ࡱ>    ~ _Bbjbj bb0=$***Pz~"*N:&T.4..03%;4Y=GGGGGGG$PRLGu>O3@3u>u>G.0kMYYYu>^.0GYu>GYY&:hE.\ cX!"L>dGM0N>S/*ShEShEHu>u>Yu>u>u>u>u>GGYu>u>u>Nu>u>u>u>Su>u>u>u>u>u>u>u>u> : TOC \o "1-5" \h \z  HYPERLINK \l "_Toc294507298" 1 UVOD  PAGEREF _Toc294507298 \h 3  HYPERLINK \l "_Toc294507299" 1.1 TROMBOCITI  PAGEREF _Toc294507299 \h 3  HYPERLINK \l "_Toc294507300" 1.1.1 Sazrijevanje trombocita  PAGEREF _Toc294507300 \h 3  HYPERLINK \l "_Toc294507301" 1.1.2 Morfologija trombocita  PAGEREF _Toc294507301 \h 6  HYPERLINK \l "_Toc294507302" 1.1.3 Funkcija trombocita  PAGEREF _Toc294507302 \h 10  HYPERLINK \l "_Toc294507303" 1.1.3.1 Adhezija i agregacija trombocita  PAGEREF _Toc294507303 \h 12  HYPERLINK \l "_Toc294507304" 1.1.3.2 Uloga trombocita u oksidacijskom stresu  PAGEREF _Toc294507304 \h 14  HYPERLINK \l "_Toc294507305" 1.1.3.2.1 Utjecaj NO na funkciju trombocita  PAGEREF _Toc294507305 \h 14  HYPERLINK \l "_Toc294507306" 1.1.3.3 Uloga trombocita u upali  PAGEREF _Toc294507306 \h 15  HYPERLINK \l "_Toc294507307" 1.1.4 Trombocitni receptori  PAGEREF _Toc294507307 \h 16  HYPERLINK \l "_Toc294507308" 1.1.4.1 Trombinski receptori  PAGEREF _Toc294507308 \h 16  HYPERLINK \l "_Toc294507309" 1.1.4.2 Kolagenski receptori  PAGEREF _Toc294507309 \h 17  HYPERLINK \l "_Toc294507310" 1.1.4.3 Adhezijski receptori  PAGEREF _Toc294507310 \h 17  HYPERLINK \l "_Toc294507311" 1.1.4.4 Adrenergi ki receptori  PAGEREF _Toc294507311 \h 17  HYPERLINK \l "_Toc294507312" 1.1.4.5 Purinergi ki receptori  PAGEREF _Toc294507312 \h 18  HYPERLINK \l "_Toc294507313" 1.1.4.6 TxA2  receptori  PAGEREF _Toc294507313 \h 18  HYPERLINK \l "_Toc294507314" 1.1.4.7 5HT  receptori  PAGEREF _Toc294507314 \h 18  HYPERLINK \l "_Toc294507315" 1.1.5 Bolesti trombocita  PAGEREF _Toc294507315 \h 19  HYPERLINK \l "_Toc294507316" 1.1.5.1 Poremeaji broja trombocita  PAGEREF _Toc294507316 \h 19  HYPERLINK \l "_Toc294507317" 1.1.5.2 Poremeaji funkcije trombocita  PAGEREF _Toc294507317 \h 20  HYPERLINK \l "_Toc294507318" 1.1.5.3 Laboratorijske pretrage za praenje funkcije trombocita  PAGEREF _Toc294507318 \h 22  HYPERLINK \l "_Toc294507319" 1.2 ANTIAGREGACIJSKI LIJEKOVI  PAGEREF _Toc294507319 \h 24  HYPERLINK \l "_Toc294507320" 1.2.1 Inhibitor ciklooksigenaze: Acetilsalicilna kiselina  PAGEREF _Toc294507320 \h 24  HYPERLINK \l "_Toc294507321" 1.2.1.1 Netrombocitni izvori TxB2  PAGEREF _Toc294507321 \h 28  HYPERLINK \l "_Toc294507322" 1.2.2 Antagonisti ADP-receptora: Tienopiridini  PAGEREF _Toc294507322 \h 29  HYPERLINK \l "_Toc294507323" 1.2.3 Antagonisti GPIIb/IIIa receptora  PAGEREF _Toc294507323 \h 29  HYPERLINK \l "_Toc294507324" 1.3 INTERINDIVIDUALNA VARIJABILNOST NA ANTIAGREGACIJSKU TERAPIJU ACETILSALICILNOM KISELINOM  PAGEREF _Toc294507324 \h 30  HYPERLINK \l "_Toc294507325" 1.4 AKUTNI KORONARNI SINDROM  PAGEREF _Toc294507325 \h 31  HYPERLINK \l "_Toc294507326" 1.4.1 Aortokoronarno premotenje  PAGEREF _Toc294507326 \h 32  HYPERLINK \l "_Toc294507327" 2 CILJ ISTRA}IVANJA  PAGEREF _Toc294507327 \h 34  HYPERLINK \l "_Toc294507328" 3 MATERIJAL I METODE  PAGEREF _Toc294507328 \h 35  HYPERLINK \l "_Toc294507329" 3.1 MATERIJAL  PAGEREF _Toc294507329 \h 35  HYPERLINK \l "_Toc294507330" 3.1.1 Ispitanici  PAGEREF _Toc294507330 \h 35  HYPERLINK \l "_Toc294507331" 3.1.2 Uzorkovanje krvi  PAGEREF _Toc294507331 \h 36  HYPERLINK \l "_Toc294507332" 3.2 METODE  PAGEREF _Toc294507332 \h 36  HYPERLINK \l "_Toc294507333" 3.2.1 Broj trombocita i MPV  PAGEREF _Toc294507333 \h 36  HYPERLINK \l "_Toc294507334" 3.2.2 Agregacija trombocita  PAGEREF _Toc294507334 \h 36  HYPERLINK \l "_Toc294507335" 3.2.3 Koncentracija TxB2 u serumu  PAGEREF _Toc294507335 \h 38  HYPERLINK \l "_Toc294507336" 3.2.4 Statisti ke metode  PAGEREF _Toc294507336 \h 39  HYPERLINK \l "_Toc294507337" 4 REZULTATI  PAGEREF _Toc294507337 \h 41  HYPERLINK \l "_Toc294507338" 4.1 ISPITANICI  PAGEREF _Toc294507338 \h 41  HYPERLINK \l "_Toc294507339" 4.2 BROJ TROMBOCITA  PAGEREF _Toc294507339 \h 44  HYPERLINK \l "_Toc294507340" 4.3 MPV - SREDNJI VOLUMEN TROMBOCITA  PAGEREF _Toc294507340 \h 45  HYPERLINK \l "_Toc294507341" 4.4 VRIJEDNOSTI AGREGACIJE TROMBOCITA ODREENE IMPEDANCIJSKOM METODOM  PAGEREF _Toc294507341 \h 46  HYPERLINK \l "_Toc294507342" 4.5 KONCENTRACIJA TROMBOKSANA TxB2  PAGEREF _Toc294507342 \h 47  HYPERLINK \l "_Toc294507343" 4.6 POVEZANOST BROJA TROMBOCITA I MPV -a S U INKOVITOSTI TERAPIJE ASK-om  PAGEREF _Toc294507343 \h 48  HYPERLINK \l "_Toc294507344" 4.7 DIJAGNOSTI KA ZNA AJNOST REZULTATA  PAGEREF _Toc294507344 \h 50  HYPERLINK \l "_Toc294507345" 5 RASPRAVA  PAGEREF _Toc294507345 \h 51  HYPERLINK \l "_Toc294507346" 6 ZAKLJU CI  PAGEREF _Toc294507346 \h 58  HYPERLINK \l "_Toc294507347" 7 SA}ETAK  PAGEREF _Toc294507347 \h 60  HYPERLINK \l "_Toc294507348" 8 SUMMARY  PAGEREF _Toc294507348 \h 62  HYPERLINK \l "_Toc294507349" 9 LITERATURA  PAGEREF _Toc294507349 \h 64  HYPERLINK \l "_Toc294507350" 10 }IVOTOPIS  PAGEREF _Toc294507350 \h 71  HYPERLINK \l "_Toc294507351" TEMELJNA DOKUMENTACIJSKA KARTICA  PAGEREF _Toc294507351 \h 72  HYPERLINK \l "_Toc294507352" BASIC DOCUMENTATION CARD  PAGEREF _Toc294507352 \h 73  POPIS KRATICA KORITENIH U TEKSTU AAengl. Arachidonic acid, arahidonska kiselinaADP adenozindifosfatAIM akutni infarkt miokardaAKS akutni koronarni sindromASK acetilsalicilna kiselinaATP adenozintrifosfatAUCengl. Area under the curve, povrina ispod krivuljeCABG engl. Coronary artery bypass grafting, aorto-koronarno premotenjeCDengl. Cluster of differentiation, diferencijacijska skupina antigenaCOX engl. Cyclo-oxygenase, ciklooksigenazaDIK diseminirana intravaskularna koagulacijaDNAengl. Deoxyribonucleic acid, deoksiribonukleinska kiselinaDTS engl. Dense tubular system, gusti tubularni sustavET esencijalna trombocitemijaGM-CSFengl. Granulocyte-Macrophage-Colony Stimulating Factor , granulocitno-makrofagni faktor stimulacije rastaGPglikoproteinHIT heparinom inducirana trombocitopenija HUS hemoliti ko-uremi ni sindromILinterleukinKML kroni na mijeloi na leukemijaMplreceptor za trombopoetinMPVengl. Mean platelet volumen, srednji volumen trombocitaNO duaikov-oksid NSAID engl. Nonsteroidal anti inflammatory drug, nesteroidni antiupalni lijekOCS engl. Open canalicular system, otvoreni kanalikularni sustavPAI engl. Plasminogen activator inhibitor,inhibitor aktivatora plazminogenaPAR engl. Protease-activated receptor, proteazom aktivirani receptoriPFengl. Platelet factor, trombocitni faktorPGG2 prostaglandin G2PGH2 prostaglandin H2POCTengl. Point of care tetsing, pretrage uz bolesnikaPRV policitemija rubra veraPVprotrombinsko vrijemeROSreaktivni kisikovi spojeviSCFengl. Steam Cell Factor, faktor stimulacije mati nih stanicaTF engl. Tissue factor, tkivni faktor TGF-(engl. Transforming Growth Factor-(, transformirajui faktor rasta (tPA engl. Tissue plasminogen activator, tkivni aktivator plazminogenaTPO TRAPtrombopoetin peptid koji simulira aktivaciju trombocita trombinomTTP tromboti na trombocitopeni na purpuraTxtromboksanVWFVon Willebrandov faktor 1 UVOD TROMBOCITI Sazrijevanje trombocita Trombociti su diskoidne stanice bez jezgre, promjera 2-3 (m. Cirkuliraju u krvi 7-10 dana prije nego li budu odstranjeni od strane makrofaga. Nastaju u koatanoj sr~i fragmentiranjem citoplazme zrelih megakariocita, a iz jednog megakariocita nastaje 2000 3000 trombocita. Njihovo nastajanje pod kontrolom je trombopoetina (TPO), hormona koji se uglavnom stvara u jetri i bubregu, ali i mnogih drugih citokina (IL-3, GM-CSF, SCF, IL-1(, IL-11).(1) (Slika1.) Trombociti sisavaca potje u od citoplazme megakariocita, poliploidne hematopoetske stanice koja nastaje iz pluripotentne mati ne stanice, zajedni ke ishodiane stanice za mijeloidnu i limfoidnu lozu. Pluripotentna mati na stanica diferencira se u nezrele prethodne stanice mijelopoeznog i limfopoeznog sustava ijom proliferacijom i sazrijevanjem nastaju funkcionalno zrele krvne stanice: eritrociti, granulociti, monociti, limfociti T i B, NK stanice i trombociti. Iz mijeloidne mati ne stanice CFU-GEMM (engl. Colony Forming Unit  Granulocyte-Erythroid-Monocyte-Macrophag; stani na jedinica koja stvara granulocitno-eritrocitno-monocitno-megakariocitne kolonije) stvaraju se najprije nezrele stanice, a onda i zreli megakariociti. Primitivne megakariocitne kolonije BFU Meg (engl. Burst Forming Unit-Megacaryocites) usmjerene su u megakariocitnu diferencijaciju i imaju veliki proliferacijski potencijal pa stvaraju 100-500 megakariocita po stanici. Za rast kolonija va~ni su proliferacijski faktori (IL-3, GM-CSF, SCF, IL-1(, a naro ito IL-11 i TPO). Kolonije CFU-Meg stvaraju diferenciranije stanice megakariocitopoeze koje imaju manji proliferacijski potencijal; samo 4-32 megakariocita.(1) Prva morfoloaki prepoznatljiva stanica megakariocitne loze je megakarioblast; stanica s izrazito velikom poliploidnom jezgrom i oskudnom citoplazmom koja prethodi promegakariocitu i megakariocitu. Na povraini stanica megakariocitne loze prisutan je specifi an stani ni biljeg CD41. CD-biljezi su stani ni antigeni na povraini stanica koji su zajedni ki svakoj jedinki unutar iste vrste. Karakteristi ni su za pojedinu stani nu lozu i za stupanj njene diferencijacije (zrelosti). Otkrie monoklonskih protutijela koja su specifi na za odreene stani ne biljege doprinjelo je razvoju suvremenih analiti kih metoda kao ato je proto na citometrija, a koje nam slu~e za mjerenje fizikalnih i kemijskih osobina stanica. Sazrijevanjem stanica smanjuje se sposobnost proliferacije, ali se poveava sposobnost sinteze DNA bez diobe. Stoga zreli megakariociti imaju mnogo vei volumen od ostalih stanica u koatanoj sr~i i otpuataju u krvnu cirkulaciju veliki broj trombocita. Zreli megakariociti sadr~e trombocitni faktor 4 (PF4), von Willebrandov faktor (VWF), trombospondin i trombomodulin. (1) Cijeli proces diferencijacije mati ne stanice hematopoeze do nastanka zrelih trombocita traje prosje no 10 dana. Mladi trombociti nakon izlaska iz koatane sr~i provedu oko 36 sati u slezeni u kojoj se ina e i pohranjuje treina nastalih trombocita. Za stvaranje trombocita va~na su dva gena: NFE-2 gen ima glavnu ulogu u stvaranju trombocita, a TUBB1 ((1-tubulin) ima ulogu u stvaranju protrombocita i odreivanju diskoidnog oblika trombocita. (1) Najva~niji proliferacijski faktor koji regulira trombocitopoezu je trombopoetin. Sintetiziraju ga jetra, bubreg, koatana sr~ i slezena. Njegovu sintezu kontrolira THPO gen smjeaten na 3. kromosomu. Za odr~avanje normalne hematopoeze TPO je vezan na svoj receptor Mpl, pa je razina slobodnog cirkulirajueg TPO niska. U stanjima trombocitopenije dolazi do redukcije vezanja TPO na Mpl pa raste koncentracija slobodnog TPO u krvi i on djeluje na stanice koje sadr~e Mpl-receptor, a to su megakariociti, trombociti i prethodne nezrele mati ne CD34+ stanice poti ui njihovo sazrijevanje. Za taj proces potrebni su i drugi faktori; IL-6, IL-11 i SCF koji takoer stimuliraju specifi ne stadije sazrijevanja megakariocita, ali samo ako djeluju sinergisti ki s TPO i IL-3. Postoje i citokini koji inhibiraju trombocitopoezu, a to su PF4, interferoni i TGF-(.(1)  Slika 1. Razvoj megakariocitne loze (slika preuzeta iz literature br. 48) Morfologija trombocita Trombociti koji normalno cirkuliraju u krvi su neaktivni, diskoidnog su oblika i nje~ne povraine. Na vanjskoj povraini nalazi se nekoliko otvorenih kanalikularnih sustava koji ulaze duboko u unutraanjost trombocita. Strukturno se trombociti sastoje od etiri cjeline: membranskog sustava, zone organela, periferne zone i strukturalne zone, a svaka od njih ima posebnu ulogu u aktivaciji trombocita.(Slika 2.)  Slika 2. Graa trombocita (slika preuzeta iz literature br. 2) Periferna zona sastoji se od fosfolipidne membrane koja je s vanjske strane prekrivena pahuljastim omota em - glikokaliksom, a s unutarnje tankom submembranoznom regijom. Glikokaliks sadr~i glikoproteine, proteine i mukopolisaharide koji slu~e kao receptori za tvari male molekulske mase (ADP, serotonin, acetil-kolin, prostaglandin) i kao receptori za makromolekule, trombin, fibrinogen, VWF, kolagen i faktore koagulacije V i VIII. Neki od povrainskih proteina su receptori za tvari koje uzrokuju aktivaciju trombocita. Glikokaliks sadr~i 30-ak glikoproteina od kojih su za funkciju trombocita najva~niji glikoproteini (GP) Ib, IIb i IIIa. ( Slika 3.)  Slika 3. Glikoproteini na povrini trombocita Glikoprotein Ib (GPIb) je receptor za VWF i nekovalentno je vezan sa GPV i GPIX. Funkcija GPV nije poznata, ali se zna da njegov nedostatak nije povezan sa pojavom krvarenja, dok je kompleks GPIb/IX odgovoran za adheziju trombocita kod oateenja krvne ~ile.(2) GPIb sastoji se od dva lanca; ( i (. (- lanac je vei i sadr~i vezna mjesta za VWF, trombin i ristocetin. Svaki trombocit sadr~i pribli~no oko 25 000 molekula GPIb.(Slika 4.)  Slika 4. Shematski prikaz glikoproteina GPIb (slika preuzeta iz literature br.2) Glikoproteini IIb i IIIa tvore kompleks (GPIIb/IIIa) koji je receptor za fibrinogen. GPIIb sastoji se od dva lanca; ( i (. (-lanac ugraen je u fosfolipidni dvosloj, dok (-lanac potje e s povraine trombocita. Dio (-lanca ini HPA-3, trombocitni aloantigen. GPIIIa sastoji se od jednog lanca koji je povezan s dijelom GPIIb unutar fosfolipidnog dvosloja. Mali dio GPIIIa ini HPA-1 antigen. Protutijela na HPA-3 i HPA-1 uzrokuju pojavu neonatalne aloimune trombocitopenije.(Slika 5.) Kompleks GPIIb/IIIa odgovoran je za agregaciju trombocita.  Slika 5. Shematski prikaz glikoproteina GPIIb/IIIa (slika preuzeta iz literature br.2) Fosfolipidna membrana trombocita sadr~i i jednu nezasienu masnu kiselinu; arahidonsku kiselinu (AA), koja je sna~an prekursor za agregaciju trombocita i konstrikciju krvnih ~ila. Strukturalna zona sastoji se od mikrotubula povezanih u mre~u proteina ija funkcija je odr~avanje diskoidne strukture neaktiviranih trombocita kao i omoguavanje promjena u obliku trombocita nakon aktivacije. Mre~a proteina sadr~i aktin, aktin-vezajui protein i nekoliko strukturnih proteina koji sa injavaju citoskelet. Aktin je najprisutniji protein u trombocitima ( ini 15-20 % ukupnih proteina u trombocitima) i povezan je s miozinom kao i s joa nekim drugim kontraktilnim proteinima, sli no kao i u glatkim miaiima samo ato je omjer aktina i miozina u glatkim miaiima 7:1, a u trombocitima 100:1. Zona organela sastoji se od mitohondrija, glikogenskog dijela koji sudjeluje kod metaboli kih aktivnosti trombocita i etiri vrste granula (gusta tijela, alfa granule, lizosomalne granule i mikroperoksisomi) koje slu~e kao skladiata proteina i drugih tvari va~nih za funkciju trombocita. Gusta tijela dobila su naziv po najveoj gustoi granula vidljivih elektronskim mikroskopom. Ona sadr~e medijatore va~ne za funkciju trombocita koji nisu proteini: ADP, ATP, kalcijeve ione i serotonin. ADP u gustim tijelima ne osigurava energiju za osnovne metaboli ke aktivnosti trombocita, ve slu~i kao pohrana energije za reakcije agregacije trombocita. Alfa granule najbrojnije su od svih granula i sadr~e dvije grupe proteina. Prvu grupu ine proteini koji su sli ni proteinima plazme, a sintetiziraju se u megakariocitu za vrijeme trombocitopoeze (VWF i faktor V). Drugu grupu ine proteini koji se apsorbiraju iz plazme na razini megakariocita i odla~u se u alfa-granule za vrijeme trombocitopoeze (fibrinogen, imunoglobulini i albumin) kao i mnogi drugi proteini kao ato su PF4, (-tromboglobulin, inhibitor aktivatora plazminogena (engl. PAI - plasminogen activator inhibitor), fibronektin, vitronektin i trombospondin. Lizosomalne granule sadr~e nekoliko hidroliti kih enzima i sli e lizosomima drugih stanica. Mikroperoksisomima do sada nije utvrena funkcija. Membranski sustav ine dva va~na sustava membrana, a to su otvoreni kanalikularni sustav (engl. OCS-open canalicular system) i gusti tubularni sustav (engl. DTS-dense tubular system). Njihova uloga je u regulaciji unutarstani ne koncentracije kalcija koja je va~na za regulaciju metabolizma trombocita i njihovu aktivaciju.(2) Funkcija trombocita Broj trombocita u krvi varira izmeu 158-424 x109/L. Trombociti sudjeluju u odr~avanju normalne hemostaze odr~avajui krv u tekuem stanju, ali i u konstrikciji i popravku oateene krvne ~ile spre avajui na taj na in gubitak krvi. Takoer sudjeluju u patofizioloakim procesima kao ato su tromboza, krvarenje, rast tumorskih stanica, upala i nastanak aterosklerotskog plaka. (Slika 6.) U prilog tome govori injenica da je za adekvatno zaustavljanje krvarenja dostatno pribli~no 10 x109/L trombocita, dok preostali trombociti sudjeluju u brojnim drugim patofizioloakim procesima. Glavna funkcija trombocita - nastanak trombocitnog ugruaka tijekom hemostaze, najviae je istra~ivana i opisivana funkcija trombocita, dok se o ostalim funkcijama trombocita manje zna. Posljednjih desetljea ine se veliki napori u razjaanjavanju i drugih, kako fizioloakih, tako i patoloakih funkcija trombocita. Sve je vei broj radova koji su ispitivali funkcije trombocita u upalnim procesima,(3) imunoloakim reakcijama,(4) oksidacijskom stresu,(5) procesu starenja,(6) neuroloakim i psihijatrijskim bolestima,(7) bolestima plua,(8) tumorskim bolestima,(9) traumatskim oateenjima,(10) alergijskim bolestima,(11) nefroloakim bolestima,(12) i dijabetesu.(13)  Slika 6. Razli ite patofizioloake funkcije trombocita Adhezija i agregacija trombocita Trombociti cirkuliraju pasivno unutar krvne ~ile koja je oblo~ena endotelom. Svaka ozljeda krvne ~ile ili oateenje endotela (uklju ujui i rupturu aterosklerotskog plaka) poticaj su trombocitima da adheriraju na mjesto oateenja, aktiviraju se, promijene svoj oblik, lu e sadr~aj svojih granula i agregiraju vezajui se jedan na drugog, stvarajui na taj na in primarni hemostatski tromb. (Slika 7.) Iako trombociti imaju sredianju ulogu upravo u primarnoj hemostazi ne smijemo zaboraviti ni njihovu ulogu u sekundarnoj hemostazi gdje osiguravaju fosfolipidnu povrainu za odvijanje nekoliko klju nih koagulacijskih reakcija. Nakon ozljede krvne ~ile trombociti se mogu aktivirati na dva razli ita na ina koji mogu djelovati zajedno, ali i nezavisno jedan od drugoga. Prvi na in uklju uje izlaganje cirkulirajuih trombocita oateenom subendotelu i kolagenu uslijed ega dolazi do adhezije trombocita na kolagen preko VWF-a koji ini most izmeu kolagena i trombocita vezajui se na trombocitni receptor GPIb/V/IX. Nadalje dolazi do otpuatanja citoplazmatskih iona kalcija koji omoguuju sekreciju trombocitnih granula pri emu (-granule lu e P-selektin koji uzrokuje adheziju monocita i granulocita na aktivirane trombocite, a guste granule otpuataju ADP koji preko trombocitnog receptora P2Y1 oslobaa fosfolipazu A2 na membrani fosfolipida. To omoguuje otpuatanje arahidonske kiseline koja pomou COX-1 stvara TxA2 koji poti e agregaciju trombocita. Oslobaanje Ca2+ uzrokuje konformacijske promjene na trombocitnom GPIIb/IIIa receptoru ato omoguuje vezanje trombocita na fibrinogen. Drugi na in omoguuje vezanje osloboenog tkivnog faktora (TF) na plazmatski FVII (od kojeg je samo 1-2 % u aktivnom obliku kao FVIIa) pri emu nastaje kompleks TF-FVIIa koji dalje aktivira FVII vezan na povraini stanice koja izra~ava TF u aktivni kompleks TF-FVIIa koji aktivira FIX i FX u FIXa i FXa. FXa mo~e aktivirati plazmatski FV dok FXa koji ostaje vezan na povraini stanice koja nosi TF ve~e se u kompleks s FVa pri emu nastaje mala koli ina trombina iz protrombina. Koli ina nastalog trombina manja je od 5% ukupne koli ine trombina koja je potrebna u procesu zgruaavanja krvi i nije dovoljna za pretvorbu fibrinogena u fibrin i samim tim za nastanak hemostatskog ugruaka. No, ta mala koli ina trombina dovoljna je za aktivaciju trombocita i njihovo lokaliziranje na mjestu izlaganja TF. Trombin se ve~e na povrainu neaktiviranih trombocita preko najmanje tri receptora: GPIb(, PAR1 i PAR 4 uzrokujui na taj na in njihovu aktivaciju. Nakon aktivacije, trombociti izlu uju hemostatski aktivne tvari meu kojima su i etiri faktora zgruaavanja koji su pohranjeni u (-granulama i citoplazmi trombocita. To su fibrinogen, FV, FXI i FXIII i oni izravno sudjeluju u nastanku stabilnog fibrinskog ugruaka.  normalno stanje aktivacija i adhezija agregacija Slika 7. Adhezija i agregacija trombocita (slika preuzeta iz literature br.25) Uloga trombocita u oksidacijskom stresu Oksidacijski stres je stanje u kojem stani na produkcija reaktivnih kisikovih spojeva (ROS-a) premaauje kapacitet stani nog antioksidativnog obrambenog sustava uzrokujui pritom disfunkciju i oateenje stani ne membrane i endotela krvnih ~ila. Oksidacijski stres prati mnoge vaskularne bolesti i jedan je od glavnih razloga smanjenju koncentracije NO budui da superoksidni radikal reagira s NO stvarajui peroksinitrit. Peroksinitrit je slabiji vazodilatator od NO, a uz to uzrokuje i nastanak lipidne peroksidacije, lan ane reakcije slobodnih radikala. Lipidna peroksidacija uzrokuje oateenje lipida na stani noj membrani, pa membrana postaje porozna i gubi svoju funkciju. Stoga lipidna peroksidacija uzrokuje aktivaciju i agregaciju trombocita. U oksidativnom stresu stimulirana je aktivacija NF-(B ato pogoduje nastanku proaterogenih citokina TNF(, interleukina IL-1 i IL-6 i kemokina koji inhibiraju nastanak NO. Endotelne stanice oslobaaju angiotenzin konvertirajui enzim (ACE) koji je antagonist NO i koji pretvara angiotenzin I u angiotenzin II uzrokujui vazokonstrikciju. Svi ovi procesi pogoduju progresiji ateroskleroze. Utjecaj NO na funkciju trombocita NO je vazodilatator i dovodi do opuatanja glatkih miaia endotela, pa tako i do cjelovitog opuatanja krvnih ~ila. Lu i se iz aktiviranih endotelnih stanica kao odgovor na djelovanje agonista trombocita, a u svrhu blokade aktivacije i agregacije trombocita. NO dovodi do aktivacije gvanilil-ciklaze, poja ane pretvorbe GTP u cGTP ato omoguuje smanjenje intracelularnog Ca2+ i inhibiciju aktivacije fosfoinozitol-3-kinaze (PI3K) koja aktivira GPIIb/IIIa fibrinogenske receptore. Jednako tako NO reducira sintezu TxA2 tvorei peroksinitrit koji reagira s tirozinskim ograncima na COX-1 inhibirajui enzim, a samim tim i pretvorbu AA u PGG2 i PGH2. (Slika 8.) NO omoguuje fosforilaciju TxA2-receptora inhibirajui njegovu funkciju. S obzirom na vrlo va~nu ulogu NO u regulaciji funkcije trombocita, smanjena produkcija ili poveana razgradnja NO va~an je imbenik u aterogenezi, a time i u nastanku cerebrovaskularnih bolesti.  Slika 8. Utjecaj NO na funkciju trombocita (slika preuzeta iz literature br.51) Uloga trombocita u upali Endotel zdravih krvnih ~ila onemoguava cirkulirajuim prokoagulantnim proteinima aktivaciju, a trombocitima aktivaciju i adheziju. Tako u odsutnosti bilo kakve vaskularne traume krv ostaje u tekuem obliku zahvaljujui antitromboti kim svojstvima endotelnih stanica. No, u slu aju upalnih stanja i pojave proupalnih citokina, ak ako joa nije doalo do oateenja krvne ~ile, dolazi do aktivacije endotela pa endotelne stanice sada poprimaju protromboti ka svojstva. Sekrecija razli itih antitromboti kih molekula kao ato su NO, ektonukleotidaze i prostaglandini je smanjena, a poja ana je ekspresija protromboti kih i proupalnih molekula. P-selektini, prisutni na aktiviranom endotelu i na povraini trombocita, mogu uzrokovati aktivaciju i adheziju trombocita i nastanak aterosklerotskog plaka ijom rupturom dolazi do nastanka AIM. Trombocitni receptori Trombocitni receptori su glikoproteini smjeateni na povraini trombocita i oni predstavljaju poveznicu trombocita s njihovom okolinom. Ti su receptori odgovorni za reaktivnost trombocita sa paletom razli itih agonista i adhezivnih proteina. Budui da trombociti nemaju jezgru i ne mogu se prilagoditi razli itim novonastalim situacijama, uvjetovanim de novo sintezom proteina, oni preko razli itih receptora daju svoj odgovor na razli ite fizioloake i patoloake dogaaje. Budui da je glavna zadaa trombocita odr~avanje hemostaze, ne iznenauje injenica da je veina njihovih receptora direktno povezana s procesom aktivacije trombocita i adhezije na oateenu krvnu ~ilu kao i stvaranja primarnog tromba. Trombinski receptori Trombin je najsna~niji fizioloaki agonist trombocita. On sudjeluje u aktivaciji trombocita, ali njegova klju na uloga je u nastajanju fibrinskog ugruaka iz topljivog fibrinogena. Trombin se ve~e na transmembranske receptore iz porodice proteazom aktiviranih receptora (engl. protease-activated receptors, PARs) koji su vezani za protein G. Poznata su etiri PAR receptora, a u ljudskim trombocitima prisutni su samo PAR1 i PAR4. (PAR2 i PAR3 prisutni su na miajim trombocitima). Hidroliti kim cijepanjem trombin aktivira PAR1 i PAR4 omoguujui prijenos signala u stanicu preko proteina G. PAR1 je visokoafinitetni receptor kojeg aktiviraju niske koncentracije trombina za razliku od PAR4 kojeg aktiviraju visoke koncentracije trombina. Kolagenski receptori Na povrini trombocita nalazi se nekoliko glikoproteinskih receptora, no smatra se da su samo GPIa/IIa i GPVI odgovorni za kolagenom induciranu aktivaciju trombocita. GP Ia/IIa receptor, integrin (2(1, uzrokuje adheziju trombocita na matriks subendotela uz prethodnu aktivaciju nekim od (ja ih( agonista npr. trombinom ili ADP-om. GPVI zajedno sa GPIb/IX/V sudjeluje u adheziji i formiranju tromba na kolagenskoj povraini u uvjetima cirkulirajue krvi. Smatra se da je polimorfizam gena za GPVI povezan s nastankom infarkta miokarda. Adhezijski receptori GPIb/V/IX omoguava trombocitima vezanje s VWF ato dovodi do usporavanja trombocita na mjestu oateenja krvne ~ile i njihovu interakciju s kolagenom. GPIIb/IIIa, integrin (IIb(3 je najzastupljeniji adhezijski receptor koji da bi postao aktivan mora proi konformacijsku promjenu uzrokovanu aktivacijom trombocita nekim od adhezijskih receptora (GPIb, GPIa/IIa), trombinskim receptorima (PAR1 i PAR4) ili ADP receptorima (P2Y12, P2Y1, P2X1). GPIIb/IIIa uglavnom ve~e fibrinogen, ali takoer i VWF, fibronektin, vitronektin i trombospondin. Njegova funkcija ovisna je o Ca 2+, a vezanje fibrinogena i VWF posreduje u agregaciji trombocita. Adrenergi ki receptori Djelovanje kateholamina posredovano je (2-adrenergi kim receptorom koji poja ava djelovanja drugih agonista i u visokoj koncentraciji izaziva agregaciju trombocita. Dopaminergi ki receptori djeluju na aktivaciju adenilat-ciklaze koja inhibira cAMP, dovodi do hidrolize fosfatidil-inozitola, aktivacije K+-kanala i promjene razine unutarstani nog Ca2+. Purinergi ki receptori Purinergi ki receptori imaju va~nu ulogu u formiranju tromba u podru ju visokog tlaka, oni posreduju u aktivaciji trombocita sekrecijom ADP-a iz sekretornih granula, a sastoje se od porodice P2X(1-7) receptora i porodice G-protein vezanih P2Y(1,2,3,4,11,12,13,14) receptora. Trombociti eksprimiraju uglavnom P2Y1, P2X1 i P2Y12 receptore, dok preostali receptori iz tih porodica nemaju zna ajniju ulogu u aktivaciji odnosno agregaciji trombocita. Vezanjem ADP-a na receptor P2Y1 dolazi do prolaznog porasta koncentracije unutarstani nog Ca2+ ato uzrokuje slabu, prolaznu agregaciju, no istodobno sinergisti ko djelovanje ADP-a preko P2Y12 receptora uvjetuje kona nu i trajnu aktivaciju i agregaciju trombocita inhibiranjem fosforilacije VASP (vazodilatator-stimulirajueg fosfoproteina) i aktiviranjem GPIIb/IIIa na povraini trombocita. TxA2  receptori TxA2-receptori pripadaju porodici G-proteina, javljaju se u dvije izoforme, ( i (, te posreduju djelovanju tromboksana na agregaciju trombocita i kontrakciju krvnih ~ila. Tromboksan A2 je vrlo moan aktivator trombocita koji nastaje pretvorbom AA u prostaglandine i dalje do TxA2 koji ima vrlo brzo djelovanje na aktivaciju trombocita. Njegov je polu~ivot u cirkulaciji samo 30 sekundi. 5HT  receptori Aktivacijom serotoninskih receptora 5HT raste citoplazmatski Ca2+. Serotonin je sam po sebi slab agonist, ali djeluje sinergisti ki s ostalim trombocitnim agonistima. Bolesti trombocita Bolesti trombocita svrstane su u dvije osnovne kategorije: kvantitativne (poremeen broj trombocita) kvalitativne (poremeena funkcija trombocita) Poremeaji broja trombocita Trombocitoza ili poviaeni broj trombocita mo~e biti: primarna (naj eae je uzrokovana mijeloproliferativnim poremeajem: esencijalna trombocitemija, PRV, KML, agnogena mijeloidna metaplazija) sekundarna ili reaktivna (posljedica je sistemne upale, maligniteta, manjka ~eljeza, krvarenja ili stanja nakon splenektomije). Trombocitopenija ili sni~en broj trombocita mo~e nastati: kao posljedica smanjenog stvaranja trombocita (aplasti na anemija, hipoplazija megakariocita, oateenje koatane sr~i zra enjem) kao posljedica poremeene raspodjele trombocita u cirkulaciji (kongestivna splenomegalija) zbog ubrzane razgradnje i potronje trombocita (DIK, HUS, TTP, imunosne trombocitopenije) kao sekundarna imunosna trombocitopenija nastala kao posljedica transfuzije krvi ili prisustva anti P1A antitijela u cirkulaciji djeteta koja nakon imunizacije majke prelaze u dijete. kao posljedica terapije lijekovima (HIT) Poremeaji funkcije trombocita Poremeaji funkcije trombocita nastaju kao posljedica nekih nasljednih bolesti (Tablica 1.), a naj eai uzrok nastanka ste enih poremeaja funkcije trombocita su razli iti lijekovi (Tablica 2.). Jedna vrsta lijekova uzima se sa svrhom inhibicije funkcije trombocita (npr. antiagregacijski lijekovi), a druga vrsta lijekova uzimanih s nekom drugom svrhom ima nuspojave koje utje u na funkciju trombocita. Tablica 1. Poremeaji funkcije trombocita Poremeaji adhezije trombocita nasljedni: von Willebrandova bolest Bernard-Soulierov sindrom ste eni: uremija Poremeaji agregacije trombocita nasljedni: Glanzmannova trombastenija ste eni : uzimanja lijekova disproteinemija Poremeaji oslobaanja granula iz trombocita nasljedni: sivi trombocit Hermansky-Pudlak sindrom Chediac-Higashijev sindrom ste eni: kardiopulmonarne premosnice mijeloproliferativni poremeaji (ET, PRV, KML)  Tablica 2. Lijekovi koji utje u na funkciju trombocita Nesteroidni i antiupalni lijekovi - aaspirin indometacin ibuprofen sulindac naproxen enilbutazonTienopiridini tiklopidin klopidogrelAntagonisti GPIIa/IIIbabciximab ebtifibatid tirofibanLijekovi koji poveavaju razinu trombocitnog cAMP ili c GMPprostaciklin iloprost dipiridamol teofilin NOAntikoagulansi i fibrinoliticiheparin streptokinaza tPA urokinazaKardiovaskularni lijekovinitroglicerin izosorbid-dinitrat propanolol blokatori Ca-kanala quinidin ACE-inhibitori verapamil diltiazemVolumni ekspanderi dextran hidroksietil-starchPsihotropni lijekovi, anestetici imipramin amitriptilin nortriptilin klorpromazin prometazin flufenazin haloperidol halotan butakain prokainCitostaticimitomicin daunorubicin karmustinOstali lijekoviantihistaminici radiografski kontrasti klofibrat Laboratorijske pretrage za praenje funkcije trombocita Premda trombociti imaju vodeu ulogu u odr~avanju normalne hemostaze i kod patoloakog krvarenja i tromboze, donedavno su se pretrage za praenje funkcije trombocita koristile uglavnom kod bolesnika s patoloakim krvarenjima. Kako je znanje o ulozi trombocita u razvoju ateroskleroze postajalo sve razumljivije, a sama ateroskleroza postala vodei uzrok pobola i smrtnosti u razvijenim zemljama, tako su se razvijale i nove pretrage s ciljem praenja funkcionalne aktivnosti trombocita, a posebice kod bolesnika na antiagregacijskoj terapiji. Ateroskleroza je multi imbeni na bolest pa ne mo~emo o ekivati da e praenjem samo jednog imbenika, pa bio on i najva~niji za njezin nastanak, biti isklju ene sve mogue komplikacije. No, mo~emo ih zna ajno smanjiti. Dobro je poznato da se u dijabetesu, hiperlipidemiji ili hipertenziji prate koncentracije glukoze, lipida ili krvni tlak kao mjera djelotvornosti antidijabetika, statina ili antihipertenziva. No, kod antiagregacijske terapije praenje terapije je zanemareno iako se zna da postoji zna ajna interindividualna varijabilnost u odgovoru trombocita na djelovanje ASK.(14) Poveava se broj bolesnika koji su na antiagregacijskoj terapiji. Sve je vei udio i rizik od nastanka krvarenja kod takvih bolesnika, osobito nakon razli itih trauma i operacijskih zahvata. Zato postoji sve vea potreba za testiranjem i procjenom funkcije trombocita kako bi se optimalnim, individualiziranim doziranjem lijeka izbjeglo krvarenje, a da se istovremeno sprije i i pojava tromboze. Sve to zajedno doprinjelo je razvoju novih POCT-analizatora na kojima se mo~e odrediti funkcionalna aktivnost trombocita iz pune krvi, brzo, jednostavno i u svakom laboratoriju. Iako antiagregacijski lijekovi inhibiraju agregaciju trombocita in vivo, smatra se da je mjerenjem funkcionalne aktivnosti trombocita in vitro mogue procijeniti stupanj inhibicije agregacije trombocita tj. stupanj djelovanja antitrombocitnog lijeka, te na taj na in predvidjeti rizik od nastanka tromboti kog stanja. Ukoliko se za odreivanje funkcionalne aktivnosti trombocita ne koristi pretraga koja je specifi na za djelovanje odreenog lijeka mo~e se dobiti poveana ostatna aktivnost trombocita koja e nam ukazati na postojanje rezistencije na lijek, a da u stvarnosti to nije tako. Zato je vrlo va~no primijeniti specifi nu pretragu koja mjeri farmakoloako djelovanje upotrijebljenog lijeka i koja e nam dati to an podatak o djelotvornosti lijeka, ili pak o rezistenciji na lijek. Funkcionalnu aktivnost trombocita mo~emo ispitati pomou razli itih pretraga: 1. Vrijeme krvarenja po Duke-u pretraga je invazivna, nereproducibilna i nedovoljno osjetljiva. Rezultat ovisi o mnogim imbenicima: broju trombocita, plazmatskim faktorima, eritrocitima, stijenci krvne ~ile, grai ko~e i dubini uboda reza. 2. Kapacitet primarne hemostaze odreuje se na analizatoru PFA-100 koji mjeri vrijeme nastanka trombocitnog ugruaka oponaaanjem in vivo uvjeta fizioloake brzine protoka krvi u arterijama u prisutnosti kolagena i adrenalina, te kolagena i ADP-a. Pretraga je brza, jednostavna, automatizirana i ne zahtijeva pripremu uzorka jer se izvodi iz pune krvi, no slabo je osjetljiva. Uo eno je da vrijeme zgruaavanja s adrenalinom nije produ~eno kod svih bolesnika na aspirinskoj terapiji iako im je koncentracija TxB2 sni~ena. 3. Pretrage agregacije trombocita mo~emo podijeliti u dvije skupine: pretrage koje se izvode iz plazme bogate trombocitima (PRP, engl. platelet-rich plasma) i pretrage koje se izvode iz pune krvi. Pretraga svjetlosne ili opti ke agregometrije (LTA, engl. Light transmittance aggregometry) predstavlja (zlatni standard( u odreivanju funkcionalne aktivnosti trombocita. Mjeri se porast prijenosa svjetla kroz suspenziju trombocita nakon dodatka trombocitnog agonista. Metoda je predanaliti ki dugotrajna i zahtjevna. Potrebna je velika koli ina krvi, a za samu analizu potrebno je pripremiti plazmu bogatu trombocitima ato se izvodi samo u specijaliziranim laboratorijima. Pretrage agregacije trombocita iz pune krvi mogu se izvoditi na IMPACT-analizatoru. To je POCT-analizator koji mjeri adheziju i agregaciju trombocita simulirajui meudjelovanje trombocita iz pune krvi s izvanstani nim subendotelom u uvjetima fizioloake brzine protoka krvi. Metoda je brza i jednostavna, izvodi se iz malog volumena pune krvi, ali joa uvijek nije u airoj upotrebi. RFPA-Verify Now je POCT-analizator koji mjeri aglutinaciju estica oblo~enih fibrinogenom nakon aktivacije trombocita agonistom. Metoda je brza, jednostavna, automatizirana i ne zahtijeva pripremu uzorka. ChronoLog Aggregometar je analizator koji impedancijskom agregometrijom mjeri porast otpora prolaza struje izmeu dvije elektrode uronjene u uzorak pune krvi nakon dodatka AA. Multiple platelet function analyzer (Multiplate) joa je jedan analizator koji mjeri promjenu impedancije izmeu dvije elektrode na kojima je dodatkom agonista (AA, kolagena, ristocetina, ADP-a ili TRAP-a) doalo do agregacije trombocita. Metoda je brza, jednostavna i izvodi se iz pune krvi, a istovremeno se mo~e analizirati pet uzoraka. 4. Proto na citometrija omoguava praenje prisutnih aktiviranih trombocita u cirkulaciji nakon dodatka AA in vitro. Metoda je jednostavna, zahtijeva malu koli inu uzorka, ali je oprema vrlo skupa, kao i izobrazba laboratorijskog osoblja. 5. Tromboelastografija je POCT-pretraga pogodna za operacijske sale, daje kompletan uvid u nastanak i fibrinolizu ugruaka. To je brza i jednostavna metoda, ali pripada grupi globalnih testova. 6. Specifi ne pretrage za odreivanje funkcionalne aktivnosti trombocita mjere urinske ili serumske metabolite TxA2. Mjerenje koncentracije urinskog 11-dehidroTxB2 pretraga je izravno ovisna o COX-1 i odra~ava in vivo nastanak TxA2, no njegova koncentracija ovisna je o bubre~noj funkciji i sudjelovanju nekih drugih stanica (endotelnih stanica, makrofaga) u nastanku 11-dehidroTxB2. Mjerenje koncentracije serumskog TxB2 pretraga je izravno ovisna o COX-1 i prema nekim autorima mogua specifi na pretraga praenja terapije ASK-om. ANTIAGREGACIJSKI LIJEKOVI Budui da trombociti imaju va~nu ulogu u nastanku i progresiji arterijske tromboze, jedna od airoko primjenjivanih terapijskih mogunosti u njenoj prevenciji i lije enju je primjena antiagregacijskih ili antitrombocitnih lijekova s ciljem inhibitornog djelovanja na funkciju trombocita. Antiagregacijski lijekovi razlikuju se po mehanizmu djelovanja u tri glavne skupine: inhibitori COX-1, antagonisti receptora ADP i antagonisti glikoproteinskog kompleksa GPIIb/IIIa.(15) Svaka od navedenih skupina lijekova selektivno inhibira odreeni put aktivacije trombocita. Inhibitor ciklooksigenaze: Acetilsalicilna kiselina Joa u doba starih Grka, oko 400 god. prije naae ere, gr ki lije nici Galen i Hipokrat uo ili su da kora vrbe ima analgetsko i antipiretsko djelovanje. Primjena je bila iskustvena, oni naravno nisu znali koja tvar poti e to djelovanje. U 18. i 19. stoljeu razvojem znanosti i kemijske tehnologije izolirana je tvar iz vrbine kore s analgetskim i antipiretskim u inkom i nazvana je salicin ato na latinskom zna i vrba. U 1832. godini sintetizirana je salicilna kiselina, a 1897. Felix Hoffman, njema ki kemi ar, sintetizirao je acetilsalicilnu kiselinu koja je dobila ime aspirin. Uo eno je da je acetilsalicilna kiselina podnoaljivija za ~eludac od salicilne kiseline, pa po inje njena airoka primjena kao antipiretika i analgetika. (Slika 9.)  Slika 9. Shematski prikaz salicilne i acetilsalicilne kiseline (slika preuzeta iz literature br.16) Dr. Lawrence L. Craven 1948. propisao je aspirin skupini od 400 bolesnika i uo io da nijedan od njih nije dobio infarkt miokarda, te je postavio hipotezu da aspirin atiti od koronarne tromboze.(16) Tijekom godina provedena su brojna istra~ivanja o djelovanju ASK na nastanak koronarne tromboze i razjaanjen je njen mehanizam djelovanja. ASK je postala airoko upotrebljivan lijek s posebnim naglaskom na njen antiagregacijski u inak i od tada je airoko primjenjivana u prevenciji i lije enju akutnog koronarnog sindroma, tijekom koronarnih intervencija, u dugotrajnoj prevenciji kardiovaskularnih i cerebrovaskularnih bolesti, a navodi se i mogunost primarne prevencije u osoba s visokim rizikom za nastanak kardiovaskularnih bolesti (diabetes mellitus, PRV). Procijenjuje se da se godianje proizvede 40 000 tona ASK, a to odgovara broju od 125 biliona 325-mg tableta koje se utroae za prevenciju kardiovaskularnih i cerebrovaskularnih bolesti.(17) ASK se nakon peroralnog unosa brzo reapsorbira iz ~eluca i mo~e se detektirati u plazmi ve nakon 20 min, a vranu koncentraciju u plazmi posti~e nakon 30-40 min. Unutar 60 min po inje njeno inhibitorno djelovanje na agregaciju trombocita. ASK se u jetri pomou esteraza brzo razgrauje u inaktivni salicilat ija se koncentracija joa dugo mo~e detektirati u krvi i urinu pa se smatra da ASK posti~e svoj antitrombocitni u inak ve u portalnoj cirkulaciji, prije nego li se u jetri razgradi.(17) Na temelju brojnih istra~ivanja uo eno je da ve mala doza od 30 mg/dan ASK redovito uzimana tokom duljeg vremena smanjuje agregaciju trombocita za viae od 95% smanjujui tako i mogunost nastanka tromboti kog dogaaja za 25%.(18) Va~no je da doza ASK bude ato manja da se izbjegne nastanak gastrointestinalnog krvarenja, a da istovremeno bude dovoljno velika za postizanje antiagregacijskog u inka. Osim antiagregacijskog u inka koji se temelji na djelovanju na COX  1 i COX  2 ASK ima i brojne druge u inke koji nisu povezani s COX. (Tablica 3.) Tablica 3. Djelovanje acetilsalicilne kiseline neovisno o COX- aktivnosti U INAK NA TROMBOCITE djelomi na inhibicija ADP, P2Y1 i P2Y2 receptora blokiranje aktivacije NF-(B koja olakaava inhibiciju aktivacije trombocita posredovane neutrofilima ANTIUPALNI U INAKinhibicija otpuatanja reaktivnih kisikovih spojeva inhibicija nastajanja malondialdehida inhibicija nastajanja antitijela na oksidirani LDL - kolesterol inhibicija otpuatanja elastazeANTIOKSIDACIJSKI U INAKinhibicija nastanka oksidiranog LDL  kolesterola odstranjivanje hidroksilnih radikala poticanje sinteze feritina poticanje sinteze duaikovog oksida acetiliranje proteina i njihova zaatita od oksidacijeOSTALI U INCI spre avanje adhezije neutrofila i monocita inhibicija angiogeneze inhibicija ( - karboksilacije faktora zgruaavanja (II, VII, IX i X)Kod oateenja krvne ~ile in vivo zbog oslobaanja TF ili pri aktivaciji trombocita nekim od agonista in vitro (kolagen, trombin, epinefrin, ADP) dolazi do aktivacije fosfolipaze A2 koja oslobaa AA iz fosfolipidne membrane trombocita. AA se djelovanjem prostaglandin (PG) H-sintaze -1 i -2 takoer poznate i kao COX-1 i COX-2 prevodi u PGH2 koji se dalje pretvara u TxA2 koji je vazokonstriktor i agonist agregacije trombocita. (Slika 10.)  Slika 10. Put sinteze TxA2 na fosfolipidnoj membrani ASK inhibira djelovanje COX-1 i COX-2 ireverzibilnom acetilacijom serinskih ostataka na lancu enzima (Ser529 na COX-1 i Ser516 na COX-2) zbog ega enzimi gube svoju funkciju pa ne nastaje TxA2. S obzirom da trombociti nemaju jezgru oni ne mogu sintetizirati zna ajne nove koli ine COX, pa antiagregacijsko djelovanje ASK traje tijekom cijelog ~ivota trombocita, dakle oko 10 dana. ASK djeluje na COX-1 otprilike 166 puta djelotvornije nego na COX-2, pa i vrlo male zaostale aktivnosti COX-2 mogu inducirati agregaciju trombocita. Uz to, endotelne stanice i monociti mogu sintetizirati COX-2 i doprinjeti visokoj koncentraciji TxA2 unato  inhibiciji COX-1 terapijskim dozama ASK. TxA2 je in vivo nestabilan, brzo se hidrolizira na TxB2 i 11-dehydro TxB2. Budui da i do 30 % 11-dehydro TxB2 mo~e potjecati iz netrombocitnih izvora (npr. iz upalnih stanica) u razli itoj literaturi(19) se navodi da je serumski TxB2 najspecifi nija pretraga za mjerenje farmakoloakog u inka ASK na agregaciju trombocita. Netrombocitni izvori TxB2 Kod nekih bolesnika uo ene su visoke koncentracije TxB2 usprkos terapiji ASK. Budui da ASK inhibira djelovanje COX-1 u trombocitima za cijelog njihovog ~ivota u cirkulaciji, pretpostavlja se da postoje zna ajni ne-trombocitni izvori nastanka TxB2. Zaista, TxB2 mogu stvarati cirkulirajui monociti, endotelne stanice i makrofagi unutar aterosklerotskog plaka koji sintetiziraju COX-2, a ona pretvara AA u PGG2 i PGH2 i dalje do TxB2. U zdravlju aktivnost COX-2 je nemjerljiva, ali u stanjima poviaenih citokina, endotoksina i tumorskog rasta mo~e biti poja ano eksprimirana u razli itim tkivima, uklju ujui ateroskleroti ne ~ile. Osim upalnih faktora poja anu sintezu TxB2 mogu uzrokovati i reaktivni kisikovi radikali jer oni kataliziraju nastajanje F2-izoprostana koji su prekursori nastanku Tx, a nastanak F2- izoprostana iz AA neovisan je o COX-1, dakle ne podlije~e ni djelovanju ASK. Poviaene razine F2-izoprostana naene su kod puaa a, bolesnika s hiperkolesterolemijom i naro ito dijabetesom, pa je prema nekim autorima(13,20) uobi ajna doza od 100 mg/dan ASK preniska za bolesnike s dijabetesom. Antagonisti ADP-receptora: Tienopiridini Tienopiridini su prolijekovi koji se putem citokroma-P450 u jetri metaboliziraju do aktivnih metabolita. Oni kompetitivno i ireverzibilno inaktiviraju trombocitni receptor P2Y12 koji je receptor za ADP, pa posljedi no tome izostaje i agregacija trombocita. Stariji lijek iz te skupine, tiklopidin, danas je uglavnom zamijenjen lijekom nove generacije  klopidogrelom (Zylt, Plavix, Pigrel), zbog zna ajno manje ne~eljenih u inaka lijeka.(15) Trenutno je u zavranoj fazi klini kih ispitivanja i jedan novi lijek iz skupine tienopiridina, prasugrel, koji bi u odnosu na tiklopidin i clopidogrel mogao imati 10-100 puta ja i antiagregacijski u inak, br~i po etak i dulje trajanje djelovanja.(15) Antagonisti GPIIb/IIIa receptora Ovi lijekovi izazivaju vrlo brzu i potpunu inhibiciju agregacije te se ne koriste u prevenciji ve isklju ivo u lije enju akutnog infarkta miokarda  daju se intravenski u invazivnim kardioloakim postupcima (perkutana koronarna intervencija sa ili bez stenta, koronarna angioplastika) u kombinaciji s heparinom. Lijekovi iz ove skupine su: Abciximab, tirofiban (Aggrastat) i eptifibatide (Integrilin). INTERINDIVIDUALNA VARIJABILNOST NA ANTIAGREGACIJSKU TERAPIJU ACETILSALICILNOM KISELINOM Pretrage ispitivanja funkcije trombocita posljednjih se godina po inju sve viae primjenjivati za praenje djelotvornosti antiagregacijske terapije, a s ciljem individualizacije terapije (optimizacije doze i vrste lijeka ili kombinacije lijekova). Takav pristup mo~e osigurati uravnote~enu terapiju bez rizika od tromboze, ili druge krajnosti - rizika od krvarenja. Rezultati dosadaanjih istra~ivanja pokazali su da ista doza lijeka u razli itih osoba rezultira razli itim koncentracijama lijeka i/ili aktivnih metabolita, odnosno, da su razli iti u inci istog antitrombocitnog lijeka, pa se govori o tzv. interindividualnoj varijabilnosti ili rezistenciji na antitrombocitnu terapiju.(21) Pojam rezistencije ili neu inkovitog odgovora na lijek podrazumijeva nesposobnost lijeka da djeluje na svoj farmakoloaki cilj zbog razli itih uzroka, pa tako ASK-rezistencija predstavlja nesposobnost ASK da inhibira COX-1-ovisnu proizvodnju TxA2. U novijoj literaturi (18,21) pojam rezistencije na lijek podijeljen je na dva entiteta: klini ka rezistencija na lijek  podrazumijeva nastanak tromboti kog stanja unato  antitrombocitnoj terapiji laboratorijska rezistencija na lijek  podrazumijeva in vitro dokaz o neodgovarajuem djelovanju lijeka na agregaciju trombocita pri emu nije nu~no nastao tromboti ki dogaaj Uzroci nastanka neu inkovitosti terapije ASK-om mogu biti razli iti: farmakogeneti ki, okoliani, intrferirajui. Isti u se: smanjena bioraspolo~ivost ASK zbog neredovitog uzimanja lijeka ili uzimanja preniskih doza lijeka, slaba apsorpcija ASK iz probavnog sustava, interferencija ASK s drugim lijekovima (nesteroidnim upalnim lijekovima-NSAID) koji kompeticijom spre avaju acetilaciju i inhibiciju COX-1, nepotpuna supresija stvaranja TxA2, ubrzano stvaranje novih trombocita ija sposobnost sinteze TxA2 nije inhibirana ASK, stresom inducirana ekspresija COX-2 u trombocitima, polimorfizmi u genima za receptore i enzime (COX-1, COX-2, TxA-sintazu) i vraanje sposobnosti cirkulirajuim trombocitima koji su bili pod utjecajem ASK da sintetiziraju TxA2 iz prostaglandina H2 osloboenog iz endotelnih stanica i monocita. S obzirom na multi imbeni nu prirodu aterotromboze ispravnijim se smatra pojam interindividualna varijabilnost u odgovoru na lijek nego pojam rezistencije na lijek. Danas je u uporabi nekoliko razli itih pretraga temeljenih na razli itim metodologijama za praenje u inka terapije ASK-om, no usporedivost razli itih pretraga pokazuje vrlo slabu povezanost, pa je tako u razli itim studijama incidencija neu inkovitosti terapije ASK-om iznosila 5,5-61%. Nasuprot tome studije u kojima je mjerena koncentracija TxB2 konzistentnije su i navode incidenciju neu inkovitosti terapije ASK-om od 1,0-1,7%.(19) Neu inkovit odgovor na lijek ili  rezistencija na lijek mo~e se dokazati samo pretragom koja specifi no mjeri farmakoloako djelovanje tog lijeka. Neki radovi podupiru izglede da bi pretraga odreivanja koncentracije TxB2 mogla biti specifi na pretraga za praenje terapije ASK-om, dok kao (zlatni analiti ki standard( joa uvijek vrijedi opti ka agregometrija; metoda koja je predanaliti ki dugotrajna i zahtjevna, a provodi se samo u specijaliziranim laboratorijima.(19) Idealna pretraga za praenje antiagregacijske terapije trebala bi biti jeftina, jednostavna za izvoenje, brza, reproducibilna, to na i standardizirana tako da se rezultati dobiveni u razli itim laboratorijima mogu usporeivati. Budui da ni jedna pretraga nije ostvarila rutinsku primjenu, praenje antiagregacijske terapije za sada se u najveoj mjeri provodi samo u istra~iva ke svrhe. AKUTNI KORONARNI SINDROM Akutni koronarni sindrom (AKS) predstavlja niz klini kih stanja u rasponu od nestabilne angine do akutnog infarkta miokarda (AIM). U patofizioloakoj podlozi (osnovi) AKS-a je ateroskleroza ili kroni na upalna bolest stijenke krvne ~ile, ijom progresijom dolazi do rupture aterosklerotskog plaka i posljedi ne tromboze koja se o ituje akutnim infarktom miokarda. Ateroskleroza naj eae zahvaa srednje i velike arterije, a nastaje kao posljedica oateenja ili disfunkcije endotela krvne ~ile. Kao mogui uzrok oateenja ili disfunkcije endotela navodi se hiperkolesterolemija, poviaena koncentracija LDL-kolesterola, slobodni radikali, dim cigarete, hiperhomocisteinemija, glikozilirane molekule te virusne i bakterijske infekcije. Disfunkcija endotela podrazumijeva poveanje permeabilnosti endotela za lipoproteine (naro ito oksidirane LDL estice koje su vrlo aterogene i ijom fagocitozom od strane makrofaga nastaju tzv. spu~vaste stanice koje ine najraniju morfoloaki prepoznatljivu ateroskleroti nu leziju  masnu prugu). Osim spu~vastih stanica masna pruga sadr~i i citokine i upalne medijatore koji daljnjim razvojem upale vode nastanku fibroznog plaka. U razvijenim zemljama svijeta uo eno je da masna pruga nastaje ve u najranijoj dobi tj. kod djece. Endotel krvne ~ile postaje sve tanji i disfunkcionalan ato poveava izlo~enost spu~vastih stanica i makrofaga cirkulirajuoj krvi, a to pak omoguava interakciju tih elemenata s trombocitima uzrokujui njihovu aktivaciju, a onda i adheziju i agregaciju pri emu nastupa tromboza. Tromboza zapo inje rupturom aterosklerotskog plaka i izlaganjem trombogenog materijala u subendotel, a u ovom procesu trombociti imaju klju nu ulogu. Na mjestu nastanka tromboze smanjena je opskrba sr anog miaia krvlju, odnosno kisikom i nutritijentima zbog ega nastupa ishemija sr anog miaia tj. akutni infarkt miokarda esto sa smrtnim posljedicama. Kardiovaskularne bolesti vodei su uzrok pobola i smrtnosti u razvijenim zemljama, pa tako i u Hrvatskoj (svaki drugi stanovnik umire od bolesti srca i krvnih ~ila).(22) imbenici rizika za nastanak aterosklerotskog plaka su: ~ivotna dob (vei rizik s poveanjem godina starosti), spol ( eae obolijevaju muakarci, a ~ene nakon menopauze), puaenje, aeerna bolest, hiperlipidemija, sjedila ki na in ~ivota, hipertenzija i stres, a va~nu ulogu imaju i genski imbenici u kombinaciji sa imbenicima okoliaa.(23) Prijaanjih godina smatralo se da je ateroskleroza bolest nakupljanja lipida. U danaanje vrijeme postalo je o ito da je upravo upala stijenke krvne ~ile klju an imbenik koji dovodi do nastanka ateroskleroze, nestabilnosti ateroma i rupture aterosklerotskog plaka koja je praena lokalnom trombozom, a to je preduvjet za nastanak AIM.(22) Aortokoronarno premoatenje Aortokoronarno premoatenje - CABG (engl. coronary artery bypass grafting) je operativna metoda lije enja sr ane bolesti koja je nastala kao posljedica za epljenja koronarne arterije (uslijed ateroskleroze) ime je sprije en dotok kisika i nutritijenata sr anom miaiu. Sama metoda sastoji se od premoatenja za epljene koronarne arterije zdravom krvnom ~ilom koja potje e iz ruke, noge ili prsa bolesnika, a koja povezuje aortu i koronarnu arteriju uspostavljajui normalan dotok krvi u sr ani miai. Bolesnicima kojima je u injeno aortokoronarno premoatenje do~ivotno je u terapiju uvedena ASK kako bi se izbjegao ili barem smanjio rizik od ponovnog za epljenja koronarnih arterija i ponovnog nastanka akutnog infarkta miokarda. Antitrombocitna terapija ASK-om ve u dozi od samo 30 mg/dan tokom duljeg vremenskog perioda kod takvih bolesnika smanjuje rizik ponovnog nastanka za epljenja koronarne arterije za 25%.(24,25,26) CILJ ISTRA}IVANJA Aterotromboti ke bolesti glavni su uzrok smrtnosti u razvijenim zemljama. Antiagregacijska terapija zato je vrlo va~na u preventivi i terapijskom lije enju navedenih bolesti. Unato  antitrombocitnoj terapiji kod zna ajnog broja bolesnika doalo je do ponovljenog tromboti nog dogaaja. Cilj ovog magistarskog rada je odrediti individualnu varijabilnost funkcije trombocita kod bolesnika na antiagregacijskoj terapiji ASK-om dvjema razli itim metodama. Specifi ni ciljevi su: Ispitati funkcionalnu aktivnost trombocita u bolesnika s CABG na antiagregacijskoj terapiji ASK-om i kod zdravih ispitanika s dvije razli ite metodologije: metodom impedancijske agregometrije iz pune krvi metodom mjerenja koncentracije TxB2 u serumu enzimimunokemijskom metodom Usporediti vrijednosti funkcionalne aktivnosti trombocita dobivenih dvjema metodama. Odrediti stupanj laboratorijske rezistencije na terapiju ASK-om tj. udio bolesnika s neu inkovitim odgovorom na antiagregacijsku terapiju ASK-om pomou specifi ne metode mjerenja koncentracije TxB2 u serumu i usporediti ga s onim dobivenim metodom impedancijske agregometrije. Ispitati postoji li korelacija izmeu broja trombocita i vrijednosti agregacije trombocita odreene impedancijskom agregometrijom, i broja trombocita i koncentracije TxB2 u serumu. Ispitati postoji li korelacija izmeu srednjeg volumena trombocita (MPV-a) i vrijednosti agregacije trombocita odreene impedancijskom agregometrijom, i MPV-a i koncentracije TxB2 u serumu. MATERIJAL I METODE MATERIJAL Ispitanici U istra~ivanje je uklju eno 110 bolesnika koji su nakon u injenog CABG-a upueni na kardioloaki odjel Specijalne bolnice za medicinsku rehabilitaciju u Krapinskim Toplicama na trotjednu rehabilitaciju. Kriteriji uklju ivanja bolesnika bili su: ASK u dozi ( 150mg/dan uzimanje lijeka najmanje 30 dana prije uklju ivanja u istra~ivanje odsutnost infekta (CRP < 5 mg/L) terapija bez steroidnih antireumatika terapija bez lijekova iz skupine tienopiridina Vaenje krvi obavljeno je pri dolasku na rehabilitaciju. Pri intervjuiranju bolesnika u svrhu dobivanja informiranog pristanka uo eno je da petoro bolesnika ne troai redovito ASK, pa su oni isklju eni iz istra~ivanja. Skupina 1  105 bolesnika s medijanom starosti 65 godine (40  85 godina) koji su zadovoljili gore navedene kriterije. Skupina 0  30 zdravih ispitanika s medijanom starosti 40 godina (21  75 godina) koji posljednjih 30 dana nisu uzimali nikakve lijekove. Svi ispitanici upoznati su sa svrhom istra~ivanja i potpisali su informirani pristanak o sudjelovanju u istra~ivanju, a Eti ko povjerenstvo Specijalne bolnice za medicinsku rehabilitaciju u Krapinskim Toplicama odobrilo je istra~ivanje. Uzorkovanje krvi Kod obje skupine ispitanika uzimana je venska krv iz kubitalne vene ujutro izmeu 7.00 i 9.00 sati. Krv za odreivanje TxB2 uzimana je u epruvete bez antikoagulansa, centrifugirana je 10 min na 1000 g, a serum je pohranjen na -80(C dva mjeseca do trenutka odreivanja kada su svi uzorci istovremeno odmrznuti. Krv za odreivanje agregacije trombocita uzimana je u epruvete koje su sadr~avale heparin kao antikoagulans, a analiza se izvodila unutar 30 do 180 min od venepunkcije krvi (preme preporukama proizvoa a Multiplate analizatora). Krv za odreivanje broja trombocita i MPV-a uzimana je u epruvete s K-EDTA kao antikoagulansom i analizirana unutar dva sata. METODE Broj trombocita i MPV Broj trombocita i MPV odreeni su na hematoloakom broja u Cell Dyn 1800 (Abbott Diagnostics, Abbott park, IL, USA). Referentni interval za broj trombocita iznosi 158 - 424 x 109/L. (27) Referentni interval za MPV iznosi 6,8  10,4 fL. (27) Agregacija trombocita Agregacija trombocita mjerila se impedancijskom agregometrijom iz pune krvi (uz heparin kao antikoagulans) na analizatoru Multiplate (ASPI-test, Dynabyte medical Munchen, Germany). Na elo pretrage je sljedee: dodatkom agonista (arahidonske kiseline: ASPI-test) uzorku pune krvi dolazi do aktivacije trombocita koji izra~avaju tendenciju adhezije i agregacije na elektrode senzora u mjernoj posudici, a mjerenje promjene elektri nog otpora na elektrodi daje mjeru agregacije trombocita izra unatu kao povraina ispod agregacijske krivulje (AUC) u dogovornim jedinicama (U). Svaka mjerna posudica ima ugraene dvije nezavisne senzorske jedinice koje se sastoje od po dvije srebrom presvu ene bakrene elektrode i analizator mjeri promjenu impedancije svakog senzora zasebno. Ra unalo analizatora izra unava koeficijent korelacije mjerenja na oba senzora, pa ako je koeficijent manji od 0,98 analizator daje oznaku i preporu a se ponavljanje mjerenja. AA sama po sebi nije agonist trombocita ve se ona pomou COX-1 i COX-2 pretvara u TxA2 koji jeste agonist trombocita i koji omoguuje njihovu agregaciju. Budui da ASK inhibira djelovanje COX-1 i COX-2 ovom pretragom omoguena je osjetljiva i specifi na detekcija djelovanja aspirina, andola i drugih lijekova koji kao djelatnu tvar sadr~e ASK. Vrijednost od 10 AU/min predstavlja aktivnost od 1 U. Grani na vrijednost dijagnosti ke odluke prema proizvoa u je 30 U ato zna i da se za vrijednosti ( 30 U potvruje da je terapija ASK-om djelotvorna.  Slika 11. Prikaz grafa agregacije trombocita na analizatoru Multiplate za osobu koja nije na terapiji ASK-om, AUC = 128 U  Slika 12. Prikaz grafa agregacije trombocita na analizatoru Multiplate za bolesnika na terapiji ASK-om, AUC = 25 U, terapija je djelotvorna. Koncentracija TxB2 u serumu Koncentracija TxB2 u serumu odreivala se kompetitivnim EIA testom tvrtke Cayman Chemical, USA. Princip odreivanja je sljedei: TxB2 iz seruma natje e se sa TxB2-AChE konjugatom za ograni en broj veznih mjesta na TxB2-ze jem antiserumu. 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"68<>@z|~$dha$$dha$$ & F-dh`a$)j.@":>@46ƹV&(08ػټ|||xphSmHsHh hSH*mHsHhSaJmHsHhSmHsHhS5\mHsHh?h aJmHsHh?hSaJmHsHhSCJmHsH jhS hSH* hS5\hSh mHsHhS5\mHsHhSmHsHhSmHsH'Ĺƹ:ֻػZPR  ,QR_$dh[$\$a$$`a$$a$$dha$$dha$f<>|]^_h  %() |~>@TVjnĽUhS5H*\ hSH*hSmHsHj-0hS5U\ hS5hS hS5\jӲhS5U\ j<hSmHsHhSH*mHsHh mHsHhSmHsHhSB*mHphsH9,.vx$0dh^`0a$$dh[$\$a$ $dh`a$$dha$ta stalna, a koncentracija TxB2 iz seruma varira, koli ina vezanog TxB2-AChE obrnuto je proporcionalna koli ini vezanog TxB2 iz seruma. Kompleks ze jeg seruma na koji je vezan TxB2 (bilo TxB2-AChE ili TxB2 iz seruma) ve~e se na anti miaja IgG-At kojima su oblo~ene ja~ice reakcijske posudice. Viaak nevezanih komponenti se ispere, a koncentracija vezanog TxB2-AChE konjugata odredi se dodatkom Ellman-ovog reagensa i obrnuto je proporcionalna koncentraciji TxB2 u serumu. Ellmanov reagens sastoji se od acetiltiokolina i 5,5(-ditio-bis-(2-nitrobenzojeve kiseline). AChE hidrolizira acetiltiokolin u tiokolin, a neenzimatska reakcija tiokolina i 5,5(-ditio-bis-(2-nitrobenzojeve kiseline) daje 5-tio-2-nitrobenzojevu kiselinu ija se opti ka gustoa mjeri na 412 nm. Opti ka gustoa mjerila se na aparatu Humareader tvrtke HUMAN, GmbH, Wisbaden, Germany. Vrijednosti koncentracije TxB2 izra unate su pomou analiti kog alata tvrtke Cayman Chemical koja je ujedno i proizvoa  testa za EIA odreivanje koncentracije TxB2. Statisti ke metode Za prikaz rezultata i statisti ku analizu koriateni su programski alati Microsoft Excel i MedCalc v.11.5.1. Dobiveni podatci opisani su deskriptivnom analizom, a skupovi podataka ispitani su na normalnost raspodjele Kolmogorov-Smirnovljevim statisti kim testom. Sve varijable koje su koriatene u statisti koj analizi, a slijedile su normalnu Gaussovu raspodjelu opisane su aritmeti kom sredinom ( EMBED Equation.3 ) i standardnom devijacijom (SD), dok su varijable koje nisu imale normalnu raspodjelu opisane medijanom i rasponom. Budui da se radilo o nezavisnim varijablama za njihovu usporedbu koriaten je parametrijski nezavisni t-test, odnosno neparametrijski Mann-Whitneyev test i 2 test za usporedbu kategori kih podataka. Povezanost izmeu pojedinih varijabli (budui da su varijable numeri ke) opisani su pomou Pearsonovog koeficijenta korelacije r za podatke koji su slijedili normalnu raspodjelu i pomou Spearmanovog koeficijenta korelacije za podatke koji nisu slijedili normalnu raspodjelu. Koeficijenti korelacije vrednovani su prema Coltonu kako je navedeno u Tablici 4. Koeficijenti korelacije r testirani su na razinu statisti ke zna ajnosti uz p vrijednost (vrijednosti p ( 0,05 smatrane su statisti ki zna ajnima).(28) Analizom ROC-krivulje (engl. Reciever Operating Characteristic Curve) odreena je dijagnosti ka zna ajnost (osjetljivost i specifi nost) rezultata dobivenih za izmjerene koncentracije TxB2. Izra unavanjem povraine ispod krivulje, AUC, (engl. Area Under the Curve) stje e se dojam o u inkovitosti u injenog testa. Odnos izmeu povraine ispod krivulje i dijagnosti ke to nosti prikazan je u Tablici 5. Tablica 4. Tuma enje koeficijenta korelacije r RPovezanost0 do 0,25 ili 0 do  0,25nema povezanosti0,26 do 0,50 ili  0,26 do  0,50 slaba povezanost0,51 do 0,75 ili  0,51 do  0,75umjerena do dobra povezanost0,76 do 1 ili  0,76 do -1vrlo dobra do izvrsna povezanost Tablica 5. Odnos povraine ispod ROC-krivulje i dijagnosti ke to nosti povraina ispod ROC-krivuljedijagnosti ka to nost0,9  1,0izvrsna0,8  0,9vrlo dobra0,7  0,8dobra0,6  0,7dovoljna0,5  0,6loaa< 0,5nedovoljna REZULTATI ISPITANICI U istra~ivanje je ukupno bilo uklju eno 110 bolesnika i 30 zdravih ispitanika, no ustanovljeno je da petoro bolesnika nije redovito uzimalo terapiju ASK pa su oni isklju eni iz statisti ke obrade prema kriterijima uklju enja. Demografske osobine bolesnika i zdravih ispitanika navedene su u Tablici 6. Bolesnici su se statisti ki zna ajno razlikovali od zdravih ispitanika i po dobi (p < 0,0001) i po spolu (p < 0,0001). Tablica 6. Demografske osobine bolesnika (skupina 1) i zdravih ispitanika (skupina 0). SKUPINA 1 (N = 105)SKUPINA 0 (N = 30)DOB, medijan (raspon)65 (40  86)40 (21  76)p < 0,0001Muakarci, N(%)88 (83,8%)4 (13,3%)p < 0,0001}ene, N(%)17 (16,2%)26 (86,7%)p < 0,0001 Tablica 7. Vrijednosti broja TRC, MPV-a, agregacije TRC i koncentracije TxB2 kod bolesnika s CABG (skupina 1) i zdravih ispitanika (skupina 0). SKUPINA10 EMBED Equation.3 SDRaspon EMBED Equation.3 SDRasponpTRC(x109/L)22060,288-46027044,3180-3700,0001MPV10,21,257,7-14,79,90,977,3-11,60,4144MedijanMinimun MaximumMedijanMinimumMaksimumagreg.TRC(U)2891129176128< 0,0001TxB2(pg/ml)28,82,13004,76022,62174,814543,2< 0,0001 Tablica 8. Udio laboratorijske rezistencije na terapiju ASK-om u skupini bolesnika s CABG (N = 105) broj i udio bolesnika s neu inkovitom terapijom ASK-omrezistAbroj bolesnika s impedancijskom agregometrijom >30 U (granicu odluke od 30 U postavio je proizvoa  analizatora)44 (41,9%)rezistBbroj bolesnika s konc. TxB2 >343,4 pg/ml (granica odluke od 343,4 pg/ml izra unata kao 95 %-tno smanjenje srednje vrijednosti konc. TxB2 u skupini zdravih ispitanika) 9 (8,6%)rezistCbroj bolesnika s konc. TxB2 >654,5 pg/ml (granica odluke od 654,5 pg/ml izra unata je kao srednja vrijednost konc. TxB2 u skupini bolesnika uveana za dvije standardne devijacije prema preporuci Frelingera i sur.) (24) 9 (8,6%)rezistDbroj bolesnika s konc. TxB2 >3100 pg/ml (granicu odluke od 3100 pg/ml predlo~ili Frelinger i sur.) (40) 0 (0%)  EMBED Excel.Chart.8 \s  Slika 13. Laboratorijska rezistencija na terapiju ASK-om odreena impedancijskom agregometrijom (rezistA) i odreena mjerenjem koncentracije TxB2 (rezistB, rezistC i rezistD). BROJ TROMBOCITA Broj trombocita kod bolesnika s CABG statisti ki se zna ajno razlikuje (p = 0,0001) i ima airi raspon vrijednosti (88 x 109/L - 460 x 109/L) od broja trombocita kod zdravih ispitanika (180 x 109/L - 380 x 109/L). (Slika 14.)  Slika 14. Broj trombocita u bolesnika s CABG (skupina 1) i zdravih ispitanika (skupina 0). (p = 0,0001) MPV - SREDNJI VOLUMEN TROMBOCITA Usporeujui vrijednosti MPV-a kod bolesnika s CABG (skupina 1) i zdravih ispitanika (skupina 0) vidljivo je da nema statisti ki zna ajne razlike (p = 0,4144). (Slika 15.)  Slika 15. Vrijednosti MPV-a u bolesnika s CABG (skupina 1) i zdravih ispitanika (skupina 0).(p = 0,4144) VRIJEDNOSTI AGREGACIJE TROMBOCITA ODREENE IMPEDANCIJSKOM METODOM Vrijednosti agregacije trombocita izmjerene impedancijskom metodom na Multiplate analizatoru izmeu bolesnika (skupina 1) i zdravih ispitanika (skupina 0) statisti ki se zna ajno razlikuju (p ( 0,0001) i prikazane su na Slici 16.  Slika 16. Vrijednosti agregacije trombocita kod bolesnika s CABG (skupina 1) i zdravih ispitanika (skupina 0). (p ( 0,0001). Crveni kvadratii prikazuju vrijednosti agregacije trombocita koje su tri puta vee od interkvartilnog raspona. KONCENTRACIJA TROMBOKSANA TxB2 Koncentracija TxB2 izmjerena u serumu bolesnika s CABG (skupina 1) statisti ki je zna ajno manja (p ( 0,0001) od koncentracije TxB2 izmjerene u serumu zdravih ispitanika (skupina 0). (Slika 17.)  Slika 17. Koncentracija TxB2 izmjerena u bolesnika s CABG (skupina 1) i kod zdravih ispitanika (skupina 0). (p ( 0,0001). Crveni kvadratii prikazuju vrijednosti koncentracije TxB2 koje su tri puta vee od interkvartilnog raspona. POVEZANOST BROJA TROMBOCITA I MPV -a S U INKOVITOSTI TERAPIJE ASK-om Prikaz rezultata meuovisnosti MPV-a i vrijednosti agregacije trombocita, koncentracije TxB2 i broja trombocita nalazi se u Tablici 9. Prikaz rezultata meuovisnosti broja trombocita i vrijednosti agregacije trombocita i koncentracije TxB2 nalazi se u Tablici 10. U Tablicama 9. i 10. navedene su i vrijednosti koeficijenta korelacije r i statisti ka zna ajnost koeficijenta korelacije p. Tablica 9. Povezanost MPV-a s vrijednoau agregacije trombocita, koncentracije TxB2 i broja trombocita u skupini bolesnika s CABG agreg.TRC(U)TxB2(pg/ml)br.TRC(x109/L)MPV (fL)r p 0,217 0,0261 0,197 0,0442 - 0,307 0,0014 Tablica 10. Povezanost broja trombocita s agregacijom trombocita i koncentracijom TxB2 u skupini bolesnika s CABG. agreg.TRC(U)TxB2(pg/ml)br.TRC(x109/L)r p 0,174 0,0755 0,054 0,5839  Slika 18. Korelacija broja trombocita i srednjeg volumena trombocita kod bolesnika s CABG (r = - 0,307; p = 0,0014; R2 = 0,094).  Slika 19. Korelacija vrijednosti agregacije trombocita i koncentracije TxB2 kod bolesnika s CABG (r = 0,443; p< 0,0001; R2 = 0,196). DIJAGNOSTI KA ZNA AJNOST REZULTATA Da bi se odredila dijagnosti ka zna ajnost (osjetljivost i specifi nost) rezultata dobivenih za test odreivanja koncentracije TxB2 u injena je ROC-analiza. Rezultati ROC-analize pokazali su da odreivanje koncentracije TxB2 ima veu dijagnosti ku specifi nost nego osjetljivost. Najbolja osjetljivost i specifi nost postignuti su za grani nu vrijednost koncentracije TxB2 130,1 pg/ml. (Slika 20.)  Slika 20. ROC-krivulja za koncentraciju TxB2 (povraina ispod krivulje AUC = 0,854; p < 0,0001; dijagnosti ka osjetljivost je 64,5%; a dijagnosti ka specifi nost 95% za grani nu vrijednost koncentracije TxB2 = 130,1 pg/ml). RASPRAVA Kardiovaskularne bolesti vodei su uzrok pobola i smrtnosti u razvijenim zemljama svijeta i predstavljaju veliki socio-ekonomski problem. Budui da je u patofizioloakoj podlozi kardiovaskularnih bolesti ateroskleroza, multi imbeni na bolest ovisna o brojnim imbenicima (~ivotna dob, spol, puaenje, aeerna bolest, hiperlipidemija, sjedila ki na in ~ivota, hipertenzija, stres, genski imbenici i imbenici okoliaa), va~no je voditi ra una o svakom podjednako kako bi se izbjegao rizik od nastanka AKS odnosno AIM. No, ako je ipak doalo do nastanka AIM, a bolesnik je pre~ivio, cilj je sprije iti ponovni nastanak AIM koji bi u ponovljenom slu aju mogao biti fatalan za bolesnika. U tu svrhu bolesnicima koji su preboljeli AIM do~ivotno se u terapiju uvode antiagregacijski lijekovi (npr. ASK ili klopidogrel ili ASK + klopidogrel) jer je razli itim istra~ivanjima dokazano da se antiagregacijskom terapijom smanjuje rizik od ponovnog nastanka AIM za 25%.(24,25,26) Tokom godina primjeeno je da je kod odreenog broja bolesnika (10 - 20%)(29,40) unato  antiagregacijskoj terapiji doalo do ponovnog nastanka AIM. Uo eno je da ista vrsta i doza lijeka kod razli itih bolesnika rezultira razli itom koncentracijom lijeka i/ili aktivnih metabolita, pa se tu govori o interindividualnoj varijabilnosti na lijek. S obzirom da praenje antiagregacijske terapije do sada nije bilo rutinski primjenjivano, lije nici su pribjegavali tzv. dvojnoj terapiji (ASK + klopidogrel) da bi bolesnike dodatno zaatitili od nastanka tromboti kog stanja. No, takva dvojna terapija poveala je rizik od nastanka gastrointestinalnog krvarenja i znatno je poveala troakove lije enja. Tableta Andola 100 (koja sadr~i 100 mg ASK) 27 puta je jeftinija od jedne tablete Zylta (koja sadr~i 75 mg klopidogrela dovoljnog za dnevnu terapiju). S obzirom na poveanje rizika od nastanka krvarenja i poveanje troakova lije enja ula~u se znatni napori za pronalazak pogodne pretrage za praenje funkcionalne aktivnosti trombocita. Danas je u uporabi nekoliko razli itih pretraga temeljenih na razli itim metodologijama za praenje antiagregacijske terapije ASK-om, no usporedivost tih pretraga vrlo je mala. Razli itim pretragama dobiveni su i razli iti podatci o incidenciji nedjelotvornosti terapije ASK-om koji su u rasponu 1,7%  61%. Cilj ovog magistarskog rada bio je odrediti stupanj nedjelotvornosti terapije ASK-om dvjema razli itim metodama: metodom impedancijske agregometrije, i mjerenjem koncentracije TxB2 u serumu za koju se smatra da bi mogla biti specifi na pretraga za praenje terapije ASK-om budui da specifi no mjeri farmakoloako djelovanje ASK. Vrlo je malo radova u svijetu, a u Hrvatskoj ni jedan, koji su mjerenjem koncentracije TxB2 u serumu procijenjivali funkcionalnu aktivnost trombocita. Budui da se tom pretragom prati farmakoloako djelovanje ASK, autori tih radova Frelinger i sur.(24), Cattaneo(30), Cox i sur.(31), Eriksson i sur.(32), Muir i sur.(33) smatraju da bi upravo odreivanje koncentracije TxB2 u serumu mogla postati najbolja pretraga za praenje terapije ASK-om. U dosadaanjim istra~ivanjima Push i sur.(29) i Karon i sur.(34) uo ili su da je nedjelotvoran odgovor na terapiju ASK-om mnogo manji kod zdravih ispitanika nego kod bolesnika s koronarnim bolestima. Objaanjenje je vrlo jednostavno: bolesnici s koronarnim bolestima imaju u svojoj anamnezi itav niz drugih bolesti (npr. dijabetes, hiperlipoproteinemiju, hipertenziju) pa u svojoj terapiji osim ASK imaju i itav niz drugih lijekova (antidijabetike, statine, antihipertenzive). Puno razli itih lijekova vjerojatno meusobno interferira ato se odra~ava i na djelotvornost ASK. Interesantno je da se lije enje dijabetesa, hiperlipoproteinemije ili hipertenzije laboratorijski prati odreivanjem glukoze i lipida u krvi ili mjerenjem krvnog tlaka, te se prema dobivenim rezultatima mijenja doza ili vrsta lijeka. Kod antiagregacijske terapije to nije tako. Paradoks je to vei ato se zna da postoji zna ajna interindividualna varijabilnost u djelovanju ASK. Laboratorijsko praenje terapije ASK-om do sada nije bilo uobi ajeno. Jedan od razloga je nepostojanje klini ki primjenjive pretrage koja bi bila brza, jednostavna za izvoenje, reproducibilna, to na i standardizirana. Naae istra~ivanje obuhvatilo je 105 bolesnika s CABG koji su bili na terapiji ASK d" 150 mg/dnevno i 30 zdravih ispitanika koji nisu uzimali nikakve lijekove. Svima je odreena funkcionalna aktivnost trombocita pomou obje metode: impedancijskom agregometrijom i mjerenjem koncentracije TxB2 u serumu. Skupina bolesnika i skupina zdravih ispitanika zna ajno su se razlikovale i po spolu (p < 0,0001) i po dobi (p < 0,0001). Neujedna enost skupina mogue je utjecala na naae rezultate koji se u potpunosti ne podudaraju s literaturnim. Skupina zdravih ispitanika poslu~ila je u ovom istra~ivanju za izra unavanje grani ne vrijednosti iznad koje smo smatrali da terapija ASK-om nije djelotvorna. Na temelju dosadaanjih istra~ivanja Frelingera i sur.(24), Lordkipanidzea i sur.(25) i Zimmermanna i sur.(35) zaklju eno je da je cilj redovite terapije od 30 100 mg/dnevno ASK postii smanjenje funkcionalne aktivnosti trombocita za ( 95%. Samo u tom slu aju mo~e se smatrati da je terapija ASK-om uspjeana i da je nastanak ponovnog tromboti kog dogaaja smanjen za 25%. Izra unavanjem srednje vrijednosti koncentracije TxB2 u skupini zdravih ispitanika izra unato je i 95%-tno smanjenje nastanka TxB2 pa je granica odluke izra unata na taj na in iznosila 343,4 pg/ml. Za sve bolesnike s koncentracijom TxB2 ( 343,4 pg/ml smatrali smo da je terapija ASK-om nedjelotvorna. Frelinger i sur.(24) su u svom radu objavljenom 2006. godine granicu odluke za koncentraciju TxB2 izra unali kao srednju vrijednost koncentracija TxB2 svih bolesnika na terapiji ASK-om (N=700) uveanu za dvije standardne devijacije. Izra unavanjem granice odluke na takav na in za naau skupinu ispitanika granica je postavljena na koncentraciju TxB2 od 654,5 pg/ml. Isti autori su 2009. godine objavili rad(40) u kojem su koristei podatke bolesnika iz prethodnog rada(24) pomou ROC-analize izra unali granicu odluke za koncentraciju TxB2 koja je iznosila 3100 pg/ml. Primijenivai vrijednost od 3100 pg/ml kao granicu odluke za naau skupinu bolesnika nijedan bolesnik nema koncentraciju TxB2 ( 3100 pg/ml ato zna i da u naaoj skupini bolesnika (N=105) nema bolesnika kod kojeg terapija ASK-om nije djelotvorna. Is itavanjem iz grafa (Slika 17.) vidljivo je da je u promatranom uzorku od 105 bolesnika s CABG njih 9 imalo koncentraciju TxB2 ( 343,4 pg/ml ato je 8,6%. To zna i da kod 8,6% bolesnika terapija ASK-om nije djelotvorna. Isto tako, ukoliko kao granicu odluke odredimo 654,5 pg/ml, ponovno istih 9 bolesnika ima koncentraciju TxB2 veu od granice odluke ato ukazuje da terapija ASK-om kod tih 8,6% bolesnika nije djelotvorna. Ako pak za granicu odluke uzmemo vrijednost od 3100 pg/ml tada postotak bolesnika kod kojih terapija ASK-om nije djelotvorna iznosi 0%. Analizom vrijednosti agregacije trombocita (Slika 16.) vidljivo je da je u uzorku od 105 bolesnika njih 44 imalo vrijednost agregacije trombocita ( 30 U ato je 41,9%. To zna i da kod 41,9% bolesnika terapija ASK-om nije djelotvorna. Svi bolesnici su na terapiji ASK-om pa je za o ekivati da je funkcionalna aktivnost trombocita zbog inhibicije COX-1 suprimirana, tako da je vrijednost njihove agregacije trombocita s ASPI- testom (Slika 16.) i koncentracija TxB2 (Slika 17.) zna ajno manja nego kod zdravih ispitanika koji nisu na nikakvoj terapiji (p < 0,0001). Rezultati su sukladni o ekivanjima. Broj trombocita u skupini bolesnika ima airi raspon i statisti ki se zna ajno razlikuje (p = 0,001) od broja trombocita u skupini zdravih ispitanika. (Slika 14.) Poznato je da na broj trombocita utje u brojni lijekovi, transfuzija krvi, splenomegalija, metoda operativnog lije enja. To su sve stanja koja prate bolesnike s CABG. Zimmermann i sur.(35) su u svom istra~ivanju primijetili da je kod bolesnika kojima je u injen CABG nakon nekoliko dana doalo do porasta broja trombocita (reaktivna trombocitoza). Poja ano stvaranje trombocita utje e na slabiji u inak ASK na agregaciju trombocita. Polu~ivot ASK u plazmi je oko 30 min, a budui da se trombociti poja ano stvaraju, nastaju i nove koli ine COX-1 koja nije inhibirana ASK-om, a koja uzrokuje nastanak TxA2 i dovodi do agregacije trombocita. Kod takvih bolesnika preporu a se viae od jedne doze 100mg ASK na dan i praenje antiagregacijske terapije.(35) U Tablici 10. vidljivo je da nema povezanosti izmeu broja trombocita i agregacije trombocita (r = 0,174; p = 0,0755), ni povezanosti izmeu broja trombocita i koncentracije TxB2 (r =0,054; p = 0,5839). Izmeu broja trombocita i MPV-a postoji slaba negativna povezanost (r = - 0,307), koja je statisti ki zna ajna (p = 0,0014). (Slika 18.) Izmeu vrijednosti MPV-a kod bolesnika s CABG i zdravih ispitanika nema statisti ki zna ajne razlike (p = 0,4144). (Slika 15.) Usporeujui vrijednosti MPV-a s vrijednostima agregacije trombocita odreene impedancijskom metodom na Multiplate analizatoru vidljivo je da nema povezanosti (r = 0,217; p = 0,0261). (Tablica 9.) Usporeujui vrijednosti MPV-a s koncentracijom TxB2 takoer nema povezanosti (r = 0,197; p = 0,0442). (Tablica 9.) To upuuje na zaklju ak da bez obzira na veli inu trombocita ASK inhibira ( 95% aktivnosti COX-1. Problem nastaje ako je bolesnik zaista rezistentan na djelovanje ASK, a uz to ima i povean MPV. Veliki trombociti sadr~e viae gustih tijela i oni su metaboli ki i enzimatski mnogo aktivniji od malih trombocita i imaju povean tromboti ki potencijal ato poveava rizik nastanka agregacije trombocita. (36) U radovima Aksua i sur.(36), Erharta i sur.(37) i Martina i sur.(38) navodi se da je aspirinska rezistencija u kombinaciji s poveanim MPV-om zna ajno loaiji prognosti ki parametar za ponovni AIM i smrtni ishod. Takoer se navodi da je MPV u novije vrijeme postao biljeg kardiovaskularnih bolesti. Poviaen je u AIM, nestabilnoj angini pektoris i akutnom ishemi nom mo~danom udaru. S obzirom da u skupini naaih bolesnika ima osoba koje imaju laboratorijsku rezistenciju na ASK i istovremeno povean MPV bilo bi korisno prospektivno ih pratiti kroz neko du~e vrijeme. Na taj na in ustanovilo bi se da li je u skupini bolesnika s ASK-rezistencijom i poveanim MPV-om bio i poveani rizik od ponovnog nastanka tromboti kog stanja. Svim tim bolesnicima, koji su impedancijskom agregometrijom na Multiplate analizatoru imali poveanu vrijednost agregacije trombocita, preventivno je iz eti kih razloga u terapiju uveden i klopidogrel da bi se dodatno smanjila agregacijska sposobnost trombocita. Izmeu vrijednosti agregacije trombocita i koncentracije TxB2 postoji slaba povezanost (r = 0,443), koja je statisti ki zna ajna (p < 0,0001). (Slika 19.) Upravo slaba povezanost i usporedivost izmeu pretraga za praenje funkcionalne aktivnosti trombocita predstavljaju problem kod pronala~enja pretrage koja bi bila airoko primjenjivana u praenju antiagregacijske terapije. U svrhu procjene dijagnosti ke vrijednosti testa za odreivanje koncentracije TxB2 u injena je statisti ka ROC-analiza. ROC-analiza slu~i nam za odreivanje dijagnosti ke osjetljivosti i specifi nosti pojedinog testa. Dijagnosti ka osjetljivost daje nam postotak bolesnih otkrivenih odreenim testom, a dijagnosti ka specifi nost daje postotak zdravih osoba kod kojih je odreeni test stvarno negativan. U idealnom slu aju dijagnosti ka osjetljivost i specifi nost trebale bi biti 100%. No, u praksi to nije tako. Na osnovu dijagnosti ke osjetljivosti i specifi nosti odabire se grani na vrijednost odluke kod koje emo s najveom sigurnoau moi razlikovati dvije meusobno potpuno razli ite skupine, zdrave i bolesne. Kod izrade ROC-analize za odreivanje koncentracije TxB2 oslanjali smo se na vrijednosti agregacije trombocita dobivene impedancijskom agregometrijom na Multiplate analizatoru kojeg smo smatrali (zlatnim standardom(. To nije potpuno ispravan na in izrade ROC-analize. U naaem laboratoriju ne postoji aparat za opti ku agregometriju koji uistinu predstavlja (zlatni standard( za odreivanje funkcionalne aktivnosti trombocita. Stoga smo vrijednosti agregacije trombocita dobivene impedancijskom agregometrijom preuzeli kao (zlatni standard( tim viae ato su ti rezultati odlu ivali o promjeni terapije. Rezultati ROC-analize pokazali su da test odreivanja koncentracije TxB2 ima vrlo dobru dijagnosti ku to nost (AUC = 0,854; p < 0,0001). Dijagnosti ka osjetljivost testa je 64,5%; a dijagnosti ka specifi nost 95% uz grani nu vrijednost koncentracije TxB2 = 130,1 pg/ml. To zna i da e kod granice odluke od 130,1 pg/ml 35,5% ispitanika imati la~no pozitivne vrijednosti testa. Takav rezultat bio je i o ekivan budui da je grani na vrijednost odluke koja je izra unata na tri prethodno opisana razli ita na ina bila znatno viaa (343,4 pg/ml, 654,5 pg/ml, odnosno 3100 pg/ml). S obzirom na nedostatke ovog istra~ivanja kao ato su neusklaenost bolesnika i zdravih ispitanika po dobi i po spolu, kao i izrada ROC-analize koja se oslanjala na rezultate dobivene testom koji ne predstavlja (zlatni standard( u odreivanju agregacije trombocita, udio bolesnika s laboratorijskom rezistencijom na ASK-u izra unat na osnovu granice odluke dobivene naaim izra unom neato je vei od literaturnih podataka (8,6% koliko je dobiveno u naaem istra~ivanju, prema 1,0-1,7% koliko navodi Cattaneo u svojim istra~ivanjima(19) ). Ukoliko za granicu odluke preuzmemo vrijednost koncentracije TxB2 koju su izra unali Frelinger i sur.(40) a koja iznosi 3100 pg/ml tada je udio naaih bolesnika s laboratorijskom rezistencijom na terapiju ASK-u ak i manji (0% koliko je dobiveno u naaem istra~ivanju, prema 1,0-1,7% koliko navodi Cattaneo). To mo~emo objasniti mnogo manjim brojem bolesnika kojeg smo obuhvatili u istra~ivanju (N=105) u odnosu na broj bolesnika kojeg su obuhvatili Frelinger i sur. (N=700). Po naaem mialjenju grani na vrijednost odluke za impedancijsku agregometriju na Multiplate analizatoru koju je postavio proizvoa  je preniska (agregacija TRC < 30 U), jer gotovo je nevjerojatno da kod 41,9% bolesnika terapija ASK-om nije djelotvorna. Na temelju dosadaanjih brojnih istra~ivanja i ovog naaeg istra~ivanja joa uvijek se ne nazire rjeaenje problema oko odabira optimalne pretrage za praenje antiagregacijske terapije. Treba proi joa dosta vremena i izvraiti brojna prospektivna istra~ivanja koja e napokon razrijeaiti dilemu je li uope potrebno praenje antiagregacijske terapije, i ako jeste, koja pretraga je najto niji pokazatelj agregacije trombocita (brza, jednostavna, jeftina, reproducibilna i standardizirana). A ato za sada govori praksa? U nedostatku istinskog klini kog saznanja o djelotvornosti ASK kod pojedinog bolesnika lije nici su pribjegli uvoenju dvojne terapije, pa uz ASK bolesnicima uvode i klopidogrel. S jedne strane, bolesnika su dodatno zaatitili, budui da klopidogrel djeluje na spre avanje agregacije trombocita preko P2Y12 receptora koji je receptor za ADP. S druge strane, bolesnike koji ve uzimaju nekoliko razli itih lijekova dodatno se optereuje s joa jednim. `toviae, pri tom se ne zna kakva je djelotvornost tog lijeka s obzirom na razli ite interferencije. Ne zna se ni hoe li uvoenje joa jednog antiagregacijskog lijeka dovesti do ne~eljenih posljedica - gastrointestinalnog krvarenja. Ali, zna se da je klopidogrel oko 27 puta skupliji lijek od ASK, ato automatski namee opravdanost uporabe relativno skupih POCT-analizatora (PFA-100, Multiplate,Verify Now) u praenju antiagregacijske terapije. No, tu se sada ponovno javlja dilema. Kako usporeivati rezultate dobivene na razli itim analizatorima kada je pokazano da razli iti analizatori daju i razli ite postotke laboratorijske rezistencije na ASK? (41) Dakle, pred pretragom praenja agregacije trombocita dug je put standardizacije kakvog je proala i pretraga PV (protrombinsko vrijeme) ija standardizacija ni nakon mnogo godina joa uvijek nije u potpunosti rijeaena. ZAKLJU CI Funkcionalna aktivnost trombocita odreena impedancijskom agregometrijom na analizatoru Multiplate u bolesnika s CABG koji su na antiagregacijskoj terapiji ASK-om manja je od funkcionalne aktivnosti trombocita kod zdravih ispitanika. Funkcionalna aktivnost trombocita odreena mjerenjem koncentracije TxB2 u serumu EIA metodom u bolesnika s CABG koji su na antiagregacijskoj terapiji ASK-om manja je od funkcionalne aktivnosti trombocita kod zdravih ispitanika. Vrijednosti funkcionalnih aktivnosti trombocita dobivenih prethodno spomenutim dvjema razli itim metodama slabo koreliraju (r = 0,443; p < 0,0001). Stupanj laboratorijske rezistencije dobiven specifi nim testom, mjerenjem koncentracije TxB2 u serumu, manji je (8,6%, odnosno 0%) od stupnja laboratorijske rezistencije dobivene odreivanjem agregacije trombocita impedancijskom agregometrijom na analizatoru Muliplate (41,9%). Broj trombocita ne korelira s vrijednoau agregacije trombocita odreene metodom impedancijske agregometrije na analizatoru Multiplate (r = 0,174; p = 0,0755). Broj trombocita ne korelira s koncentracijom TxB2 u serumu odreenom EIA metodom (r = 0,054; p = 0,5839). Vrijednost MPV-a ne korelira s vrijednoau agregacije trombocita odreene metodom impedancijske agregometrije na analizatoru Multiplate (r = 0,217; p = 0,0261). Vrijednost MPV-a ne korelira s koncentracijom TxB2 u serumu odreenom EIA metodom (r = 0,197; p = 0,0442). ROC-analiza TxB2 pokazala je vrlo dobru dijagnosti ku to nost (AUC = 0,854; p < 0,0001). Granica odluke od 130,1 pg/ml TxB2 ima dijagnosti ku osjetljivost 64,5% i dijagnosti ku specifi nost 95%. Tako niska granica odluke za TxB2 i relativno niska dijagnosti ka osjetljivost posljedica su koriatenja metode koja ne predstavlja (zlatni standard( za odreivanje agregacije trombocita. OPI ZAKLJU AK: Funkcionalna aktivnost trombocita u bolesnika koji su na terapiji ASK-om znatno je manja od one u skupini zdravih ispitanika koji nisu na terapiji. Stupanj laboratorijske rezistencije na ASK odreen s dvije razli ite metode, impedancijskom agregometrijom trombocita na analizatoru Multiplate i EIA metodom odreivanja koncentracije TxB2, pokazuje slabu povezanost. Test odreivanja koncentracije TxB2 u serumu mogao bi biti bolji jer mjeri specifi no farmakoloako djelovanje ASK. Postotak laboratorijske rezistencije dobiven tim testom puno je manji (8,6% odnosno 0%) od onog dobivenog impedancijskom agregometrijom (41,9%) ato je realnije, jer upravo je nevjerojatno da bi ak 41,9% bolesnika bilo rezistentno na ASK. Potrebno je u initi daljnja istra~ivanja koja bi napokon razrijeaila dilemu je li uope potrebno praenje antiagregacijske terapije, i ako jeste, koja pretraga bi bila najprimjerenija (brza, jednostavna, jeftina, reproducibilna i standardizirana). SA}ETAK Uvod: U patofizioloakoj podlozi akutnog koronarnog sindroma je kroni na upalna bolest stijenke krvne ~ile (ateroskleroza) ijom progresijom dolazi do rupture aterosklerotskog plaka i posljedi ne tromboze koja se o ituje akutnim infarktom miokarda. Cilj: Pomou dvije razli ite metode, impedancijskom agregometrijom trombocita na analizatoru Multiplate i EIA metodom odreivanja koncentracije TxB2 u serumu, odrediti da li je antiagregacijska terapija ASK-om u bolesnika s CABG djelotvorna. Usporediti stupnjeve laboratorijske rezistencije na ASK dobivene pomou te dvije metode i ispitati postoji li povezanost izmeu broja trombocita i MPV-a s laboratorijskom rezistencijom na ASK odreenom pomou tih metoda. Ispitanici: Ispitano je 105 bolesnika s CABG (medijan dobi bio je 65 godina, a raspon dobi 40  86 godina) koji su bili na terapiji ASK ( 150 mg/dan i 30 zdravih ispitanika (medijan dobi bio je 40 godina, a raspon dobi 21  76 godina) koji nisu bili na terapiji ASK. Skupina zdravih ispitanika poslu~ila je za izra un grani ne vrijednosti koncentracije TxB2 iznad koje smo smatrali da terapija ASK-om nije djelotvorna. Metode: Agregacija trombocita odreivana je metodom impedancijske agregometrije na analizatoru Multiplate iz pune krvi, a koncentracija TxB2 u serumu odreivana je EIA metodom. Rezultati: Razli itim metodama dobiveni su i razli iti postotci laboratorijske rezistencije na ASK. Metodom impedancijske agregometrije na analizatoru Multiplate postotak ASK-rezistencije bio je 41,9%, a odreivanjem koncentracije TxB2 u serumu taj postotak je iznosio 8,6% tj. 0%. Korelacija izmeu te dvije metode je slaba (r = 0,443, p < 0,0001). Nema povezanosti izmeu broja trombocita i impedancijske agregometrije (r = 0,174; p = 0,0755), kao ni izmeu broja trombocita i koncentracije TxB2 (r = 0,054; p = 0,5839). Nema povezanosti izmeu MPV-a i impedancijske agregometrije (r = 0,217; p = 0,0261), kao ni izmeu MPV-a i koncentracije TxB2 (r = 0,197; p = 0,0442). ROC-analiza TxB2 uspostavljena u odnosu na metodu impedancijske agregometrije dala je granicu odluke od 130,1 pg/ml pri emu je dijagnosti ka osjetljivost 64,5%, a dijagnosti ka specifi nost 95%. Zaklju ak: Funkcionalna aktivnost trombocita u bolesnika koji su na terapiji ASK-om znatno je manja od one u skupini zdravih ispitanika koji nisu na terapiji. Stupanj laboratorijske rezistencije na ASK odreen s dvije razli ite metode, impedancijskom agregometrijom trombocita na analizatoru Multiplate i EIA metodom odreivanja koncentracije TxB2 u serumu, pokazuje slabu povezanost, iako ima naznaka da bi pretraga odreivanja koncentracije TxB2 u serumu mogla biti bolja. Potrebno je u initi daljnja istra~ivanja koja bi napokon razrijeaila dilemu je li uope potrebno praenje antiagregacijske terapije, i ako jeste, koja pretraga je najidealnija (brza, jednostavna, jeftina, reproducibilna i standardizirana). SUMMARY Background: The pathophysiological basis of acute coronary syndrome is a chronic inflammatory disease of the blood vessel wall (atherosclerosis), whose progression leads to atherosclerotic plaque rupture and subsequent thrombosis, which is manifested by acute myocardial infarction. Objective: two different methods, impedance platelet aggregometry, by means of the Multiplate analyzer, and the EIA method of determining the concentration of TxB2 in serum, are used, to determine whether the antiaggregation ASA therapy in patients with CABG surgery is effective. To compare the levels of laboratory ASA- resistance obtained using these two methods and to examine the relation between platelet count and MPV with laboratory resistance to aspirin given by these methods. Subjects: The study included 105 patients with CABG (median age was 65 years, and the age range 40 -86 years) who were treated with ASA ( 150 mg / day and 30 healthy volunteers (median age was 40 years and age range 21-76 years) who were treated with ASA. A group of healthy subjects served to calculate the concentration of TxB2 threshold above which we believed that the ASA therapy is not effective. Methods: Platelet aggregation was measured by the method of impedance aggregometry with the Multiplate analyzer from whole blood, a concentration of TxB2 in serum was determined by EIA method. Results: Different methods have been performed with different percentages of laboratory resistance to ASA. The percentage of ASA- resistance was 41,9%, with the impedance aggregometry with the Multiplate analyzer, and by determining the concentration of TxB2 in serum, this percentage was only 8.6% and 0%. The correlation between these two methods was weak (r = 0,443; p <0,0001). There was no correlation between platelet count and impedance aggregometry (r = 0,174; p = 0,0755) nor between platelet count and concentration of TxB2 (r = 0,054; p = 0,5839). There was no correlation between MPV and impedance aggregometry (r = 0,217; p = 0,0261), nor between the MPV and the concentration of TxB2 (r = 0,197; p = 0,0442). ROC analysis of TxB2 established in relation to the method of impedance aggregometry made a decision limit of 130,1 pg / ml while the diagnostic sensitivity of 64,5% and diagnostic specificity of 95%. Conclusion: The functional activity of platelets in patients who were treated with ASA was significantly lower than those in the control group not on therapy. 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FitzGerald GA. Antithrombotic Trialists( Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction and stroke in high risk patients. Oxford: BMJ 2002;324:71-86. Michelson AD, Cattaneo M, Eikelboom JW, Gurbel P, Kottke-Marchant K, Kunicki TJ, i sur. Aspirin resistance: position paper of the Working Group on aspirin resistance. J Thromb Haemost 2005;3:1309-11. Ritter J, Ferro A, Gkaliagkonsi E. Platelet-derived nitric oxide signaling and regulation. Circul Res 2007;101:654-62. }IVOTOPIS Roena sam 3. velja e 1972. godine u Krapini gdje sam zavraila osnovnu akolu i matemati ku gimnaziju. Akademske godine 1990./91. upisala sam Farmaceutsko-biokemijski fakultet Sveu iliata u Zagrebu, smjer medicinska biokemija. Diplomirala sam 1995. godine s temom pod naslovom "Odreivanje koncentracije Tc-99 u prvom eluatu Mo-99/Tc-99m radionuklidskog generatora pomou scintilacijskog broja a." Rad je izraen na Zavodu za nuklearnu medicinu Instituta "Ruer Boakovi" pod stru nim vodstvom dr. sc. }eljka Kuatera. Pripravni ki sta~ obavila sam u Medicinsko-biokemijskom laboratoriju Specijalne bolnice za medicinsku rehabilitaciju u Krapinskim Toplicama, gdje sam nakon polo~enog stru nog ispita primljena u stalan radni odnos. Poslijediplomski studij iz medicinske biokemije na Farmaceutsko-biokemijskom fakultetu Sveu iliata u Zagrebu upisala sam 1999. godine. Od 2008. godine na specijalisti kom sam usavraavanju iz medicinske biokemije u KBC "Sestre milosrdnice" u Zagrebu. lan sam Hrvatskog druatva medicinskih biokemi ara kao i Hrvatske komore medicinskih biokemi ara. Aktivno sam sudjelovala na nekoliko domaih i stranih znanstvenih i stru nih skupova kao autor i koautor posterskih radova. TEMELJNA DOKUMENTACIJSKA KARTICA Sveu iliate u Zagrebu Farmaceutsko-biokemijski fakultet Magistarski rad FUNKCIONALNA AKTIVNOST TROMBOCITA U BOLESNIKA NA ANTIAGREGACIJSKOJ TERAPIJI ACETILSALICILNOM KISELINOM Anita Klasi Kardiovaskularne bolesti vodei su uzrok pobola i smrtnosti u razvijenim zemljama svijeta i predstavljaju veliki socioekonomski problem. U patofizioloakoj podlozi kardiovaskularnih bolesti je ateroskleroza, kroni na upala stijenke krvne ~ile, ijom progresijom dolazi do rupture aterosklerotskog plaka i posljedi ne tromboze koja se o ituje akutnim infarktom miokarda. Cilj ovog magistarskog rada bio je pomou dvije razli ite metode, impedancijskom agregometrijom trombocita na analizatoru Multiplate i EIA metodom odreivanja koncentracije TxB2 u serumu, odrediti da li je antiagregacijska terapija ASK-om djelotvorna, te postoji li povezanost izmeu broja trombocita i MPV-a s laboratorijskom rezistencijom na ASK odreenom pomou tih metoda. Metodom impedancijske agregometrije postotak ASK-rezistencije bio je 41,9%, a EIA odreivanjem koncentracije TxB2 taj postotak je iznosio 8,6% odnosno 0%. Korelacija izmeu te dvije metode je slaba (r = 0,443, p < 0,0001). Nema povezanosti izmeu broja trombocita i agregacije trombocita odreene na Multiplate analizatoru (r = 0,174; p = 0,0755), kao ni izmeu broja trombocita i koncentracije TxB2 (r = 0,054; p = 0,5839). Nema povezanosti izmeu MPV-a i impedancijske agregometrije (r = 0,217; p = 0,0261), kao ni izmeu MPV-a i koncentracije TxB2 (r = 0,197; p = 0,0442). ROC-analiza TxB2 uspostavljena u odnosu na metodu impedancijske agregometrije dala je granicu odluke od 130,1 pg/ml pri emu je dijagnosti ka osjetljivost 64,5%, a dijagnosti ka specifi nost 95%. Stupanj laboratorijske rezistencije na ASK odreen impedancijskom agregometrijom i EIA metodom odreivanja koncentracije TxB2 pokazuje slabu povezanost, iako ima naznaka da bi test odreivanja koncentracije TxB2 mogao biti bolji. Potrebno je u initi daljnja istra~ivanja koja bi napokon razrijeaila dilemu je li uope potrebno praenje antiagregacijske terapije, i ako jeste, koji test je najidealniji (brz, jednostavan, jeftin, reproducibilan i standardiziran). (71 stranica, 20 slika, 10 tablica, 83 literaturna navoda, jezik izvornika hrvatski) Rad je pohranjen u knji~nici Farmaceutsko-biokemijskog fakulteta u Zagrebu, A. Kova ia1. Klju ne rije i: AIM, ateroskleroza, agregacija trombocita Mentori: doc. dr. sc. Nada Vrki, Klini ki zavod za kemiju KBC Sestre milosrdnice Zagreb doc. dr. sc. Duako Cerovec, SB za medicinsku rehabilitaciju Krapinske Toplice Ocjenjiva i: dr.sc. Roberta Petlevski, prof. dr.sc. Nada Vrki, doc. dr.sc. Renata Zadro, prof. Rad prihvaen: 18. svibnja 2011. BASIC DOCUMENTATION CARD University of Zagreb Faculty of Pharmacy and Biochemistry Master of Science Thesis FUNCTIONAL ACTIVITY OF PLATELETS IN PATIENTS ON ASA ANTIAGGREGATION THERAPY Anita Klasi Cardiovascular diseases are the leading cause of morbidity and mortality in developed countries in the world and are a major socioeconomic problem. In the pathophysiological basis of cardiovascular disease is atherosclerosis, chronic inflammation of blood vessel walls, whose progression leads to atherosclerotic plaque rupture and subsequent thrombosis, which is manifested by acute myocardial infarction. The aim of this thesis was using two different methods, impedance platelet aggregation by means of the Multiplate analyzer, and the EIA method of determining the concentration of TxB2 in serum, to determine whether ASA antiaggregation therapy is effective, and the relation between platelet count and MPV, with laboratory resistance to ASA specified using these impedance methods. By the aggregometry method, the percentage of ASA- resistance was 41,9%, a concentration of TxB2 EIA determining this percentage was 8,6% and 0%. The correlation between these two methods was weak (r = 0,443; p <0 , 0001). There is no correlation between platelet count and platelet aggregation determined on Multiplate analyzer (r = 0,174; p = 0,0755), nor between the number of platelets and the concentration of TxB2 (r = 0,054; p = 0,5839). There is no connection between MPV and the impedance aggregometry (r = 0,217; p = 0,0261), nor between MPV and TxB2 concentrations (r = 0,197; p = 0,0442). ROC analysis of TxB2 established in relation to the method of impedance aggregometry made a decision limit of 130,1 pg / ml, while the diagnostic sensitivity of 64,5% and diagnostic specificity of 95%. The degree of laboratory ASA- resistance is determined with impedance aggregometry and determining the concentration of TxB2 EIA shows a weak correlation, although there are indications that a test to calculate the concentration of TxB2 could be better. It is necessary to do further research to finally resolve the dilemma whether it is necessary to monitor antiplatelet therapy, and, if so, which test is most ideal (fast, easy, inexpensive, reproducible and standardized). (71 pages, 20 photos, 10 tables, 83 literary references, the original is in the Croatian language). The paper is stored in the Central Library of the Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacic 1. Key words: acute myocardial infarction, atherosclerosis, platelet aggregation Mentors: Docent Vrki Nada, PhD, Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb Docent Duako Cerovec, PhD, Special Hospital for Medical Rehabilitation, Krapinske Toplice Reviewers: Professor Roberta Petlevski, PhD Docent Nada Vrki, PhD. Professor Renata Zadro,PhD. 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