Disintegrin-like/cysteine-rich (DC) proteins have long been regarded just as products of proteolysis of P-III snake venom metalloproteinases (SVMPs). However, here we demonstrate that a DC protein from the venom of Vipera ammodytes (Vaa; nose-horned viper), VaaMPIII-3, is encoded per se by a P-III SVMP-like gene that has a deletion in the region of the catalytic metalloproteinase domain and in part of the non-catalytic disintegrin-like domain. In this way, we justify the proposal of the introduction of a new subclass P-IIIe of SVMP-derived DC proteins. We purified VaaMPIII-3 from the venom of Vaa in a series of chromatographic steps. A covalent chromatography step based on thiol-disulphide exchange revealed that VaaMPIII-3 contains an unpaired Cys residue. This was demonstrated to be Cys6 in about 90% and Cys19 in about 10% of the VaaMPIII-3 molecules. We further constructed a three-dimensional homology model of VaaMPIII-3. From this model, it is evident that both Cys6 and Cys19 can pair with Cys26, which suggests that the intramolecular thiol-disulphide exchange has a regulatory function. VaaMPIII-3 is an acidic 21-kDa monomeric glycoprotein that exists in at least six N-glycoforms, with isoelectric points ranging from pH 4.5 to 5.1. Consistent with the presence of an integrin-binding motif in its sequence, SECD, VaaMPIII-3 inhibited collagen-induced platelet aggregation. It also inhibited ADP- and arachidonic-acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination and the blood coagulation cascade.

Platelets play pivotal roles in thromboembolic diseases, such as myocardial infarction and ischemic stroke. In patients envenomed by the snake Vipera a. ammodytes (Vaa), pronounced and transient thrombocytopenia without bleeding is observed. We previously showed that Vaa-snaclec-3/2, the snake venom C-type lectin-like protein, mediates this effect ex vivo. Here, we extended our study of the antithrombotic potential of this protein in vivo using a mouse model of ferric chloride (FeCl₃)-induced carotid artery thrombosis. Prior to inducing thrombus formation, the mice received 1, 5, 10, 20, or 50 μg/kg Vaa-snaclec-3/2 intravenously. Afterward, the arterial blood flow was monitored with a perivascular Doppler probe. Additionally, the platelet count in the peripheral venous blood; tail bleeding time; and liver, lung, kidney, spleen, and heart histology were evaluated. The lowest dose of Vaa-snaclec-3/2 that we showed to cause severe thrombocytopenia and completely inhibit FeCl₃-induced thrombus formation was 20 µg/kg. This dose prolonged the median tail bleeding time from 86.5 to 153.5 s but did not induce acute spontaneous hemorrhage, as demonstrated by histological analysis. Histology revealed no signs of apoptosis, necrosis or other degenerative changes in the inspected organs of mice exposed to 20 μg/kg Vaa-snaclec-3/2. Platelet clusters were observed only in the lungs, which appear to be the primary site of platelet sequestration and the cause of thrombocytopenia. Taken together, our findings highlight the high potential of Vaa-snaclec-3/2 as a safe and effective antithrombotic agent for the transient prevention of thrombosis in acute clinical settings.

Investigating modes of action of natural toxins and venoms frequently inspire new therapies. Namely, many toxins precisely target specific receptors or pathways in organisms. For example, glucagon-like peptide-1-based drugs to treat diabetes and obesity, like Ozempic, were developed based on a peptide toxin from the venom of lizard Gila monster. In the venom of the nose-horned viper, we discovered and characterized the protein VaaSPH-1, which strongly inhibits blood coagulation via the intrinsic pathway. All current therapies for the prevention of venous thrombosis carry a high risk of severe bleeding as a side effect. Although numerous efforts have been made to improve treatment, no major breakthroughs have yet been achieved. The structure and mode of action of VaaSPH-1 are unique, making it a promising basis for the development of a safer anticoagulant drug for the treatment of venous thromboembolism that includes deep vein thrombosis and pulmonary embolism. Here, we describe our strategy for the development of innovative low molecular mass anticoagulant based on the structure of VaaSPH-1. The most promising candidate molecules are already in the process of patent protection. Protein iz strupa modrasa obeta razvoj inovativnega zdravila za zdravljenje venske trombembolije Izvleček: Preučevanje načinov delovanja naravnih toksinov in strupov pogosto navdahne razvoj novih načinov zdravljenja. Številni toksini namreč zelo natančno prizadenejo specifične receptorje ali signalne poti v organizmih. Na primer učinkovine homologne glukagonu podobnemu peptidu-1 za zdravljenje sladkorne bolezni in debelosti, kot je Ozempic, so bile razvite na osnovi peptidnega toksina iz strupa kuščarja gilske pošasti (Gila monster). V strupu modrasa smo odkrili in opisali protein VaaSPH-1, ki močno zavira strjevanje krvi po intrinzični poti. Vse trenutno dostopne terapije za preprečevanje venske tromboze so tvegane za pojav hudih krvavitev kot stranskega učinka. Kljub številnim prizadevanjem za izboljšanje zdravljenja te patologije večjega preboja še ni bilo. Struktura in način delovanja VaaSPH-1 sta edinstveni, zato ta molekula predstavlja obetavno osnovo za razvoj varnejše antikoagulantne učinkovine za zdravljenje venske trombembolije, ki vključuje trombozo globokih ven in pljučno embolijo. V članku je opisana strategija razvoja inovativnega nizkomolekularnega antikoagulanta na osnovi strukture VaaSPH-1. Najobetavnejše kandidatne molekule so že v postopku pridobivanja patentne zaščite. Ključne besede: kačji strup; hemostaza; inhibitor intrinzične tenaze; antagonist FIXa; antikoagulant; venska tromboza