Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation

Costa, Caroline Rabelo; Belchor, Mariana Novo; Rodrigues, Caroline Fabri Bittencourt; Toyama, Daniela de Oliveira; Oliveira, Marcos Antônio de; Novaes, Danielle P.; Toyama, Marcos Hikari

doi:10.3390/ijms19082405

Cited by 17 publications

(25 citation statements)

References 50 publications

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“…The recombinant collinein-1 was able to induce a discreet paw edema in mice 30 min after injection, leading to an increase in paw thickness of less than 15%. Some serine proteases have been previously tested for induction of paw edema in mice, such as BpirSP41 and BpirSP27 from Bothrops pirajai [55], BpSP-I from B. pauloensis [56], TLBm from B. marajoensis [57] and Cdtsp 2 from C. d. durissus [58], in which all of them induced mild to moderate edema formation. The edema induced by snake venoms depends mainly on the release of pro-inflammatory mediators, such as arachidonic acid metabolites (prostaglandins and leukotrienes), lipoxygenase products, histamines, serotonin and nitric oxide [59, 60].…”

Section: Discussionmentioning

confidence: 99%

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

Boldrini-França

Pinheiro-Júnior²,

Arantes³

2019

J. Venom. Anim. Toxins incl. Trop. Dis

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Background: The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus , named rCollinein-1. Methods: Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins. Results: When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro , which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis Conclusion: These results suggest that the recombinant enzyme has no potential as a thrombolytic agent but can be applied in the prevention of thrombus formation in some pathological processes and as molecular tools in studies related to hemostasis.

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Section: Discussionmentioning

confidence: 99%

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

Boldrini-França

Pinheiro-Júnior²,

Arantes³

2019

J. Venom. Anim. Toxins incl. Trop. Dis

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“…SVSPs cause interference and imbalances of the hemostatic system by promoting specific proteolysis at various key points of the coagulation cascade [ 97 , 98 ]. Furthermore, SVSPs have been demonstrated to induce significant edema via the metabolism of arachidonic acid (AA), involving protease-activated receptors (PARs), protein kinase c (PKC), phospholipase C (PLC), and cyclooxygenase-2 (COX-2) receptors, and also induce a significant increase in malondialdehyde (MDA) levels [ 99 ]. Costa et al [ 99 ] demonstrated that SVSPs may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress.…”

Section: Neurotherapeutics and Preconditioning In Surgical Brain Injumentioning

confidence: 99%

“…Furthermore, SVSPs have been demonstrated to induce significant edema via the metabolism of arachidonic acid (AA), involving protease-activated receptors (PARs), protein kinase c (PKC), phospholipase C (PLC), and cyclooxygenase-2 (COX-2) receptors, and also induce a significant increase in malondialdehyde (MDA) levels [ 99 ]. Costa et al [ 99 ] demonstrated that SVSPs may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. SVSPs trigger inflammatory cascades that have been implicated in SBI pathophysiology that SVMPs and PLA 2 might not successfully trigger.…”

Section: Neurotherapeutics and Preconditioning In Surgical Brain Injumentioning

confidence: 99%

Surgically-induced brain injury: where are we now?

et al. 2019

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Neurosurgical procedures cause inevitable brain damage from the multitude of surgical manipulations utilized. Incisions, retraction, thermal damage from electrocautery, and intraoperative hemorrhage cause immediate and long-term brain injuries that are directly linked to neurosurgical operations, and these types of injuries, collectively, have been termed surgical brain injury (SBI). For the past decade, a model developed to study the underlying brain pathologies resulting from SBI has provided insight on cellular mechanisms and potential therapeutic targets. This model, as seen in a rat, mouse, and rabbit, mimics a neurosurgical operation and causes commonly encountered post-operative complications such as brain edema, neuroinflammation, and hemorrhage. In this review, we elaborate on SBI and its clinical impact, the SBI animal models and their clinical relevance, the importance of applying therapeutics before neurosurgical procedures (i.e., preconditioning), and the new direction of applying venom-derived proteins to attenuate SBI.

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“…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.…”

Section: Serinoproteasesunclassified

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

Polli¹

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Despite their toxicity, snake venoms are composed of a series of organic and non-organic components that show activities against virus and bacteria, as well as therapeutic activities in thrombotic disorders. Thus, the characterization of biomolecules present in venoms has shown to be important to develop alternative therapies. Bhalternin, a toxin from Bothrops alternatus, has been described as a thrombin-like enzyme and has a therapeutic potential in cases of thrombotic disorders. So, the present study had as objective to perform cloning and expression of Bhalternin in bacterial and yeast systems. The mature Bhalternin coding sequence was cloned into pGS-21a e pPICZα A vectors and, after confirmation of the nucleotide sequences by Sanger Method, the plasmids that were constructed, pPGSBH2 e pPαABH9, were utilized in the transformation of Escherichia coli (strain BL21-Codon-Plus(DE3)-RIPL) and the Pichia pastoris (strain X-33), respectively. Different colonies, which showed presence of plasmid DNA by Polymerase Chain Reaction, were purified, submitted to the expression process and, then, evaluated for Bhalternin expression by protein eletrophoresis and Western blotting. In the bacterial system, a protein band corresponding to the expected molecular weight of Bhalternin expressed as fusion protein with glutathione Stransferase (GST) was observed in the supernatant and in the precipitate of bacterial lysate. However, in yeast system, it was not possible to identify a protein band corresponding to the secreted form of Bhalternin. However, in ELISA it was observed levels of expression in yeast system. Still, the coagulation test was carried out and, although a bacterial sample obtained by treating the lysate precipitate with 3 M urea showed activity, the result was inconclusive, since a sample obtained with the control clone (transformed with the vector) also showed activity. In the azocaseinolytic test, all results samples showed activity, including samples obtained with control clones. In the case of yeast expression system, the results seem to indicate low expression of Bhalternin, even though codons optimized for P. pastoris expression were used. On the other hand, in bacterial system, solubilization of Bhalternin with urea was necessary and the treatment may have caused changes in its activities. Therefore, further tests should be performed in order to obtain Bhalternin as soluble form and to evaluate its therapeutic potential in thrombotic disorders, as well as, its antiviral, antibacterial, and antiparasitic activities.

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Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation

Cited by 17 publications

References 50 publications

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

Surgically-induced brain injury: where are we now?

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

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