20.500.12556/RUL-189829 ​Izolacija in lastnosti proteinov iz strupa modrasa (Vipera ammodytes ammodytes), ki povzročajo prehodno in reverzibilno trombocitopenijo funkcionalnih trombocitov ​Isolation and characterization of proteins from long-nosed viper (Vipera ammodytes ammodytes) venom that induce transient and reversible thrombocytopenia of functional platelets Uvod: V Centru za klinično toksikologijo in farmakologijo Univerzitetnega kliničnega centra Ljubljana letno zdravimo 5–10 bolnikov po ugrizu modrasa (Vipera ammodytes ammodytes; Vaa). Pri bolnikih po ugrizu modrasa smo večkrat opazili pojav izrazite, vendar prehodne trombocitopenije brez klinično pomembne krvavitve. Mehanizem tega procesa še ni bil pojasnjen. Namen: Namen doktorske disertacije je bil raziskati mehanizem trombocitopenije po zastrupitvi z modrasovim strupom. Metode: Doktorsko disertacijo smo razdelili na klinične, biokemijske, ex vivo in in vivo raziskave. V kliničnem delu raziskave smo pri bolnikih po ugrizu modrasa analizirali pogostost pojava trombocitopenije, z njo povezano klinično sliko ter lastnosti trombocitov med trombocitopenijo in po njeni razrešitvi. Ocenili smo tudi vpliv različnih vrst protistrupa na potek in resnost trombocitopenije. V biokemijskem delu raziskave smo iz modrasovega strupa izolirali več proteinov snaclec, jih strukturno in funkcijsko opredelili ter z ex vivo poskusi preučili njihove učinke na trombocite. Na podlagi pregleda literature smo namreč sklepali, da utegnejo snacleci pomembno prispevati k razvoju trombocitopenije. V sklepnem delu raziskave smo učinke izbranega snacleca, Vaa-snaclec-3/2, opredelili v in vivo modelu ter preverili njegov antitrombotični potencial. Rezultati: V kliničnem delu disertacije smo pokazali, da se trombocitopenija po ugrizu modrasa pojavlja pogosteje kot po ugrizu gada, kar je najverjetneje posledica razlike v sestavi strupa obeh vrst kač. Trombocitopenija je bila prehodna in reverzibilna po aplikaciji protistrupa ter neposredno odvisna od koncentracije prostega strupa v krvi, kar potrjuje ključno vlogo strupnih komponent pri njenem nastanku. Zvišanje števila trombocitov po intravenski aplikaciji protistrupa je sovpadalo z znižanjem koncentracije strupa v obtoku. Kljub hudi trombocitopeniji so trombociti pri prizadetih bolnikih ohranili polno funkcionalnost. Rotacijska tromboelastometrija je po ponovni normalizaciji števila trombocitov podala rezultate v mejah referenčnih vrednosti. To kaže na ohranitev polne funkcionalnosti hemostaze, v skladu z rezultati agregometrije. Rezultati pretočne citometrije so pokazali, da med trombocitopenijo in po njej ni prišlo do povečane izpostavljenosti P-selektina na zunanji membrani trombocitov, ki sicer označuje njihovo aktivacijo. Naši izsledki kažejo, da trombocitopenija ni posledica agregacije trombocitov, temveč bolj verjetno njihove aglutinacije brez aktivacije. V biokemijskem delu disertacije smo izolirali več snaclecev iz modrasovega strupa, pri čemer je le heterodimerni snaclec Vaa-snaclec-3/2 brez vpliva na koagulacijske teste specifično zaviral z ristocetinom-inducirano aglutinacijo trombocitov. Ex vivo je dodatek Vaa-snacleca-3/2 polni krvi povzročil izrazito trombocitopenijo. Pod mikroskopom smo pri tem opazili tvorbo skupkov (aglutinatov) manjšega števila trombocitov. Pokazali smo, da se Vaa-snaclec-3/2 koncentracijsko-odvisno veže na trombocitni receptor GPIb, ne pa na receptorje GPIIb/IIIa, GPIX ali GPVI, pri tem pa ne sproži aktivacije trombocitov. V in vivo modelu tromboze karotidne arterije pri miši je Vaa-snaclec-3/2 izval izrazito, a prehodno trombocitopenijo, š čimer je zavrl nastanek tromba ob poškodbi žilne stene. Aplikacija fragmentov F(ab’)₂ protiteles, usmerjenih proti modrasovemu strupu, je trombocitopenijo učinkovito odpravila. Klinični primer bolnice s ponavljajočimi se epizodami trombocitopenije po ugizu modrasa in aplikacijami fragmentov Fab protistrupa je dodatno potrdil, da je trombocitopenija odvisna od koncentracije strupa. Potrdil je tudi reverzibilnost trombocitopenije ob uporabi imunoterapije, ki ohrani tudi popolno funkcionalnost trombocitov. Zaključek: Izrazita trombocitopenija je pogost pojav ob zastrupitvi z modrasovim strupom. Zaplet je ob aplikaciji fragmentov Fab ali F(ab’)₂, usmerjenih proti celotnemu strupu modrasa, hitro razrešljiv, trombociti pa ostanejo funkcionalni. Med več snacleci v strupu modrasa le Vaa-snaclec-3/2 povzroči trombocitopenijo. Vzrok za to je vezava Vaa-snaclec-3/2 na trombocitni receptor GIb, ki izove aglutinacijo trombocitov brez njihove aktivacije. V disertaciji smo celostno pojasnili mehanizem trombocitopenije pri zastrupitvi z modrasovim strupom. Komponento strupa Vaa-snaclec-3/2 smo identificirali kot ključni molekulski dejavnik opazovanega procesa in s tem izpostavili njegov velik terapevtski potencial pri medicinskih postopkih, kjer je potreben prehoden in hitro razrešljiv antitrombotičen učinek. Introduction: At the Centre for Clinical Toxicology and Pharmacology of the University Medical Centre Ljubljana, we treat 5–10 patients annually due to bites by nose-horned viper (Vipera ammodytes ammodytes; Vaa). In patients bitten by Vaa, we have repeatedly observed the occurrence of pronounced but transient thrombocytopenia without clinically significant bleeding. The mechanism underlying this process has not yet been elucidated. Aim: The aim of this doctoral dissertation was to investigate the mechanism of thrombocytopenia following envenomation with Vaa venom. Methods: The doctoral dissertation was divided into clinical, biochemical, ex vivo, and in vivo studies. In the clinical part of the study, we analyzed the frequency of thrombocytopenia in patients bitten by the Vaa, the associated clinical presentation, and platelet characteristics during thrombocytopenia and after its resolution. We also assessed the impact of different types of antivenom on the course and severity of thrombocytopenia. In the biochemical part of the study, several snaclec proteins were isolated from Vaa venom, structurally and functionally characterized, and their effects on platelets were investigated using ex vivo experiments. Based on a review of the literature, we hypothesized that snaclecs might play an important role in the development of thrombocytopenia. In the final part of the study, the effects of a selected snaclec, Vaa-snaclec-3/2, were characterized in an in vivo model, and its antithrombotic potential was evaluated. Results: In the clinical part of the dissertation, we demonstrated that thrombocytopenia occurs more frequently after Vaa bites than after Vipera berus berus bites, most likely due to differences in venom composition between the two snake species. Thrombocytopenia was transient and reversible following antivenom administration and was directly dependent on the concentration of free venom in the blood, confirming the key role of venom components in its development. The increase in platelet count after intravenous antivenom administration coincided with a decrease in circulating venom concentration. Despite severe thrombocytopenia, platelets in affected patients retained full functionality. Rotational thromboelastometry performed after normalization of platelet count yielded results within reference ranges. This indicates preserved overall hemostatic function, in agreement with aggregometry results. Flow cytometry results showed no increased exposure of P-selectin on the external platelet membrane during or after thrombocytopenia, which would otherwise indicate platelet activation. Our findings suggest that thrombocytopenia is not the result of platelet aggregation, but rather of platelet agglutination without activation. In the biochemical part of the dissertation, several snaclecs were isolated from Vaa venom. Among them, only the heterodimeric snaclec Vaa-snaclec-3/2 specifically inhibited ristocetin-induced platelet agglutination without affecting coagulation tests. Ex vivo, the addition of Vaa-snaclec-3/2 to whole blood caused profound thrombocytopenia. Microscopic examination revealed the formation of small clusters (agglutinates) consisting of a smaller number of platelets. We demonstrated that Vaa-snaclec-3/2 binds to the platelet receptor GPIb in a concentration-dependent manner, but not to the GPIIb/IIIa, GPIX, or GPVI receptors, and does not trigger platelet activation. In an in vivo mouse model of carotid artery thrombosis, Vaa-snaclec-3/2 induced profound but transient thrombocytopenia, thereby inhibiting thrombus formation following vascular wall injury. Administration of F(ab’)₂ antibody fragments directed against Vaa venom effectively reversed thrombocytopenia. A clinical case of a patient with recurrent episodes of thrombocytopenia following Vaa bites and repeated administrations of Fab antivenom fragments further confirmed that thrombocytopenia is dependent on venom concentration. It also confirmed the reversibility of the process with immunotherapy, which preserves full platelet functionality. Conclusion: Profound thrombocytopenia is a common finding in Vaa venom envenomation. This complication is rapidly reversible after administration of Fab or F(ab’)₂ fragments directed against whole Vaa venom, and platelets remain functional. Among the several snaclecs present in Vaa venom, only Vaa-snaclec-3/2 induces thrombocytopenia. The cause is the binding of Vaa-snaclec-3/2 to the platelet receptor GPIb, which triggers platelet agglutination without activation. In this dissertation, we comprehensively elucidated the mechanism of thrombocytopenia in Vaa venom envenomation. The venom component Vaa-snaclec-3/2 was identified as the key molecular factor underlying the observed process, highlighting its considerable therapeutic potential in medical procedures requiring a transient and rapidly reversible antithrombotic effect. Modras kačji strup reverzibilna trombocitopenija Vaa-snaclec-3/2 antitrombotik Nose-horned viper snake venom reversible thrombocytopenia Vaa-snaclec-3/2 antithrombotic true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-10-16 13:31:12 2026-10-16 13:31:27 2026-10-17 04:16:19 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 43331 9242.pdf 9242.pdf 1 82D05ADE55CB203F8F1BE7ECE6498F5C 4fdcd458e260ae90a748e25eba0bdd392b589fb5b8c7216ce326553d3c1c719e f537f576-c954-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=250694 Medicinska fakulteta 0 0 0