PRETREATMENT WITH LOW DOSES OF ACENOCOUMAROL INHIBITS THE DEVELOPMENT OF ACUTE ISCHEMIA/REPERFUSION-INDUCED PANCREATITIS

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1 JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 215, 66, 5, Z. WARZECHA 1, P. SENDUR 1,2, P. CERANOWICZ 1, M. DEMBINSKI 3, J. CIESZKOWSKI 1, B. KUSNIERZ-CABALA 4, R. TOMASZEWSKA 5, A. DEMBINSKI 1 PRETREATMENT WITH LOW DOSES OF ACENOCOUMAROL INHIBITS THE DEVELOPMENT OF ACUTE ISCHEMIA/REPERFUSION-INDUCED PANCREATITIS 1 Deprtment of Physiology, Jgiellonin University Medicl College, Crcow, Polnd; 2 Deprtment of Anesthesiology nd Intensive Therpy, Jgiellonin University Medicl College, Crcow, Polnd; 3 The Second Deprtment of Generl Surgery, Jgiellonin University Medicl College, Crcow, Polnd; 4 Deprtment of Clinicl Biochemistry, Jgiellonin University Medicl College, Crcow, Polnd; 5 Deprtment of Pthology, Jgiellonin University Medicl College, Crcow, Polnd Cogultive disorders re known to occur in cute pncretitis nd re relted to the severity of this disese. Vrious experimentl nd clinicl studies hve shown protective nd therpeutic effect of heprin in cute pncretitis. Aim of the present study ws to determine the influence of cenocoumrol, vitmin K ntgonist, on the development of cute pncretitis. Studies were performed on mle Wistr rts weighing g. Acenocoumrol t the dose of 5, 1 or 15 µg/kg/dose or vehicle were dministered once dy for 7 dys before induction of cute pncretitis. Acute pncretitis ws induced in rts by pncretic ischemi followed by reperfusion. The severity of cute pncretitis ws ssessed fter 5-h reperfusion. Pretretment with cenocoumrol given t the dose of 5 or 1 µg/kg/dose reduced morphologicl signs of cute pncretitis. These effects were ccompnied with decrese in the pncretitis-evoked increse in serum ctivity of lipse nd serum concentrtion of pro-inflmmtory interleukin-1β. Moreover, the pncretitis-evoked reductions in pncretic DNA synthesis nd pncretic blood flow were prtilly reversed by pretretment with cenocoumrol given t the dose of 5 nd 1 µg/kg/dose. Administrtion of cenocoumrol t the dose of 15 µg/kg/dose did not exhibit ny protective effect ginst ischemi/reperfusion-induced pncretitis. We concluded tht pretretment with low doses of cenocoumrol reduces the severity of ischemi/reperfusion-induced cute pncretitis. Key words: vitmin K ntgonist, ischemi/reperfusion-induced pncretitis, cenocoumrol, interleukin-1bet, lipse ctivity, pncretic blood flow, DNA synthesis, D-Dimer INTRODUCTION Inflmmtion nd cogultion re closely linked processes. Inflmmtion in response to severe infection or trum results in systemic ctivtion of cogultion. Inflmmtion promotes clot formtion, reduces the ctivity of nturl nticogulnts nd inhibits fibrinolysis (1, 2). Pro-inflmmtory cytokines ply min role in ctivtion of cogultion by incresing expression of tissue fctor on monocytes nd endothelium, leding to thrombin formtion (3). Severe infection nd inflmmtion led to hemosttic bnormlities, rnging from significnt increse in lbortory sensitive tests for cogultion ctivtion to gross ctivtion of cogultion tht my led to disseminted intrvsculr cogultion (4). Similrly, cogultion results not only in thrombosis but lso in ctivtion of inflmmtory process. Fctor X, thrombin nd the complex of tissue fctor-fctor VII exhibit pro-inflmmtory ctivities. Thrombin, s well s the complex tissue fctor-fctor VII, cn stimulte protese ctivted receptors (PARs) (5, 6). This in turn cn induce the expression of dhesion molecules tht fcilitte leukocyte-medited injury (1). Moreover, thrombin ctivtes production of monocyte chemotctic protein-1 (MCP-1) nd pro-inflmmtory interleukin-6 in fibroblsts, epithelil cells nd mononucler cells (7). Inflmmtory response is lso induced by CD4 lignd relesed from ctivted pltelets. CD4 lignd stimultes synthesis of tissue fctor nd increses level of proinflmmtory interleukin-6 nd -8 (1). Animl nd clinicl studies hve shown tht cogultive disorders occur in cute pncretitis nd re relted to the severity of this disese (8-13). Acute pncretitis ctivtes the hemosttic system with formtion of thrombi within blood vessels nd cogultive disorders my rnge from scttered intrvsculr thrombosis to severe disseminted intrvsculr cogultion (13). Altertions in plsm concentrtion of D- Dimer nd ntithrombin III re proposed to be useful nd ccurte mrker in vlution of cute pncretitis severity nd prediction for poor outcome of this disese t dmission (11, 12). Vrious experimentl studies hve shown nti-inflmmtory effect of heprin, including the protective nd therpeutic effect of heprin in cute pncretitis. (14-19).

2 732 Also there re clinicl studies indicting tht pretretment with heprin reduces frequency of post-ercp pncretitis (2, 21). Administrtion of heprin given together with insulin is recommended s stndrd tretment in hyperlipidemi-induced pncretitis (22-24). Moreover, low moleculr weight heprin hs been shown to be effective in the prevention of encephlopthy in ptients with severe cute pncretitis (25). The present study ws designed to determine the influence of pretretment with cenocoumrol, vitmin K ntgonist, on the development of ischemi/reperfusion-induced cute pncretitis. Animls nd tretment MATERIALS AND METHODS Studies were performed on mle Wistr rts weighing g nd were conducted following the experimentl protocol pproved by the First Locl Commission of Ethics for the Cre nd Use of Lbortory Animls in Crcow. Animls were housed in cges with wire mesh bottoms, with norml room temperture nd 12-hour light-drk cycle. Rts were fsted with free ccess to wter for 16 h before induction of cute pncretitis, erlier food nd tp wter were vilble d libitum. Experiments were crried out in two seprte series. The first series of studies were performed to determine pproprite dose of cenocoumrol cusing n increse of interntionl normlized rtio (INR) to rnge between 2.5 to 3.5. This vlue of INR is recommended in the most clinicl conditions relted to cogultion disorders (26). Thirty six nimls were rndomly divided into six equl experiment groups: [1] control rts treted with sline (queous solution of.9% NCl); [2 6] rts treted with cenocoumrol (Acenocumrol WZF, Wrszwskie Zkldy Frmceutyczne Polf S.A., Wrsw, Polnd) given t the dose of 5, 1, 15, 3 or 6 µg/kg/dose. Sline or cenocoumrol were dministered intrgstriclly once dy for 7 dy. The second series of studies were performed to ssess the influence of pretretment with cenocoumrol on the development of ischemi/reperfusion-induced cute pncretitis. Eighty nimls were rndomly divided in eight equl experimentl groups: [1] control rts treted with sline before shm-opertion; [2] rts treted with sline before induction of cute pncretitis; [3 5] rts treted with cenocoumrol given t the dose of 5, 1 or 15 µg/kg/dose before shm-opertion; [6 8] rts treted with cenocoumrol given t the dose of 5, 1, 15 before induction of cute pncretitis. Sline or cenocoumrol were dministered intrgstriclly once dy for 7 dy before induction of cute pncretitis or shm-opertion. Acenocoumrol t the dose of 5, 1 or 15 µg/kg/dose ws given intrgstriclly once dy for 7 dys before induction of cute pncretitis. Before induction of cute pncretitis rts were nesthetized with ketmine (5 mg/kg i.p., Bioketn, Vetoquinol Biowet, Gorzow Wielkopolski, Polnd). Acute pncretitis ws induced by severe pncretic ischemi followed by reperfusion s described previously in detils (27). Briefly, the splenic inferior rtery ws occluded for 3 min nd fter tht microvsculr clips were removed to obtin pncretic reperfusion. The bdominl cvity for time of reperfusion ws closed by suture. In shmoperted control rts, longitudinl lprotomy nd mobiliztion of the celic rtery without clmping ws performed. Determintion of pncretic blood flow In ccordnce with experimentl group, rts were nesthetized with ketmine gin fter 5-h pncretic reperfusion or 5 hours fter shm-opertion. The bdominl cvity ws opened nd pncretic blood flow ws mesured by lser Doppler flowmeter using PeriFlux 41 Mster monitor (Perimed AB, Jrfll, Sweden), s described previously (28, 29). Dt were presented s percentge of pncretic blood blow obtined in shm-operted sline-treted rts without induction of cute pncretitis. Biochemicl nlysis After the mesurement of pncretic blood flow, rteril blood ws tken from the bdominl ort. INR ws determined in fresh blood, using Alere INRtio 2 PT/INR Monitoring Systems nd Alere INRtio PT/INR Monitoring System Test Strips (Alere Sn Diego, Inc, Sn Diego, Cliforni, USA). Plsm D-Dimer concentrtion ws determined using n immunoturbidimetric ssy (Innovnce D-Dimer Assy, Siemens Helthcre GmbH, Mrburg, Germny) on utomtic cogultion nlyzer BCS XP System (Siemens Helthcre Dignostics, Erlngen, Germny). Serum lipse nd mylse ctivity ws determined with Kodk Ectchem DT II System nlyzer (Estmn Kodk Compny, Rochester, NY, USA) using Lip nd Amyl DT Slides (Vitros DT Chemistry System, Johnson & Johnson Clinicl Dignostic, Inc., Rochester, NY, USA). Serum concentrtion of interleukin-1β (IL-1β) ws mesured using the Rt IL-1β Pltinum Elis (Bender MedSystem GmbH, Vienn, Austri). Determintion of pncretic DNA synthesis After the blood withdrwl, the pncres ws crefully dissected out from its ttchment to the stomch, duodenum, nd spleen. Ft nd peripncretic tissue were trimmed wy. Smples of pncretic tissue were tken for study of DNA synthesis nd morphologicl exmintion. The rte of DNA synthesis ws mesured by incubtion of minced pncretic tissue t 37 C for 45 min in 2 ml of medium contining 8 µci /ml of [ 3 H]thymidine ([6-3 H]-thymidine, 2 3 Ci/mmol, Institute for Reserch, Production nd Appliction of Rdioisotopes, Prgue, Czech Republic), s described previously (3, 31). The incorportion of lbeled thymidine into DNA ws determined by counting.5 ml DNA-contining superntnt in liquid scintilltion system. DNA synthesis ws expressed s [ 3 H]thymidine disintegrtions per minute per microgrm DNA (dpm/µg DNA). Histologicl exmintion of pncretic dmge Morphologicl exmintion of pncretic tissue ws performed in hemtoxilin nd eosin stined slides s describe previously in detil (32). The histologicl grding of edem ws mde using scle rnging from to 3 ( = no edem, 1 = interlobulr edem, 2 = interlobulr nd moderte intrlobulr edem, nd 3 = interlobulr edem nd severe intrlobulr edem). Leukocytic infiltrtion ws lso grded from to 3 ( = bsent, 1 = scrce perivsculr infiltrtion, 2 = moderte perivsculr nd scrce diffuse infiltrtion, 3 = bundnt diffuse infiltrtion). Grding of vcuoliztion ws bsed on the pproprite percentge of cinr cells involved: = bsent, 1 = less thn 25%, 2 = 25 5% nd 3 = more thn 5% of cinr cells. Hemorrhgi ws grded: = no hemorrhgi, 1 = 1 2 hemorrhgic foci per slide, 2 = 3 5 hemorrhgic foci per slide, 3 = more thn 5 hemorrhgic foci per slide. Necrosis ws grded: = no necrosis, 1 = less thn 15% of pncretic cells involved, 2 = % of cells involved, 3 = more thn 35 % of cells involved. Results of histologicl exmintion hve been expressed s predominnt histologicl grding in ech experimentl group of nimls.

3 733 Sttisticl nlysis Results, except histologicl dt, hve been expressed s mens ± S.E.M. In the first series of studies, we used six rts in ech experimentl group; wheres in the second series of studies ech experimentl group ws composed of ten nimls. Sttisticl nlysis ws mde by nlysis of vrince followed by Tukey s multiple comprison test. A difference with P vlue of less thn.5 ws considered significnt. The first series of studies RESULTS Intrgstric dministrtion of cenocoumrol once dy for 7 dys cused dose-dependent increse in INR in intct rts (Fig. 1). Acenocoumrol given t the dose of 5, 1 or 15 µg/kg/dose cused round two-, three- nd five-fold increse in INR, respectively. INR ws non-detectble in rts treted with cenocoumrol given t the dose of 3 or 6 µg/kg/dose becuse obtined vlues were out of the Alere INRtio 2PT/INR Monitoring System detection rnge. Results obtined in the first series of study prompted us to use cenocoumrol t the dose of 5, 1 nd 15 µg/kg/dose t the second series of study. The second series of studies In control sline-treted shm-operted rts without induction of cute pncretitis, INR reched vlue of 1.11 ±.9 (Fig. 2). Morphologicl fetures showed tht the pncres in this group of nimls exhibits regulr histology without dmge (Tble 1; Fig. 9A). In rts without induction of cute pncretitis, dministrtion of cenocoumrol given for 7 dys t the dose of 5, 1 or 15 µg/kg/dose incresed INR to similr vlues s in the first series of studies (Fig. 2). In those rts, cenocoumrol given t the dose 5 or 1 µg/kg/dose ws without significnt effect on serum lipse ctivity; wheres cenocoumrol given t the dose of 15 µg/kg/dose, significntly incresed serum lipse ctivity by round 5% (Fig. 3). In rts without induction of cute pncretitis, dministrtion of cenocoumrol t the doses used did not significntly ffect serum ctivity of mylse (Fig. 4). Administrtion of cenocoumrol given lone t the dose of 5 or 1 µg/kg/dose tended to increse serum concentrtion of pro-inflmmtory interleukin-1β (IL-1β), but this effect ws sttisticlly insignificnt (Fig. 5). In the cse of cenocoumrol given lone t the dose of 15 µg/kg/dose, this effect reched sttisticl significnce. In rts without induction of cute pncretitis, dministrtion of cenocoumrol t the doses used did not significntly ffect pncretic blood flow (Fig. 6), pncretic DNA synthesis (Fig. 7) or plsm D-Dimer Tble 1. Influence of ischemi/reperfusion-induced pncretitis () nd pretretment with cenocoumrol given t the dose of 5, 1 or 15 µg/kg/dy, pplied lone or in their combintion (cenocoumrol plus ) on morphologicl signs of pncretic dmge EDEMA (-3) INFLAMMATORY INFILTRATION (-3) VACUOLIZATION (-3) NECROSIS (-3) HEMORRHAGES (-3) CONTROL ACENOCOUMAROL 5-1 ACENOCOUMAROL 1-1 ACENOCOUMAROL ACENOCOUMAROL ACENOCOUMAROL ACENOCOUMAROL Numbers represent the predominnt histologicl grding in ech group ,b,c 5 INR ND ND C ACN 5 ACN 1 ACN 15 ACN 3 ACN 6 Fig. 1. Influence of tretment with cenocoumrol (ACN) for 7 dys t the dose of 5, 1, 15, 3 nd 6 µg/kg/dy on interntionl normlized rtio (INR). Men ± S.E.M., N = 6 in ech group of rts. P <.5 compred to control (C); b P <.5 compred to ACN 5: c P <.5 compred to ACN 1. ND = nondetectble.

4 INR C ACN 5 ACN 1 ACN 15 ACN 5 ACN 1 ACN 15 Fig. 2. Influence of ischemi/ reperfusion-induced pncretitis () nd pretretment with cenocoumrol (ACN) given t the dose of 5, 1 or 15 µg/kg/dy pplied lone or in their combintion (cenocoumrol plus ) on interntionl normlized rtio (INR). Men ± S.E.M., N = 1 in ech group of rts. P <.5 compred to control (C). 8 7 LIPASE (U/L) ,b,b C ACN 5 ACN 1 ACN 15 ACN 5 ACN 1 ACN 15 Fig. 3. Influence of ischemi/ reperfusion-induced pncretitis () nd pretretment with cenocoumrol (ACN) given t the dose of 5, 1 or 15 µg/kg/dy, pplied lone or in their combintion (cenocoumrol plus ) on serum lipse ctivity. Men ± S.E.M., N = 1 in ech group of rts. P <.5 compred to control (C); b P <.5 compred to lone AMYLASE (U/L) ,b,b,c C ACN 5 ACN 1 ACN 15 ACN 5 ACN 1 ACN 15 Fig. 4. Influence of ischemi/ reperfusion-induced pncretitis () nd pretretment with cenocoumrol (ACN) given t the dose of 5, 1 or 15 µg/kg/dy, pplied lone or in their combintion (cenocoumrol plus ) on serum mylse ctivity. Men ± S.E.M., N = 1 in ech group of rts. P <.5 compred to control (C); b P <.5 compred to lone; c P <.5 compred to ACN 5 plus.

5 735 INTERLEUKIN-1β (pg/ml) ,b,b C ACN 5 ACN 1 ACN 15 ACN 5 ACN 1 ACN 15 Fig. 5. Influence of ischemi/ reperfusion-induced pncretitis () nd pretretment with cenocoumrol (ACN) given t the dose of 5, 1 or 15 µg/kg/dy, pplied lone or in their combintion (cenocoumrol plus ) on serum interleukin-1β concentrtion. Men ± S.E.M., N = 1 in ech group of rts. P <.5 compred to control (C); b P <.5 compred to lone BLOOD FLOW (% of control) ,b,b C ACN 5 ACN 1 ACN 15 ACN 5 ACN 1 ACN 15 Fig. 6. Influence of ischemi/ reperfusion-induced pncretitis () nd pretretment with cenocoumrol (ACN) given t the dose of 5, 1 or 15 µg/kg/dy, pplied lone or in their combintion (cenocoumrol plus ) on pncretic blood flow. Men ± S.E.M. N = 1 in ech group of rts. P <.5 compred to control (C); b P <.5 compred to lone. 7 6 DNA SYNTHESIS (dpm/µg DNA) ,b,b C ACN 5 ACN 1 ACN 15 ACN 5 ACN 1 ACN 15 Fig. 7. Influence of ischemi/ reperfusion-induced pncretitis () nd pretretment with cenocoumrol (ACN) given t the dose of 5, 1 or 15 µg/kg/dy, pplied lone or in their combintion (cenocoumrol plus ) on pncretic DNA synthesis. Men ± S.E.M., N = 1 in ech group of rts. P <.5 compred to control (C); b P <.5 compred to lone.

6 D-DIMER (µg/ml) ,b,b,b C ACN 5 ACN 1 ACN 15 ACN 5 ACN 1 ACN 15 Fig. 8. Influence of ischemi/ reperfusion-induced pncretitis () nd pretretment with cenocoumrol (ACN) given t the dose of 5, 1 or 15 µg/kg/dy, pplied lone or in their combintion (cenocoumrol plus ) on plsm D-Dimer concentrtion. Men ± S.E.M., N = 1 in ech group of rts. P <.5 compred to control (C); b P <.5 compred to lone. concentrtion (Fig. 8). Morphologicl fetures showed tht cenocoumrol given lone cused in hlf of cses slight interlobulr edem of the pncres (Tble 1; Fig. 9B). Moreover, the highest dose of cenocoumrol, 15 µg/kg/dose led to pper single hemorrhgic foci in pncretic tissue. In the rest of cses, cenocoumrol given lone ws without effect on pncretic morphology (Tble 1). Pncretic ischemi followed by 5-h reperfusion induced cute hemorrhgic pncretitis in ll tested rts (Tble 1; Fig. 9C). In morphologicl exmintion, moderte inter- nd intrlobulr edem ws ccompnied with scre or moderte perivsculr nd scre diffuse leukocytic infiltrtion. Vcuoliztion ws observed in to less thn 25% of cinr cells. Necrosis ws observed in ll cses of cute pncretitis but involved less thn 15% of pncretic cells. Hemorrhge ws limited to 1 5 foci per slide. Histologicl findings were ssocited with biochemicl signs of cute pncretitis. Ischemi/reperfusion-induced pncretitis cused more thn 1-fold increse in serum ctivity of lipse (Fig. 3) nd more thn 13-fold increse in serum ctivity of mylse (Fig. 4). Serum concentrtion of pro-inflmmtory IL-1β reched round 34% of vlue observed in control rts (Fig. 5); wheres pncretic blood flow nd pncretic DNA syntheses were reduced by round 68 nd 52%, respectively (Figs. 6 nd 7). Moreover, ischemi/reperfusion-induced pncretitis cused significnt increse in INR (Fig. 2) nd plsm D-Dimer concentrtion (Fig. 8) by 52 nd 334%, respectively. In rts with ischemi/reperfusion-induced pncretitis, pretretment with cenocoumrol given t the dose of 5 or 1 µg/kg/dose ttenuted the development of cute pncretitis. In histologicl exmintion, this effect ws found s reduction in pncretic edem, inflmmtory infiltrtion, vcuoliztion of cinr cells, necrosis nd hemorrhges (Tble 1; Fig. 9D). Also, in those rts, cenocoumrol given t the dose of 5 or 1 µg/kg/dose significntly decresed serum ctivity of lipse (Fig. 3) nd mylse (Fig. 4), nd reduced serum concentrtion of proinflmmtory IL-1β (Fig. 5). Moreover; those doses of cenocoumrol prtly reversed the pncretitis-evoked decrese in pncretic blood flow (Fig. 6) nd pncretic DNA synthesis (Fig. 7). Pretretment with cenocoumrol t the dose of 1 µg/kg/dose trended to improve beneficil effects of cenocoumrol given t the doses of 5 µg/kg/dose; however difference between those effect ws not sttisticlly significnt, prt from the effect on serum ctivity of mylse. In contrst to effects of low doses of cenocoumrol, cenocoumrol given t the dose of 15 µg/kg/dose ws without beneficil effect on the pncretitis-evoked chnges of serum pncretic enzymes ctivity (Figs. 3 nd 4), serum concentrtion of IL-1β (Fig. 5), pncretic blood flow (Fig. 6) or pncretic DNA synthesis (Fig. 7). Morphologicl fetures showed tht pretretment with cenocoumrol given t the dose of 15 µg/kg/dose, ws without effect on the pncretitis-evoked pncretic inflmmtory infiltrtion, vcuoliztion of cinr cells or pncretic necrosis (Tble 1). Moreover, pretretment with cenocoumrol given t the dose of 15 µg/kg/dose incresed the pncretitis-evoked pncretic edem nd number of hemorrhges. Pretretment with cenocoumrol given t the dose of 5, 1 or 15 µg/kg/dose before induction of cute pncretitis cused the similr chnges in INR s in nimls without induction of pncretitis (Fig. 2). Administrtion of cenocoumrl in ll doses used cused similr nd sttisticlly significnt reduction in the pncretitis-evoked increse in plsm D-Dimer concentrtion (Fig. 8). DISCUSSION Our present study hs shown tht pretretment with low doses of cenocoumrol, leding to inhibition of cogultion, reduces the development of ischemi/reperfusion-induced cute pncretitis. Acenocoumrol is vitmin K ntgonist. Vitmin K is required to the synthesis of prothrombin nd three other plsm-clotting fctors, fctor VII, IX nd X. These clotting fctors re synthesized from biologiclly inctive precursor proteins (PIVKA - protein induced by vitmin K bsence) by γ- glutmyl-crboxylse in liver microsomes (33). Acenocoumrol, s others vitmin K ntgonists, reduces plsm levels of vitmin K-dependent clotting fctors nd thereby reduces the cogulbility of the blood (34). Previous studies hve shown close interction between inflmmtion nd cogultion (1, 4, 6). Inflmmtion results in ctivtion of cogultion, due to tissue fctor-medited thrombin genertion, decrese in the ctivity of nturl nticogulnt mechnisms nd inhibition of fibrinolysis. Similrly, ctivtion of cogultion increses the inflmmtory responses by presence of ctive clotting fctors (especilly thrombin, fctor X nd

7 737 A H C II V N E B E D II E H N Fig. 9. Representtive morphologicl fetures of the pncres observed in shm-operted sline-treted rts (pnel A), rts treted with cenocoumrol t the doses of 1 µg/kg/dose without induction of cute pncretitis (pnel B), rts with ischemi/reperfusioninduced cute pncretitis (pnel C), rts pretreted with cenocoumrol t the doses of 1 µg/kg/dose before induction of cute pncretitis (pnel D). Arrows with E men edem, with II - inflmmtory infiltrtion, with V - vcuoliztion, with N - necrosis, with H - hemorrhges. Hemtoxylin-eosin counterstin, originl mgnifiction 2. tissue fctor-fctor VII complex), meditors relesed from pltelets nd promotion of cell-cell interction (1, 4, 6). Moreover, there re studies showing tht cogultion bnormlities occurring in cute pncretitis re relted to the severity of this disese (13). Our present study is in greement with these dt nd hs shown tht inhibition of cogultion my ffect the development of cute pncretitis. We hve fond tht pretretment with low doses of cenocoumrol, inhibits the development of ischemi/reperfusion-induced cute pncretitis. Protective effect of cenocoumrol on the pncres ws mnifested by reduction in histologicl nd biochemicl signs of pncretic dmge. Morphologicl fetures of pncretic tissue hs shown tht pretretment with cenocoumrol reduces the pncretitisevoked pncretic edem, necrosis, hemorrhge, leukocyte infiltrtion nd vcuoliztion of pncretic cinr cells. In cute pncretitis, ctivtion of leukocytes nd relese of pro-inflmmtory cytokines re responsible for locl pncretic dmge nd development of systemic disorders such s systemic inflmmtory response syndrome (SS) nd multiple orgn filure (MOF) (35). Previous studies concerning the role of cytokines in cute pncretitis hve shown tht proinflmmtory cytokines such s IL-1β, IL-6 nd tumor necrosis fctor-α (TNF-α) re primry produced within pncres nd subsequently within distnt orgns, developing orgn dysfunction in severe cses of this disese (36). The severity of cute pncretitis is well-correlted with production of proinflmmtory cytokine (36). In erly phse of inflmmtion, the relese of IL-1β nd TNF-α precedes production of IL-6, nd this sequence of events hs been lso found in cute pncretitis (36). IL-1β plys the essentil role in the relese other members of pro-inflmmtory cytokine cscde nd the induction of systemic cute phse response (37). The role of leukocyte ctivtion nd IL-1β relese in the course of cute pncretitis hs been dditionlly evidenced by finding tht dministrtion of interleukin-1 receptor ntgonist prevents serum rise in IL6 nd TNF-α, nd decreses severity of cute pncretitis (38). Our present study indictes tht protective effect cenocoumrol in the pncres is ssocited with reduction in leukocyte inflmmtory infiltrtion of pncretic tissue nd decrese in the pncretitis-induced increse in serum IL-1β concentrtion. These chnges seem to be, t lest in prt, result of influence of cenocoumrol on thrombin formtion. Induction of cute pncretitis ctivtes cogultion (13). Acenocoumrol, s other vitmin K ntgonists, decreses the liver production of prothrombin nd reduces formtion of thrombin fter induction of cogultion. Thrombin promotes blood cogultion, but it lso serves s signling molecule by binding to protesectivted receptors (PARs) (6, 39). PARs expression hve been found on pltelets, endothelil cells nd mny immune cells including mcrophges, monocytes, dendritic cells, lymphocytes nd mst cells (6, 39, 4). In pltelets, pro-inflmmtory effect of thrombin is relted to ltertion of pltelets shpe to ctive phenotype nd relese of pltelet fctors, such s serotonin, thromboxne nd vriety of chemokines nd growth fctors. Furthermore, thrombin libertes the fibrinogen receptor GPIIbIII integrin complex nd P-selectin, s well s mobilizes the CD4 lignd to the pltelet surfce. CD4 lignd induces

8 738 endothelil cells to secrete chemokines nd to express dhesion molecules, leding to genertion of signls for recruitment nd extrvstion of leukocytes (6). Acting directly on PARs on endothelil cells, thrombin nd other proteses of the cogultion-fibrinolysis system chnge shpe of these cells into pro-inflmmtory phenotype, increse vsculr permebility, mobilize dhesive molecules nd stimulte the production of cytokines leding to the locl ccumultion of pltelets nd leukocytes (4). Moreover, thrombin is chemotctic for monocytes nd promotes the production, nd relese of proinflmmtory cytokines in immune cells (6). The increse in serum ctivity of lipse nd mylse is well estblished index of cute pncretitis severity with high sensitivity nd specificity (41). In our present study, pretretment with cenocoumrol given t the dose of 5 or 1 µg/kg/dy, hs reduced the pncretitis-evoked increse in serum ctivity of lipse nd mylse. This effect seems to be result of protective properties of cenocoumrol in the pncres, s well s, reduction in serum ctivity of pncretic enzymes is lso one of mechnism reducing the development of tissue dmge. Study performed by Keck et l. (42) hs shown tht presence of ctive pncretic digestive enzymes in the circultion up-regultes the expression of dhesion molecules on leukocytes nd endothelil cells, leding to increse in leukocyte-endothelil interction nd disturbnce pncretic microcircultion. Numerous clinicl nd experimentl studies hve shown tht pncretic ischemi plys n importnt role in the development of cute pncretitis nd to the progression of this disese to severe necrotizing pncretitis (43-46). On the other hnd, the improvement of pncretic blood flow inhibits the development of cute pncretitis nd ccelertes the recovery in this disese (47-49). In cute pncretitis evoked by pncretic ischemi followed by reperfusion, disturbnce of pncretic blood flow is primry cuse of this disese. Our present study hs shown tht pretretment with cenocoumrol, given t the low dose of 5 or 1 µg/kg/dy, improves pncretic blood flow in rts with induction of cute pncretitis nd this effect hs been ssocited with reduction of severity of pncretic dmge. This observtion indictes tht protective effect of cenocoumrol in the pncres reducing the development of ischemi/reperfusion-induced cute pncretitis involves improvement of pncretic microcircultion. However, this effect seems to be secondry one nd relted to nticogulnt ctivity of cenocoumrol. In our present study, induction of cute pncretitis by severe ischemi followed by reperfusion hs significntly incresed INR by 52% nd plsm D-Dimer concentrtion by more thn 3%. These findings indicte tht development of ischemi/reperfusion-induced cute pncretitis is ssocited with ctivtion of cogultion nd formtion of thrombi within pncretic nd systemic circultion, nd this process is followed by fibrinolysis. This conclusion is bsed on previous studies showing tht experimentl nimls (8, 13) nd ptients (1, 13, 5) with cute pncretitis develop the consumptive cogulopthy. Also, severe cute pncretitis my result in the development of disseminted intrvsculr cogultion (12, 51). D-Dimer is product of plsmin-induced degrdtion of stbilized fibrin (52, 53) nd for this reson it is recognized s mrker of fibrinolysis ctivtion (11, 13, 54). Our study hs lso shown tht pretretment with cenocoumrol dose-dependently incresed INR nd this effect reched similr rte in nimls with or without subsequent induction of cute pncretitis. On the other hnd, pretretment with cenocoumrol significntly reduced the pncretitisevoked increse in plsm D-Dimer concentrtion. These findings indicte tht pretretment with cenocoumrol reduces ctivtion of cogultion during induction of cute pncretitis nd for this reson inhibits formtion of thrombi nd reduces cretion of fibrinolysis products. DNA synthesis is n index of tissue cell vitlity nd cell prolifertion nd reduction in pncretic DNA synthesis is well-correlted with pncretic dmge in cute pncretitis (55-57). In our present study, pretretment with cenocoumrol given lone ws without significnt effect on pncretic DNA synthesis. This observtion indictes tht cnocoumrol given lone in doses used does not ffect pncretic cell vitlity nd prolifertion. On the other hnd, our present study hs shown tht pretretment with low doses of cenocoumrol before induction of cute pncretitis, ttenutes the pncretitisevoked fll in pncretic DNA synthesis. This finding is the dditionl evidence of the cenocoumrol-evoked protection of the pncres ginst the development of ischemi/reperfusioninduced pncretitis. In contrst to protective effects of low doses of cenocoumorol, our present study hs shown tht dministrtion of this vitmin K ntgonist t the dose of 15 µg/kg/dy given lone, leds to some pncretic dmge. Moreover, pretretment with cenocoumrol given t this dose, hs not shown ny protective effect ginst the development of ischemi/reperfusion-induced cute pncretitis. These findings re most likely result of excessive reduction in blood cogultion leding to excvtion of blood from blood vessels nd disturbnce of generl nd orgn circultion. This thesis is supported by our present observtion tht pretretment with cenocoumrol given t the dose of 15 µg/kg/dy cuses five-fold increse in INR. Lck of protective effect of pretretment with cenocoumrol given t the dose of 15 µg/kg/dy is lso in hrmony with previous observtion tht pretretment with high doses of cenocoumrol, such s 1 mg/kg/dy or more, increses the severity of experimentl cute pncretitis (58). Acenocoumrol given t the dose of 3 or 6 µg/kg/dose cused n increse INR bove detection rnge of the Alere INRtio 2PT/INR Monitoring System. On the other hnd, in rts with pncretitis nd pretreted with cenocoumrol given t the dose of 15µg/kg/dose D-Dimer concentrtion reched similr level s in nimls pretreted with lower doses of cenocoumrol. D-Dimer concentrtion reflects n ctivity of cogultion nd subsequent fibrinolysis. As ws shown in our present study, excessive reduction in cogultion by high dose of cenocoumrol ggrvtes the severity of ischemi/reperfusion-induced pncretitis. However, cenocoumrol reduces clot formtion nd for this reson deleterious effect of cenocoumrol given t the dose 15 µg/kg/dose ws not ssocited with increse in plsm D- Dimer concentrtion. Previous niml studies hve indicted tht pncretic ischemi my be cusl fctor in the pthogenesis of cute pncretitis development (45, 46, 59, 6). Vsculr mechnism plys n essentil role in the development of cute pncretitis in vrious clinicl setting, such s crdic (44) nd ortic (43, 61) surgery, hypovolemic shock (62), hypothermi (63) nd trnsplnttion of the pncres (64, 65). These dt nd our present observtion tht pretretment with low doses of cenocoumrol cn inhibit the development of ischemi/reperfusion-induced cute pncretitis, suggest tht pretretment cenocoumrol could be useful in the prevention of cute pncretitis development in ptients with diseses ssocited with pncretic circultion disorders. Finlly, we cn conclude tht pretretment with low doses of cenocoumrol reduces the severity of ischemi/reperfusioninduced cute pncretitis. Conflict of interests: None declred.

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