러연구자들에의해발표되었다. 7,8,10-12) 마그네슘이혈소판에작용하는기전은아직확립되지않았지만, 직접작용하거나, 13) 혹은혈관내피세포를자극하여일산화질소를분비하게함으로써간접적으로작용한다는주장도있다. 14) 이에저자들은마그네슘이보존혈소판의활성화에영향을주는지, 특히냉장온도에서어떤영향을주는지살펴보고혈소판장기보존을위한물질로서의가능성 재료및방법 1. 농축혈소판의준비와혈소판수및관련지표의측정 혈소판수가 /μl 이상인 5명의건강한성인헌혈자로부터혈소판성분채집기 (Amicus, Baxter) 를통해성분채집혈소판 ( 약 250 ml) 을얻고, 20 ml씩소분한후, 황산마그네슘 (MgSO 4 ) 을최종농도가 0 mm, 2 mm, 4 mm, 8 mm 그리고 10 mm 이되도록각각의혈소판에첨가하고, 실온과냉장보관으로나누어보관전과보관후 1일, 3일, 5일, 7일, 9일에혈소판수, glucose, ph, HCO 3, lactate를측정하였다. 혈소판수는자동혈구계산기인 Sysmex XE 2100 (TOA Medical Electronics, Kobe, Japan) 를이용하여측정하였고, 혈액가스분석기인 GemPremier 3000 (Instrumentation Laboratory, Lexington, MA) 을이용하여 ph, HCO 3, lactate, glucose를측정하였다. 2. 유세포분석 보관과정에서혈소판활성화여부를알기위해 FACSCalibur (Becton Dickinson, San Jose, CA) 를이용하여혈소판의 CD62P (P-selectin) 의발현측정은 anti-cd62p-pe (BD, San Jose, CA) 를이용하였고, phosphatidylserine의발현은 anti-annexin V-FITC (BD Pharmingen, San Diego, CA) 를이용하여측정하였다. 각검체는 phosphate-buffered saline (PBS) 로희석하여 가포함된 100μL 부유액에 anti-cd62p-pe 5μL를첨가하여빛을차단한상태에서실온 30분간방치하고, PBS로세척후 400μL가되게하였다. Anti-annexin V- FITC는실온 15분간반응시킨후, CaCl 2 가들어

3 대한수혈학회지 : 제 18 권제 3 호 있는 annexin V buffer 희석액을 400μL 넣어부유액을만들었다. 염색된각각의검체는유세포분석기에서 10,000개의세포를분석하였다. 매분석시에 IgG-Cy5 isotype control과 IgG-FITC isotype control을사용하여음성대조를확인하였다. 3. 혈소판응집능검사각검체를 4,000 rpm에서 10분간원심분리하여 platelet-poor plasma (PPP) 500μL를얻고, 나머지검체는 platelet-rich plasma (PRP) 로사용하였다. PPP와 PRP 500μL를이용하여 Chronolog 490-2A (Chrono-log Co., Havertown, PA) 기기의기저치를 설정하였고, PRP 500μL에 adenosine 5'-diphosphate (ADP) 20μL를추가하여응집반응을 5분동안관찰하고, ADP 투여후최대반응을나타낸 Amax 결과를비교하였다. 결과 1. 마그네슘유무에따른냉장보관혈소판지표의변화 냉장보관된혈소판의평균혈소판수는 771.7± /L로실온보관혈소판의 ±218.2 Fig. 1. Comparison of results derived from cold platelets treated with between magnesium and no magnesium. (A) Platelet count, (B) LD, and (C) ph

4 혈소판제제의냉장보존에있어서마그네슘의유효성 Fig. 2. Results of flow cytometric analysis using anti-cd62p-pe (A), anti-annexin V-FITC (B) /L보다매우낮았고, lactate 농도는 6.2±3.0 mg/dl로 13.8±2.5 mg/dl에비해매우낮았다. ph 는 7.1±0.1 로 6.9±0.2 에비해높았고, glucose 도 311.4±37.5 mg/dl로 124.7±101.3 mg/dl에비해높았다. 냉장보관혈소판의 CD62P와 annexin V의발현은각각 28.6±11.2%, 62.6±8.7% 로, 실온보관혈소판의 55.0±26.6%, 84.0±14.1% 보다모두낮게나타났다. 마그네슘이첨가된냉장혈소판은혈소판수 686.8± /L, lactate 6.7±3.2 mg/dl, ph 7.1±0.1, glucose 304.1±40.8 mg/dl, CD62P 27.1± 10.9%, annexin V 65.8±9.4% 를보여, 마그네슘이첨가되지않은냉장혈소판과모든지표가비슷한수치를나타내었다 (Fig. 1). 2. 마그네슘의농도에따른혈소판활성화지표의변화 마그네슘의농도를 0 10 mm까지차이를두었을때, 농도가증가함에따라냉장혈소판의 CD62P의발현은 28.6±11.2%, 26.9±10.7%, 27.3± 12.3%, 26.7±10.8%, 27.5±10.6% 로거의차이가없었으나, annexin V의발현은 62.6±8.7%, 63.4± 9.4%, 65.4±9.8%, 66.4±9.1%, 68.2±9.3% 로농도가 Fig. 3. Time- and Mg 2+ concentration-dependent changes of ADP-induced aggregation in cold temperature of platelet. 증가함에따라비례적으로증가하는소견을보였고, 보관일수에따라비교해도동일한결과를보였다 (Fig. 2). 3. 혈소판응집능의유지 마그네슘농도에따른냉장혈소판검체에대해 ADP 처리후 Amax 결과를비교하였다. Amax 는

5 대한수혈학회지 : 제 18 권제 3 호 ADP를처리하기전빛의투과도를 0% 로가정하였을때 ADP 처리후빛이가장많이투과된 % 로정의하였다. 마그네슘을처리하지않은 37 o C 상태의혈소판에비해냉장혈소판의 Amax 값이현저히높았으며, 냉장상태에서는마그네슘농도의증가에일관되지않은 Amax 값을나타내었다 (Fig. 3). 고찰혈소판은생존및보존기간이짧고, 전혈의경우얻을수있는양이제한되어있고, 성분헌혈에의한경우, 비용이많이들어공급에많은어려움을겪는대표적인혈액제제이다. 혈소판기능이유지되고, 안전하게보존기간을연장하는새로운보관법이모색된다면혈소판제제의문제는근본적으로해결될수있다. 본연구에서다른보고자와같이냉장보관혈소판은실온보관혈소판에비해혈소판의대사산물인 lactate의농도는낮은반면, 혈소판수가급격히감소함을나타내었다. 마그네슘을첨가한혈소판과첨가하지않은냉장보관혈소판의수, LD, ph는유의한차이를보이지않았지만, 마그네슘의농도가증가함에따라혈소판활성지표인 CD62P의발현이약간감소하는경향을나타내었다. 이는마그네슘이냉장상태의혈소판보존시활성을억제할수있는가능성을나타낸다. 하지만, annexin V의발현은마그네슘농도와비례하여증가하는경향을나타내어혈소판활성여부가두지표사이에불일치소견을보였다. 혈소판활성화를나타내는지표로는 CD62P와혈소판표면수용체중의하나인 glycoprotein IIb/IIa에결합하는항체인 PAC-1, LIBS-1 등이사용되고있다. 이가운데 CD62P가혈소판활성화여부를좀더민감하게반영했다는보고를기초로생각해볼때, 6) 본결과는마그네슘이냉장온도에서보 존되는혈소판의활성화를일부억제할수있음을나타낸것으로해석할수있다. 그러나, 마그네슘농도에따른결과양상이뚜렷하지않아본실험결과로는이를확실하게말할수없고, annexin V의결과와도전혀일치하지않았기때문에마그네슘농도와혈소판활성화는상관관계가있다고생각되지않았다. 혈소판의기능을살펴보기위해시행한 ADP에의한 Amax 값의비교결과에서는마그네슘농도에따라혈소판의반응이일관되지않게나타났으며, 고농도의마그네슘을처리한검체에서 ADP- Amax 값이증가되었다. 일반적으로, 37 o C 상태의혈소판에비해냉장상태의혈소판에서 ADP- Amax 값이현저히크다고알려져있으며, 15) 이를기준으로할때 4 mm의마그네슘을처리한경우혈소판의응집능이좀더유지되었다고생각되었다. 결론적으로, 혈소판의냉장보존은혈소판수와기능에큰변화를주지만, 마그네슘의특정농도에서활성화에의한기능변화를약하게할수있음을확인하였다. 하지만, 그정도가뚜렷하지않아혈소판의안정적인냉장보존을위한마그네슘의처리는임상적인적용을하기에부적절하다고생각되었다. 요약배경 : 마그네슘은혈소판의기능을억제하며, ADP에의해유도되는혈소판활성화를억제한다고알려져있다. 저자들은냉장온도에서보관된혈소판의활성화를마그네슘이억제할수있는지평가하였다. 방법 : 황산마그네슘의최종농도가 0 mm, 2 mm, 4 mm, 8 mm, 그리고 10 mm이되도록첨가된혈소판혼합물을실온과냉장온도 (4 o C) 에보관하였다. 보관후 1, 3, 5, 7, 9일에각각의혈소판

6 혈소판제제의냉장보존에있어서마그네슘의유효성 수, ph, LD, glucose, HCO 3, lactate, CD62P, annexin V, ADP에의한응집반응을측정하였다. 결과 : 냉장보관혈소판은실온혈소판에비해혈소판수와 lactate 농도가매우낮았고, ph와 Glucose는높았으며, CD62P와 annexin V의발현은낮았으나, LD농도는비슷하였다. 마그네슘농도가증가함에따라냉장혈소판의 CD62P의발현은약간감소하였으나, annexin V의발현은오히려증가하였다. ADP에의한응집반응은마그네슘농도와무관하게나타났다. 결론 : 마그네슘은냉장보관혈소판의활성화에뚜렷한영향을주지못하므로, 혈소판의안정적인냉장보관을위한보존제로적용하기에는부적절하다고생각되었다. 참고문헌 1. Walker RH, ed. Technical manual. 11th ed. Arlington, VA: American Association of Blood Banks, 1990: Hoffmeister KM, Felbinger TW, Falet H, Denis CV, Bergmeier W, Mayadas TN, et al. The clearance mechanism of chilled blood platelets. Cell 2003;112: Kim BS, Park JH, Shin SW, Kim YH, Lee KN, Kim JS, et al. The evaluation of in-vitro functional recovery and transfusion effect at frozen thawed platelet concentrates by 6% dimethyl sulfoxide and storage at 80 o C. Korean J Hematol 1995;30: Park KH, Kim BS, Bae SY, Yoon SY, Sul HR, Choi JG, et al. Comparison of the activities of platelets with different cryopreservation methods. Korean J Hematol 2003;38: Hoffmeister KM, Josefsson EC, Isaac NA, Clausen H, Hartwig JH, Stossel TP. Glycosylation restores survival of chilled blood platelets. Science 2003;301: Schaller M, Hofmann A, Connor J, Nydegger UE. Filtered platelet concentrates from pooled buffy coats show comparable storage lesions when stored for 9 d at degree C or when supplemented with thrombosol at 2 6 degree C. Eur J Haematol 2000;64: Corsonello A, Malara A, De Domenico D, Damiano MC, Mirone S, Loddo S, et al. Effects of magnesium sulphate on leptin-dependent platelet aggregation: an ex vivo study. Magnes Res 2005;18: Gawaz M, Ott I, Reininger AJ, Neumann FJ. Effects of magnesium on platelet aggregation and adhesion. Magnesium modulates surface expression of glycoproteins on platelets in vitro and ex vivo. Thromb Haemost 1994;72: Fuentes A, Rojas A, Porter KB, Saviello G, O'Brien WF. The effect of magnesium sulfate on bleeding time in pregnancy. Am J Obstet Gynecol 1995;173: Leaphart WL, Meyer MC, Capeless EL, Tracy PB. Adenosine diphosphate-induced platelet activation inhibited by magnesium in a dosedependent manner. Obstet Gynecol 1998;91: Ravn HB, Vissinger H, Kristensen SD, Husted SE. Magnesium inhibits platelet activity - an in vitro study. Thromb Haemost 1996;76: Ravn HB, Kristensen SD, Vissinger H, Husted SE. Magnesium inhibits human platelets. Blood Coagul Fibrinolysis 1996;7: Hsiao G, Shen MY, Chou DS, Lin CH, Chen TF, Sheu JR. Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na+/H+ exchanger. J Biomed Sci 2004;11: Shankar PR, Reeta KH, Bhargava VK, Grover A. A possible role for nitric oxide in the antiaggregatory effect of magnesium. Indian J Pharmacol 2001;33: Breddin HK. Can platelet aggregometry be standardized? Platelets 2005;16:

γ

경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical