Cardiovascular disease is the largest single cause of mortality and its major underlying pathology is atherosclerosis. The proliferation of vascular smooth muscle cells (VSMC) is a key event in the pathogenesis of various vascular diseases, including atherosclerosis and hypertension. Thrombin is involved in the differentiation and abnormal proliferation of VSMC associated with atherosclerosis and hypertension. Thrombin stimulation results in extracellular signal-regulated kinase (ERK 1/2) activation through transactivation of the epidermal growth factor receptor (EGFR). Based on the studies in which PD98059 used to inhibit ERK1/2, we have shown previously that ERK1/2 was involved in the regulation by thrombin of VSMC’s proliferation. In addition, heparin-binding EGF-like growth factor (HB-EGF) and matrix metalloproteinases (MMPs) have also been detected in VSMC and shown to be regulated by thrombin. ADAMs (A Disintegrin And Metalloproteinase) are transmembrane metalloproteinases, belonging to adamalysin group, that are distinct from matrix metalloproteinases (MMPs) in that, they have an extracellular disintegrin domain and cytoplasmic domain that can associate with intracellular proteins. To the present knowledge there is no study that indicates the activation of an ADAM member in thrombin-induced VSMC proliferation. In this dissertation, the role of EGFR, ERK1/2, HB-EGF, general metalloproteinases, MMP-2 and ADAM 12, as well as PKCδ in mediating the mitogenic action of Thrombin in rat VSMC was investigated. Incubation of rat VSMC with Thrombin (1 U/ml) for 5 minutes resulted in significant increase of ERK1/2 phosphorylation by 8.7 ± 0.9 fold (p<0.001), EGFR phosphorylation by 8.5 ± 1.3 fold (p<0.001) and DNA synthesis by 3.6 ± 0.4 fold (p<0.001). Separate pretreatments for 30 minutes with EGFR tyrosine kinase irreversible inhibitor, 10 μM PD169540 (PD), and 20 μM anti-HB-EGF antibody, significantly reduced thrombinstimulated EGFR and ERK1/2 phosphorylation by 81 %, 72 % and by 48 % and 61%, respectively. Furthermore, same pretreatments with PD and anti-HB-EGF antibody reduced Thrombin-induced VSMC’s proliferation by 44% and 45%, respectively. In addition, pretreatments for 30 minutes with 10 μM KB-R7785 (KB), a specific ADAM 12 inhibitor or 10 μM specific MMP2 inhibitor significantly reduced thrombin-stimulated EGFR and ERK1/2 phosphorylation by 47 %, 47 % and by 25 % and 25 %, respectively. Furthermore, same pretreatments with either KB or MMP2 inhibitor reduced thrombin-induced VSMC’s proliferation by 27 % and 45 % respectively. Pretreatment of VSMC with 5 μM rottlerin, a specific inhibitor of PKCδ, for 30 min significantly reduced EGFR and ERK1/2 phosphorylation by 58 % and 55%, respectively and reduced significantly thrombin-induced VSMC’s proliferation by 32%. These results show that thrombin-induced DNA synthesis correlates with the level of ERK1/2 activation rather than EGFR activation. Further, these results suggest that thrombin acts through EGFR and ERK 1/2 signaling pathways involving PKCδ, MMP-2 and ADAM 12, to up-regulate proliferation of VSMC. Therefore, this thesis examines recent findings in signaling mechanisms employed by thrombin in modulating the regulation of proliferation of VSMC with particular emphasis on involvement of ADAM 12 and MMP2 that have been identified as important mediators of VSMC hypertrophy and vascular diseases. These findings are critical for understanding the role of thrombin in vascular biology and vascular diseases. Since these two molecules have been acknowledged as important mediators of VSMC migration and hypertrophy, MMP-2 and ADAM 12 are identified as one of key targets for novel therapeutic interventions to minimize irreversible tissue damage associated with hypertension and atherosclerosis. A safe option to regulate in vivo synthesis of MMP-2 and ADAM 12 would be welcome. Kardiovaskularne bolesti predstavljaju najveći uzrok smrtnosti ljudske populacije, a njihova glavna i osnovna patološka komponenta je ateroskleroza. Proliferacija ili deoba glatkih mišićnih ćelija krvnog suda (VSMC) ključni je događaj u nastanku raznih vaskularnih oboljenja, uključujući aterosklerozu i hipertenziju. U procesu diferencijacije i abnormalne deobe VSMC povezanih sa hipertenzijom i aterosklerozom uključen je i trombin. Stimulisanje VSMC trombinom dovodi do aktivacije ekstracelularnim signalima regulisanih kinaza 1 i 2 (ERK1/2), preko transaktivacije receptora za epidermalni faktor rasta (EGFR). U ranijim studijama Isenović i saradnici potvrdili su na osnovu inhibicije ERK1/2 od strane PD9805 inhibitora, učešće ERK1/2 u regulaciji proliferacije VSMC izazvanoj trombinom. U nastavku, faktor rasta sličan epidermalnom faktoru rasta koji vezuje heparin (HB-EGF), protein kinaza C delta (PKCδ) i matriksne metaloproteinaze (MMP), nađene su u VSMC i pokazano je da je i njihova aktivnost regulisana trombinom. ADAM (engl. “A Disintegrin And Metalloproteinase”) transmembranske su metaloproteinaze koje pripadaju adamalizinskoj grupi i razlikuju se od matriksnih metaloproteinaza po tome što imaju vanćelijski dizintegrinski i citoplazmatski domen. C terminalni domen može da stupa u interakciju sa unutarćelijskim proteinima. Uvidom u literaturu, do sada ne postoji istraživanje koje ukazuje na prisustvo ADAM metaloproteinazne aktivnosti vezane za proliferaciju VSMC stimulisane trombinom. Predmet ove doktorske teze jeste proučavanje uloge EGFR, ERK1/2, HB-EGF, PKCδ, ukupnih metaloproteinaza, kao i specifičnih MMP-2 i ADAM 12, u posredovanju proliferativnog efekta trombina na VSMC pacova. Inkubacija VSMC pacova sa trombinom (1 U/ml) u periodu od 5 minuta rezultirala je u značajnom povećanju: fosforilacije ERK1/2 od 8.7 ± 0.9 puta (p<0.001), fosforilacije EGFR od 8.5 ± 1.3 puta (p<0.001) i sinteze DNK od 3.6 ± 0.4 puta (p<0.001). Prethodni pojedinačni tretmani ovih ćelija u trajanju od 30 minuta sa 10 μM PD169540 (PD), ireverzibilnim inhibitorom EGFR, i 20 μM antitelom protiv HB-EGF značajno su smanjili trombinom stimulisanu fosforilaciju EGFR za 81 % i 72 % i ERK1/2 za 48 % i 61 %, respektivno. Istim ovakvim pretretmanima sa PD i HB-EGF antitelom smanjena je značajno proliferacija VSMC stimulisana trombinom za 44 % i 45%. Takođe, polučasovni pretretmani VSMC u toku 30 minuta sa 10 μM KB-R7785 (KB), specifičnim ADAM 12 inhibitorom i 10 μM specifičnim MMP-2 inhibitorom značajno su smanjili fosforilacije izazvane trombinom: EGFR za 47 % i 78 % i ERK1/2 za 25 % i 25 %, respektivno. U nastavku, istim ovim pretretmanima sa KB ili MMP2 inhibitorom, smanjena je trombinom stimulisana proliferacija za 27 % i 45 %. Prethodni polučasovni tretman VSMC sa 5 μM rotlerinom, specifičnim inhibitorom za PKC delta, značajno je smanjio fosforilaciju EGFR i ERK1/2 i proliferaciju VSMC stimulisanu trombinom za 58 %, 55% i 32%, respektivno. Dobijeni rezultati pokazuju da je proliferacija VSMC pod delovanjem trombina pretežno zavisna od nivoa aktivacije ERK1/2 kinaze nego od nivoa aktivacije EGFR. Takođe, ovi rezultati ukazuju da trombin deluje preko EGFR i ERK1/2 signalnih puteva koji uključuju PKCδ, MMP-2 i ADAM 12, stimulišući proliferativni proces VSMC pacova. U zaključku, saznanje o učešću ADAM 12 molekula u proliferaciji VSMC pod delovanjem trombina je od izuzetne važnosti za bolje razumevanje uloge trombina u kardiovaskularnoj biologiji i medicini. Pošto su MMP-2 i ADAM 12 potvrđeni kao medijatori migracije i hipertrofije VSMC, oni predstavljaju potencijalne ključne „mete” u novim terapeutskim intervencijama kojima bi se smanjilo ireverzibilno oštećenje tkiva povezano sa aterosklerozom i hipertenzijom. Zasigurno bi jedna od poželjnih opcija, kao in vivo regulisane sinteze MMP-2 i ADAM 12, doprinela redukciji patološke proliferacije VSMC.