Trombositlerde glukoz bağlayıcı proteinin eldesi, kinetiği, saflaştırılması ve defibrotidin normal ve aterosklerozlu olgularda glukoz transportuna in vivo etkisi

Abstract

65 VI.OZET Normal ve aterosklerozlu kişilerde glukoz transportu ortamda 10 -s mol/1 glukoz konsantrasyonunda takip edildi. Normal şahısların trom- bositlerinde 20 dakikalık glukoz birikimi 1,8 ± 0,40 nmol glukoz/109 trom- bosit (n = 8) iken, aterosklerozlu şahısların trombositlerinde 0,57 ± 0,54 nmol glukoz/109 trombosit, +4°C de ise 0,26 ± 0,09 nmol glukoz/109 trom- bosit olarak bulundu (n = 42). Trombositlerin ozmotik şok sıvısında glukoz bağlayıcı protein aktivitesi normal şahıslarda 53,2 nmol glukoz/mg protein, triton X-100 le muameleden sonra ise 13,6 nmol glukoz/mg protein olarak saptandı. Bağla yıcı protein (NH4)2S04 presipitasyonu, Sephadeks G-25 kolon kromatogra- fisi ve izokratik HPLC ile 3-3,5 kez saflaştırıldı ve molekül ağırlığı SDS-- PAGE ile 27,000 Da, HPLC ile 25,000 Da olarak tespit edildi. Glukoz bağ layıcı proteinin Km değeri 7,5 x 10 ~5 mol/1, Vmax değeri ise 12,5 nmol/g- lukoz/mg protein olarak bulundu. Antitrombotik bir ilaç olan defibrotid (i.v. 600 mg) aterosklerozlu şahıslarda 20 dakikalık glukoz birikimini 0,57 ± 0,54 nmol glukoz/109 trombositten 2 saat sonra 0,77 ± 0,67 nmol glu koz/109 trombosite yükselttiği görüldü (n = 33). Normal vakalarda ise defib rotid uygulamasından önce ve sonra 1,8 ± 0,4 nmol glukoz/109 trombosit olarak saptandı (n = 8). 15 gün süre ile hergün defibrotid uygulaması sonucunda ise 2066 dakikalık glukoz birikimi önce; 0,30 ± 0,27 nmol glukoz/109 trombosit, 2 saat sonra 0,41 ± 0,35 nmol glukoz/109 trombosit, 15. gün sonunda ise 0,55 ± 0,47 nmol glukoz/109 trombosit olarak bulundu (n = 9). In vivo defibrotid uygulamasından sonra 66,000 Da molekül ağır lığında bir proteinin solubilize trombosit membran fonksiyonunda ortaya çıktığı görüldü.

67 VII. SUMMARY Glucose transport in normal and atherosclerotic subjects was followed at 10-5 mol/1 glucose concentration by measuring the time dependent 14C glucose accumulation. Glucose accumulation in normal subjects was found as 1.8 ± 0.40 nmol glucose per 109 platelets per 20 minutes at 37°C (n = 8). It was found as 0.26 ± 0.09 nmol glucose per 109 platelets at +4°C. In atherosclerotic patients glucose accumulation was 0.57 ± 0.54 nmol glucose per 109 platelets both at 37°C and +4°C (n = 42). In the osmotic shock fluid of platelets, glucose binding protein activity was found as 53.2 nmol glucose per mg protein in normal subjects and the platelets where membrane solubilization was done with triton X 100 had 13.6 nmol glucose per mg protein of binding activity. Binding protein was purified 3 to 3.5 times with (NH4)2S04 precipitation, Sephadex G-25 column chromatography and isocratic HPLC. Its molecular weight (Mr) was determined as 27.000 Da through SDS-PAGE and 25.000 Da through HPLC. Km value of glucose binding protein was found as 7.5 x 10 _5 mol/1 and Vmax value as 1.25 nmol glucose per mg protein. After 2 hours of defibrotide administration (600 mg i.v.) the glucose accumulation was increased from 0.57 ± 0.54 nmol glucose per 10968 platelets for 20 minutes accumulation to 0.77 ± 0.67 nmol glucose per 109 platelets for 20 minutes accumulation in atherosclerotic patients (n = 33). In normal cases it has been determined as 1.8 ± 0.4 nmol glucose per 109 platelets for 20 minutes accumulation both before and after the administration of defibrotide. According to the results of daily administration of defibrotide for 15 days, glucose transport values have been found as 0.30 ± 0.27 nmol glucose per 109 platelets for 20 minutes accumulation before the administration, 0.41 ± 0.35 nmol glucose per 109 platelets for 20 minutes accumulation after 2 hours of administration and 0.55 ± 0.47 nmol glucose per 109 platelets for 20 minutes accumulation at the end of 15 days of administration (n = 9). Upon in vivo defibrotide administration, a protein with 66,000 Da molecular weight was detected in the solubilized membrane fraction.