Ispitivanje endotelne disfunkcije i postojanja rezistencije na antitrombocitnu terapiju kod bolesnika sa tipom 2 dijabetes melitusa

Abstract

UVOD: Procesi koji obuhvataju endotelnu disfunkciju, oksidativni stres, hroničnu inflamaciju, hiperaktivnost i aktivaciju trombocita te narušavanje ravnoteže procesa koagulacije i fibrinolize od najranijih faza razvoja dijabetes melitusa tip 2 (T2DM) promovišu aterogenezu i nastanak aterotromboznih komplikacija. Kompleksan terapijski pristup u T2DM ima za cilj ne samo uspostavljanje glikoregulacije, korekciju brojnih metaboličkih poremećaja i modifikaciju pridruženih faktora rizika za nastanak ateroskleroze već i primenu antitrombocitne terapije u cilju primarne ili sekundarne prevencije aterotromboznih komplikacija. Uprkos primenjenoj antiagregacionoj terapiji, deo bolesnika doživi rekurentne aterotrombozne atake. Bolesnici sa T2DM se izdvajaju kao grupa sa posebnim rizikom za recidivantne aterotromboze što može biti uslovljeno rezistencijom na primenjenu antitrombocitnu terapiju. Praćenje efekata antitrombocitne terapije i blagovremeno identifikovanje rezistentnih bolesnika ima za cilj optimizaciju primenjene antitrombocitne terapije što može biti od izuzetnog kliničkog značaja u smislu sprečavanja progresije aterotromboznog procesa. CILJ: Proceniti i uporediti nivoe biomarkera, pokazatelja endotelne aktivacije, aktivacije i agregabilnosti trombocita u bolesnika sa bolešću arterijskih krvnih sudova u tipu 2 dijabetes melitusa u odnosu na njihove vrednosti u zdravoj populaciji. Uporediti efikasnost primenjene antitrombocitne terapije tienopiridinima u bolesnika sa tipom 2 dijabetes melitusa i bolešću arterijskih krvnih sudova u odnosu na efikasnost ove terapije u nedijabetičnoj populaciji bolesnika sa bolešću arterijskih krvnih sudova. MATERIJAL I METODE: U ispitivanje je uključeno 100 ispitanika oba pola, starosti od 33 do 70 godina života, kod kojih je prethodno utvrđeno postojanje neke od kliničkih manifestacija bolesti arterijskih krvnih sudova (IBS, CVB, PAB) koji kao antitrombocitnu terapiju uzimaju tienopiridinski preparat, klopidogrel. Od toga, 50 uključenih ispitanika imalo je dijagnozu dijabetes melitus tipa 2, a 50 su bili bolesnici bez dijabetesa. Kontrolnu grupu je činilo 30 klinički i biohemijski zdravih ispitanika, nepušača koji su prema polnoj i dobnoj strukturi odgovarali ispitivanim grupama bolesnika. Svim ispitanicima su urađena antropometrijska merenja, laboratorijska analiza uzoraka krvi na automatizovanim analizatorima sa određivanjem parametara metabolizma glukoze, lipida, parametera inflamacije, KKS, parmetara koagulacije i trombocitnih pokazatelja. Određivanje serumske koncentracije sE–selektina i sP-selektina je vršeno ELISA tehnikom (R&D Systems, Inc. Minneapolis, USA). Plazmatska koncentracija vWFAg-a određivana je imunoturbidimetrijskom metodom na koagulacionom analizatoru Siemens Healthcare Diagnostics, Nemačka. Agregabilnost trombocita je određivana impedantnom agregometrijom (Multiple Electrode Aggregometry - MEA) na Multiplate analizatoru, Dynabyte, Minhen, Nemačka. Bazalna agregabilnost trombocita procenjivana je TRAP testom, rezidualna agregabilnost trombocita pod terapijom klopidogrela ADP testom, rezidualna agregabilnost trombocita pod terapijom aspirina, ASPI testom. Individualni odgovor na primenjenu antiagregacionu terapiju je procenjivan i na osnovu procenta sniženja bazalne agregabilnosti trombocita (%SAT) nakon primenjene antiagregacione terapije što je izračunato sledećim formulama: procena antiagregacionog efekta klopidogrela:%SATadp =100 x (1-ADP/TRAP) i procena antiagregacionog efekta aspirina:%SATaspi =100 x (1-ASPI/TRAP). REZULTATI: Nivo sE-slektina je bio signifikantno viši u bolesnika sa T2DM u odnosu na bolesnike bez dijabetesa (45,1±18,1vs.31,8±10,5ng/ml; p<0,001) i kontrolnu grupu zdravih ispitanika (45,1±18,1vs.27,2±11,2ng/ml; p<0,001). Plazmatski nivo vWF Ag, bio je statistički značajno viši u bolesnika sa T2DM u odnosu na grupu ispitanika bez dijabetesa (172±75,2vs. 146±40,6%; p=0,045), kao i u odnosu na kontrolnu grupu zdravih (172±75,2vs.130±33,8%; p=0,007). Nivo sPselektina bio je statistički značajno viši kod bolesnika s T2DM u odnosu na ispitanike u grupi dijabetesa (95,2±31,8vs.84,0±21,8 ng/ml; p=0,042) i kontrolnoj grupi (95,2±31,8vs.76,7±16,2ng/ml; p=0,004). Uočeno je da je %rP statistički bio značajno viši u grupi dijabetičara u odnosu na grupu ispitanika bez dijabetesa (3,47±1,30vs.2,30±1,30%; p<0,001) i kontrolnu grupu zdravih (3,47±1,30vs.2,29±1,23%; p<0,001). Bolesnici sa T2DM imali su statistički značajno više vrednosti ADP testa (70,3±22,0vs.56,9±19,7U; p=0,002) u odnosu na bolesnike bez dijabetesa, a uočen je i značajno niži stepen procenta sniženja bazalne agregabilnosti, %SATadp, u dijabetičara u odnosu na ispitanike bez dijabetesa (31,6±12,4vs. 48,6±12,6 %; p<0,001). U grupi ispitanika sa T2DM vrednost TRAP testa statistički značajno pozitivno koreliše sa brojem neutrofila (r=0,349;p= 0,013) i NLR-om (r=0,472;p=0,001), a multivarijantnom linearnom regresionom analizom dokazana je nezavisna povezanost TRAP testa i fibrinogena (B=9,61;p=0,009). Takođe, u istoj ispitivanoj grupi postoji pozitivna povezanost ADP testa sa HOMAIR (r=0,319;p=0,024), NLR-om (r=0,515;p<0,001), hsCRP-om (r=0,356;p=0,011), kao i sa %rP (r=0,302;p=0,049). Multivarijantnom linearnom regresionom analizom dokazana je nezavisna povezanost ADP testa i ITM (B=1,43;p=0,043). %SATadp u bolesnika sa T2DM negativno je korelisao sa ITM (r= -0,381;p=0,006), OS (r= - 0,387;p=0,006), HOMA-IR (r= -0,349;p=0,013), hsCRP-om (r= -0,288; p=0,043), %rP (r= -0,302;p=0,049), sE-selektinom (r= -0,369; p=0,008) i sP-selektinom (r= - 0,374;p=0,007). U grupi dijabetičara, postoji pozitivna povezanost %rP sa ITM (r=0,365;p= 0,016), OS (r=0,435;p=0,004), HOMA-IR (r=0,409;p=0,006), hsCRP (r=0,374;p=0,014), sP-selektinom (r=0,341;p=0,025) i vWFAg-om (r=0,348;p=0,022). Takođe, sE-selektin pozitivno koreliše sa ITM (r=0,380;p =0,006), OS (r=0,380; p=0,007), HOMA-IR (r=0,339;p=0,016), hsCRP-om (r=0,351;p=0,013), a sP-selektin sa ITM (r=0,312;p=0,027), OS (r=0,395;p=0,005), HOMA-IR (r=0,286;p=0,044), hsCRP-om (r=0,369; p=0,008) i nivoom sE – selektina (r=0,560;p <0,001). Evaluirajući odgovor na terapiju klopidogrelom u podgrupama bolesnika sa dijabetesom, napravljenim prema kvartilnoj distribuciji nivoa ADP-a, tj. stepenu rezidualne agregabilnosti trombocita u toku terapije klopidogrelom, uočeno je da ukupna bazalna agregabilnost trombocita procenjena TRAP testom statistički značajno raste od prvog do četvrtog kvartila (76,50 ±19,91 vs. 94,54±16,67 vs. 112,00±10,22 vs. 128,92±15,69U;p<0,001), dok se %SATadp od prvog do četvrtog kvartila značajno smanjivao (40,44±13,33 vs. 31,20±11,82 vs. 33,16±7,03 vs. 21,53±10,16%). ZAKLJUČAK: Koncentracije cirkulišućih biomarkera endotelne aktivacije, sE – selektina i vWF Ag-a, solubilnog biomarkera trombocitne aktivacije, sP – selektina, kao i procenat retikulisanih trombocita, %rP, markera trombocitnog prometa, značajno su povišene kod bolesnika sa bolešću arterijskih krvnih sudova u tipu 2 dijabetes melitusa u odnosu na njihove koncentracije kod zdravih ispitanika i bolesnika bez dijabetesa. Bolesnici sa T2DM imali su znatno viši stepen rezistencije na antitrombocitnu terapiju klopidogrelom u odnosu na bolesnike bez dijabetesa, procenjene stepenom rezidualne agregabilnosti trombocita, ADP test, kao i procentom sniženja ukupne bazalne agregabilnosti trombocita, %SATadp, metodom impedantne agregometrije, a što je uslovilo i trend učestalijeg ponavljanja ishemijskih ataka u odnosu na bolesnike bez dijabetesa. Međusobna povezanost ispitivanih biomarkera endotelne i trombocitne aktivacije (sE – selektina, vWF Aga, sP – selektina), kao i markera prometa trombocita (%rP) sa metaboličko inflamatornim parametrima i sa indikatorima odgovora na antiagregacionu terapiju, može ukazivati na to da nepovoljan metabolički milje dijabetičara može biti jedan od doprinosnih faktora lošem odgovoru na antitrombocitnu terapiju klopidogrelom.

INTRODUCTION: Processes involving endothelial dysfunction, oxidative stress, chronic inflammation, platelet activation and the imbalance between coagulation and fibrinolysis promote atherogenesis and atherothrombotic complications at early stage of diabetes mellitus type 2 (T2DM). The complex therapeutic approach in T2DM aims not only to reestablish glycemic control and to correct a number of metabolic disorders, but also to achieve primary or secondary prevention of atherothrombotic complications. Despite the applied antiplatelet therapy, some patients experience recurrent atherothrombotic attacks. Patients with T2DM are the group at particular risk for recurrent atherothrombosis, which can be caused by antiplatelet therapy resistance. Monitoring the effectiveness of antiplatelet therapy and identification of resistant patients aims to optimize the applied antiplatelet therapy, which can be of great clinical significance in terms of preventing progression of atherotrombotic processes. AIM: Evaluate and compare the levels of biomarkers, indicators of endothelial activation, platelet activation and aggregability in patients with arterial vascular disease in type 2 diabetes mellitus compared to their values in a healthy population. Compare the effectiveness of applied antiplatelet therapy with thienopyridines in patients with type 2 diabetes mellitus and arterial vascular disease compared to the efficacy of this therapy in nondiabetic population of patients with arterial vascular disease. MATERIAL AND METHODS: The study included 100 patients, 33 to 70 years of age, with previously established existence of some of the clinical manifestations of arterial vascular disease (CAD, CVD, PAD), taking thienopyridine antiplatelet therapy with clopidogrel. 50 patients was previously diagnosed with diabetes mellitus type 2 and 50 were nondiabetic patients. Control group included 30 age and sex matched healthy participants, non-smokers. All subjects underwent anthropometric measurements and laboratory analysis of blood samples on automated analyzers with determining the parameters of glucose metabolism, lipids, inflammation parameters, complete blood count, coagulation and platelet parameters. Serum concentrations of sEselectin and sP-selectin were determined by ELISA (R&D Systems, Inc., Minneapolis, USA). vWFAg was determined by immunoturbidimetry on coagulometer Siemens Healthcare Diagnostics, Germany. Platelet aggregability was determined by impedance aggregometry (Multiple Electrode Aggregometry - MEA) on Multiplate analyzer, Dynabyte, Munich, Germany. Basal platelet aggregability was estimated by TRAP test, residual platelet aggregability during clopidogrel treatment was estimated by ADP test and during aspirin treatement by ASPI test. Individual response to antiplatelet therapy was estimated by the percentage of decrease in basal platelet aggregability (%DPA) obtained after antiplatelet therapy, calculated bypresented formulas: %DPAadp =100 x (1-ADP/TRAP)and %DPAaspi =100 x (1- ASPI/TRAP). RESULTS: Concentration of sE-selectin was significantly higher in patients with T2DM in order to non-diabetic patients (45,1±18,1vs.31,8±10,5ng/ml;p<0,001) and healthy control group (45,1±18,1vs.27,2±11,2ng/ml; p<0,001). vWF Ag was significantly higher in diabetic patients than in non-diabetics (172±75,2vs. 146±40,6%; p=0,045) and healthy controls (172±75,2vs.130±33,8%; p=0,007). sP-selectin was also significantly higher in patients with T2DM than in non-diabetics (95,2±31,8vs.84,0±21,8 ng/ml; p=0,042) and healthy controls (95,2±31,8vs.76,7±16,2ng/ml; p=0,004). %rP was significantly higher in group of patients with T2DM than in nondiabetic patients (3,47±1,30vs.2,30±1,30%; p<0,001) and healthy control group (3,47±1,30vs.2,29±1,23%; p<0,001). T2DM patients had statistically higher values of ADP test (70,3±22,0vs.56,9±19,7U; p=0,002) compared to patients without diabetes, and significantly lower %DPAadp (31,6±12,4vs. 48,6±12,6 %; p<0,001). In T2DM group of patients, level of TRAP test correlated positively with number of white blood cells (r=0,349;p= 0,013) and NLR (r=0,472;p=0,001), and multivariant linear regression analisys showed significant independent association of TRAP test with fibrinogen (B=9,61;p=0,009). Statistically significant positive correlation of ADP test with HOMA-IR (r=0,319;p=0,024), NLR (r=0,515;p<0,001), hsCRP (r=0,356;p=0,011) and %rP (r=0,302;p=0,049) was observed in patients with T2DM. Multivariant linear regression analisys showed significant independent association of ADP test with BMI (B=1,43;p=0,043). %DPAadp negatively correlated with BMI (r=-0,381;p=0,006), WC (r= - 0,387;p=0,006), HOMA-IR (r= -0,349;p=0,013), hsCRP (r= -0,288; p=0,043), %rP (r= -0,302;p=0,049), sE-selectin (r= -0,369; p=0,008) and sP-selectin (r= -0,374;p=0,007) in diabetic patients. Significant positive correlation of %rP with BMI (r=0,365;p= 0,016), WC (r=0,435;p=0,004), HOMA-IR (r=0,409;p=0,006), hsCRP (r=0,374;p=0,014), sP-selectin (r=0,341;p=0,025) and vWFAg (r=0,348;p=0,022) was found in diabetics. Also, sE-selectin positively correlated with BMI (r=0,380;p =0,006), WC (r=0,380; p=0,007), HOMA-IR (r=0,339;p=0,016), hsCRP(r=0,351;p=0,013), and sPselectin correlated positively with BMI (r=0,312;p=0,027), WC (r=0,395;p=0,005), HOMA-IR (r=0,286;p=0,044), hsCRP (r=0,369; p=0,008) and sE – selectin (r=0,560;p <0,001). Evaluating the response to clopidogrel therapy in subgrpoups of diabetic patients accoarding the quartile distribution of ADP test (clopidogrel on-treatment platelet reactivity), it is found that total basal aggregability estimated by TRAP test significantly increased from the first to the fourth quartile (76,50 ±19,91 vs. 94,54±16,67 vs. 112,00±10,22 vs. 128,92±15,69U;p<0,001) while %DPAadp decreased (40,44±13,33 vs. 31,20±11,82 vs. 33,16±7,03 vs. 21,53±10,16%). CONCLUSION: Concentration of circulating biomarkers of endothelial activation, sE-selectin and vWF Ag, soluble marker of platelet activation, sP – selectin, as well as percentage of reticulated platelets, %rP, marker of platelet turnover, were significantly higher in patients with arterial vascular disease in T2DM compared to healthy controls and non-diabetics. Patients with T2DM had significantly higher degree of resistance to antiplatelet therapy with clopidogrel compared to non diabetics, estimated by ADP test, as well as with %DPAadp, what caused more frequent recurrent ischemic attacks compared to nondiabetic patients. Correlation of biomarkers of endothelial and platelet activation (sE – selectin, vWF Ag, sP – selectin) and markers of platelet turnover (%rP) with metabolic profile indicators and poor antiplatelet therapy response suggest that altered metabolic profile can be one of contributing factors of poor antiplatelet response in diabetic patients.