Serine proteinases from Bothrops snake venom activates PI3K/Akt mediated angiogenesis

Abstract

The discovery of rapid acting and powerful angiogenic proteins are of significant interest in the treatment of various human disorders associated with insufficient angiogenesis such as ischemia, menorrhagia and delayed wound healing. Snake venoms consist of a mixture of bioactive proteins and polypeptides and are rich sources of pharmacologically important molecules. Serine proteinases are one of the abundant proteins present in Bothrops snake venoms and possess multiple biological functions including the regulation of the blood coagulation cascade. In this study, serine proteinases from Bothrops atrox (B. atrox) and Bothrops brazili (B. brazili) that modulate angiogenesis were purified and characterized. Molecular size exclusion chromatography, affinity chromatography followed by ion exchange chromatography of the serine proteinases indicated molecular masses of around 32 kDa. Serine proteinases from both the species exhibited diverse catalytic activities such as the ability to induce amidolytic, fibrinogenolytic, gelatinolytic activities and also coagulated plasma with a minimal coagulation concentration of 2.4 μg/mL. Serine proteinases facilitated the sprouting of human umbilical vein endothelial cells (HUVEC) in three-dimensional culture systems and induced tubule formation in monolayer culture systems. Serine proteinase stimulated Akt and eNOS phosphorylation in endothelial cells and addition of PI3K inhibitor LY294002 abrogated the effects of serine proteinases on sprout formation of endothelial cells in 3D collagen gels, suggesting that serine proteinase facilitated angiogenesis was mediated by PI3K/eNOS signaling axis. We also show in agarose plug assays using a mouse model, serine proteinases from Bothrops venoms significantly enhanced neovascularization. Our data suggests pro-angiogenic activity by the serine proteinases from B. atrox and B. brazili venom and further studies are warranted to explore the therapeutic applications.

Venomics and antivenomics of the poorly studied Brazil’s lancehead, Bothrops brazili (Hoge, 1954), from the Brazilian State of Pará

“…In the murine model, Peruvian B. brazili exhibited minimum hemorrhagic dose (MHD) of 7.40 μg/ mouse), minimum dermonecrotic dose (MND) of 152.15 μg/ mouse, minimum coagulant dose against plasma (MCD-P) and fibrinogen (MCD-F) of 19.20 and 1020.0 μg/mL, respectively, and minimum defibrinogenating dose (MDD) of 7.0 μg/mouse [11]. Although described as a new Bothrops from Brazil 65 years ago [15], very few studies have been reported on the toxin arsenal of the Brazil's lancehead venom, and these were mainly focused on the pharmacological effects and possible biotechnological applications of isolated toxins [21][22][23][24][25][26][27][28][29][30][31], including acidic and basic phospholipase A 2 (PLA 2 ) molecules (myotoxic Braziliase I and II, MTX I and II, brazilitoxins II and III) [23][24][25][26]; a PI-snake venom metaloproteinase (SVMP), with in vitro antiplasmodial properties [27]; coagulant thrombin-like and pro-angiogenic snake venom serine proteinase (SVSP) [28,29]; and a hyaluronidase [30].…”

Ac2-26 peptide and serine protease of Bothrops atrox similarly induces angiogenesis without triggering local and systemic inflammation in a murine model of dorsal skinfold chamber

“…Regardless the stimulatory or inhibitory effect, these mediators certainly play a role in mediating the pro-and anti-angiogenic balance, and investigating them is crucial to provide new alternatives for public health issues such as the application of bioengineering tools therapeutic strategies which success depends on angiogenesis, including tissue/organ transplantations and the treatment of diseases associated with impaired vascular irrigation (Liu et al, 2017;Said and Geoffrey, 2013;Mimura et al, 2016). Studies in this field have pointed the anti-inflammatory protein annexin A1 (AnxA1), as well as the serine protease isolated from Bothrops atrox venom (BaSp) as potent inductors of neovascularization (Bhat et al, 2016;M Yi and Schnitzer, 2009).…”

Clonagem e Expressão da Balternina, uma Serinoprotease de Bothrops alternatus (Urutu), em Sistema de Células de Bactéria e Levedura

“…Além disso, possuem também resíduos de cisteína, que são responsáveis pela formação das pontes dissulfeto. Apesar de serem glicoproteínas de cadeia única, interagem com uma grande diversidade de substratos, e possuem uma massa molecular que varia entre 26 e 67 kDa (MAMEDE, 2015;AMORIM et al, 2018).…”

“…As serinoproteases são sintetizadas como zimogênios e desempenham um papel importante na atividade patofisiológica induzida pela peçonha, pelo fato de estarem envolvidas em desordens hemostáticas, como ter influência na agregação plaquetária, no sistema fibrinolítico, na pressão sanguínea, além de poderem modular a angiogênese (AMORIM et al, 2018;COSTA et al, 2018). Ainda apresentam ação anticoagulante, pois podem ou realizar a degradação proteolítica de componentes do sistema hemostático, ou ativar e inibir de forma específica fatores da coagulação e fibrinólise.…”

“…Ainda apresentam ação anticoagulante, pois podem ou realizar a degradação proteolítica de componentes do sistema hemostático, ou ativar e inibir de forma específica fatores da coagulação e fibrinólise. Possuem também a característica de degradar preferencialmente a cadeia Bβ do fibrinogênio (PASCHOAL, 2015;BHAT et al, 2016).…”