Identification of α2ß1 integrin inhibitor VP-i with anti-platelet properties in the venom of Vipera palaestinae.

Integrins are receptors of the extracellular matrix (ECM), playing a vital role in pathophysiological processes. They bind to ECM ligands like collagens and can mediate wound healing as well as tumor metastasis and thrombosis, thus being a part of cell adhesion and migration as well as platelet aggregation. For this reason, identifying α2ß1 integrin-specific antagonists can assist in the development of drugs to treat tumor progression, angiogenesis, and cardiovascular diseases. Snake venoms have been shown to contain antagonists which target collagen-binding integrins. EMS16, rhodocetin, and VP12 are three toxins belonging to the C-type lectin-related protein family and have been proven to inhibit the α2ß1 integrin, specifically the α2 integrin A domain. To specifically isolate antagonists targeting the α2ß1 integrin A domain, we developed a protocol based on affinity chromatography. Using this novel approach, the toxin VP-i was isolated from Vipera palaestinae venom. We show that VP-i binds to the α2 integrin A domain and that it successfully inhibits adhesion of various cells to type I collagen as well as cell migration. Moreover, our results indicate that VP-i differs structurally from the previously purified VP12, although not functionally, and therefore is a further venom compound which can be utilized for drug development.

The collagen-binding integrin α2ß1 is a novel interaction partner of the Trimeresurus flavoviridis venom protein flavocetin-A.

Many snake venoms are known for their antithrombotic activity. They contain components that specifically target different platelet-activating receptors such as the collagen-binding integrin α2ß1 and the von Willebrand factor receptor GPIb. In a search for an α2ß1 integrin-blocking component from the venom of the habu snake (Trimeresurus flavoviridis), we employed two independent purification protocols. First, we used the integrin α2A domain, a major collagen-binding domain, as bait for affinity purification of an α2ß1 integrin-binding toxin from the crude venom. Second, in parallel, we used classical protein separation protocols and tested for α2ß1 integrin-inhibiting capabilities by ELISA. Using both approaches, we identified flavocetin-A as an inhibitor of α2ß1 integrin. Hitherto, flavocetin-A has been reported as a GPIb inhibitor. However, flavocetin-A inhibited collagen-induced platelet aggregation even after GPIb was blocked with other inhibitors. Moreover, flavocetin-A antagonized α2ß1 integrin-mediated adhesion and migration of HT1080 human fibrosarcoma cells, which lack any GPIb, on collagen. Protein chemical analyses proved that flavocetin-A binds to α2ß1 integrin and its α2A domain with high affinity and in a cooperative manner, which most likely is due to its quaternary structure. Kinetic measurements confirmed the formation of a strong complex between integrin and flavocetin-A, which dissociates very slowly. This study proves that flavocetin-A, which has long been known as a GPIb inhibitor, efficiently targets α2ß1 integrin and thus blocks collagen-induced platelet activation. Moreover, our findings suggest that the separation of GPIb- and α2ß1 integrin-blocking members within the C-type lectin-related protein family is less strict than previously assumed.

Vixapatin (VP12), a c-type lectin-protein from Vipera xantina palestinae venom: characterization as a novel anti-angiogenic compound.

A C-type lectin-like protein (CTL), originally identified as VP12 and lately named Vixapatin, was isolated and characterized from Israeli viper Vipera xantina palestinae snake venom. This CTL was characterized as a selective α2ß1 integrin inhibitor with anti-melanoma metastatic activity. The major aim of the present study was to prove the possibility that this protein is also a potent novel anti-angiogenic compound. Using an adhesion assay, we demonstrated that Vixapatin selectively and potently inhibited the α2 mediated adhesion of K562 over-expressing cells, with IC(50) of 3 nM. 3 nM Vixapatin blocked proliferation of human dermal microvascular endothelial cells (HDMEC); 25 nM inhibited collagen I induced migration of human fibrosarcoma HT-1080 cells; and 50 nM rat C6 glioma and human breast carcinoma MDA-MB-231 cells. 1 µM Vixapatin reduced HDMEC tube formation by 75% in a Matrigel assay. Furthermore, 1 µM Vixapatin decreased by 70% bFGF-induced physiological angiogenesis, and by 94% C6 glioma-induced pathological angiogenesis, in shell-less embryonic quail chorioallantoic membrane assay. Vixapatin's ability to inhibit all steps of the angiogenesis process suggest that it is a novel pharmacological tool for studying α2ß1 integrin mediated angiogenesis and a lead compound for the development of a novel anti-angiogenic/angiostatic/anti-cancer drug.

C-type lectin-like proteins from snake venoms.

C-type lectin-like proteins (CTLs) as found in snake venoms fulfill various physiological functions. They play a role in hemostasis and have helped elucidate mechanisms involved in blood coagulation and platelet activation. Their basic structure consists of the subunits α and ß, which form heterodimers via a typical domain-swapping motif. These heterodimers can form oligomers such as the tetrameric flavocetin-A and convulxin, which arrange into cyclic structures. Rhodocetin is a selective α2ß1 integrin antagonist consisting of four distinct subunits forming a novel cruciform structure. Along with EMS16 and VP12, rhodocetin inhibits collagen-binding to the α2A-domain. These integrin-specific antagonists are lead structures for the development of antimetastatic and antiangiogenic drugs.

Pharmacological aspects of Vipera xantina palestinae venom.

In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1ß1selective KTS disintegrin and VP12, a α2ß1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation.