ABSTRACT
The COVID-19 pandemic caused by the severe acute syndrome virus 2 (SARS-CoV-2) has been around since November 2019. As of early June 2022, more than 527 million cases were diagnosed, with more than 6.0 million deaths due to this disease. Coronaviruses accumulate mutations and generate greater diversity through recombination when variants with different mutations infect the same host. Consequently, this virus is predisposed to constant and diverse mutations. The SARS-CoV-2 variants of concern/interest (VOCs/VOIs) such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28/P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) have quickly spread across the world. These VOCs and VOIs have accumulated mutations within the spike protein receptor-binding domain (RBD) which interacts with the angiotensin-2 converting enzyme (ACE-2) receptor, increasing cell entry and infection. The RBD region is the main target for neutralizing antibodies; however, other notable mutations have been reported to enhance COVID-19 infectivity and lethality. Considering the urgent need for alternative therapies against this virus, an anti-SARS-CoV-2 equine immunoglobulin F(ab')2, called ECIG, was developed by the Butantan Institute using the whole gamma-irradiated SARS-CoV-2 virus. Surface plasmon resonance experiments revealed that ECIG binds to wild-type and mutated RBD, S1+S2 domains, and nucleocapsid proteins of known VOCs, including Alpha, Gamma, Beta, Delta, Delta Plus, and Omicron. Additionally, it was observed that ECIG attenuates the binding of RBD (wild-type, Beta, and Omicron) to human ACE-2, suggesting that it could prevent viral entry into the host cell. Furthermore, the ability to concomitantly bind to the wild-type and mutated nucleocapsid protein likely enhances its neutralizing activity of SARS-CoV-2. We postulate that ECIG benefits COVID-19 patients by reducing the infectivity of the original virus and existing variants and may be effective against future ones. Impacting the course of the disease, mainly in the more vulnerable, reduces infection time and limits the appearance of new variants by new recombination.
Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Viral , Horses , Humans , Nucleocapsid Proteins , Pandemics , Receptors, Virus/metabolism , Spike Glycoprotein, CoronavirusABSTRACT
The COVID-19 pandemic, caused by SARS-CoV-2, had its first cases identified in late 2019 and was considered a clinical pandemic in March 2020. In March 2022, more than 500 million people were infected and 6,2 million died as a result of this disease, increasingly associated with changes in human hemostasis, such as hypercoagulation. Numerous factors contribute to the hypercoagulable state, and endothelial dysfunction is the main one, since the activation of these cells can strongly activate platelets and the coagulation system. In addition, there is a dysregulation of the renin-angiotensin system due to the SARS-CoV-2 takeover of the angiotensin converting enzyme 2, resulting in a strong immune response that could further damage the endothelium. Thrombus formation in the pulmonary microvasculature structure in patients with COVID-19 is an important factor to determine the severity of the clinical picture and the outcome of this disease. This review describes the hemostatic changes that occur in SARS-CoV-2 infection, to further improve our understanding of pathogenic mechanisms and the interaction between endothelium dysfunction, kallikrein-kinins, renin angiotensin, and the Coagulation/fibrinolysis systems as underlying COVID-19 effectors. This knowledge is crucial for the development of new effective therapeutic approaches, attenuating the severity of SARS-CoV-2's infection and to reduce the deaths.
Subject(s)
COVID-19 , Hemostasis , Humans , Pandemics , Peptidyl-Dipeptidase A/metabolism , Renin-Angiotensin System/physiology , SARS-CoV-2ABSTRACT
Leptospirosis is a worldwide spread zoonosis, caused by pathogenic Leptospira. Evidences suggest that compromised hemostasis might be involved in the leptospirosis pathophysiology. In the genome of L. interrogans serovar Copenhageni, we found two genes coding for proteins which comprise von Willebrand factor (VWF) A domains (BatA and BatB). As VWF A domains exhibit multiple binding sites which contributes to human VWF hemostatic functions, we hypothesized that the L. interrogans BatA and BatB proteins could be involved in the hemostatic impairment during leptospirosis. We have cloned, expressed in Escherichia coli, and purified recombinant BatA and BatB. The influence of recombinant BatA and BatB on different in vitro hemostatic assays evaluating the enzymatic activity, platelet aggregation and fibrinogen integrity was investigated. We describe BatB as a new serine protease which is able to cleave thrombin chromogenic substrate, fibrin, fibrinogen, gelatin and casein; while BatA is active only towards fibrinogen. BatA and BatB interfere with the platelet aggregation induced by VWF/ristocetin and thrombin. Our results suggest an important role of the L. interrogans serovar Copenhageni Bat proteins in the hemostasis dysfunction observed during leptospirosis and contribute to the understanding of the leptospirosis pathophysiological mechanisms.
Subject(s)
Bacterial Proteins/metabolism , Fibrinogen/metabolism , Leptospira interrogans/enzymology , Platelet Aggregation/physiology , Serine Proteases/metabolism , Bacterial Proteins/genetics , Blood Coagulation , Factor V/metabolism , Factor Xa/metabolism , Humans , Leptospira interrogans/genetics , Leptospira interrogans/metabolism , Leptospira interrogans/pathogenicity , Recombinant Proteins/metabolism , Serine Proteases/genetics , von Willebrand Factor/metabolismABSTRACT
BACKGROUND: Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolin's roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosac's in vivo effects using a skin wound healing model in rats. METHODS: Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. RESULTS: The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. CONCLUSION: The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.
ABSTRACT
Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolins roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosacs in vivo effects using a skin wound healing model in rats. Methods Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. Results The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. Conclusion The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.
Subject(s)
Wound Healing , Apoptosis Regulatory Proteins/analysis , Apoptosis Regulatory Proteins/adverse effects , Lepidoptera/chemistryABSTRACT
Background Hemolin proteins are cell adhesion molecules from lepidopterans involved in a wide range of cell interactions concerning their adhesion properties. However, hemolin's roles in cell proliferation and wound healing are not fully elucidated. It has been recently reported that rLosac, a recombinant hemolin from the caterpillar Lonomia obliqua, presents antiapoptotic activity and is capable of improving in vitro wound healing. Therefore, this study aimed to explore rLosac's in vivo effects using a skin wound healing model in rats. Methods Circular full-thickness wounds in the rat dorsum skin were treated either with rLosac, or with saline (control), allowing healing by keeping the wounds occluded and moist. During the wound healing, the following tissue regeneration parameters were evaluated: wound closure and collagen content. Furthermore, tissue sections were subjected to histological and immunohistochemical analyses. Results The rLosac treatment has demonstrated its capacity to improve wound healing, as reflected in findings of a larger number of activated fibroblasts, proliferation of epithelial cells, increase of collagen type 1, and decrease of inflammatory infiltrate. Conclusion The findings have indicated the rLosac protein as a very promising molecule for the development of new wound-healing formulations.(AU)
Subject(s)
Skin/injuries , Wound Healing , Wounds and Injuries , Proteins , Cell Proliferation , Epithelial Cells , LepidopteraABSTRACT
Using a cDNA library made from Lonomia obliqua caterpillar bristles, we identified a transcript with a 603 bp open reading frame. The deduced protein corresponds to Lopap, a prothrombin activator previously isolated by our group from the bristles of this species. The mature protein is composed by 185 amino acids and shares similarity with members of the lipocalin family. The cDNA encoding the mature form was amplified by PCR, subcloned into pAE vector and used to transform Escherichia coli BL21(DE3) cells. As for the native Lopap, the recombinant fusion protein shows enzymatic activity, promotes prothrombin hydrolysis, generates fragments similar to prethrombin-2 and fragment 1.2 as intermediates, and generates thrombin as the final product. In addition, structural bioinformatics studies indicated several interesting molecular features, including the residues that could be responsible for Lopap's serine protease-like activity and the role of calcium binding in this context. Such catalytic activity has never been found in other members of the lipocalin family. This is the first report describing the recombinant production and biochemical characterization of a Lonomia obliqua lipocalin, as well as the structural features that could be responsible for its serine protease-like catalytic activity.
Subject(s)
Insect Proteins/chemistry , Insect Proteins/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Circular Dichroism , DNA, Complementary/genetics , Electrophoresis, Polyacrylamide Gel , Factor X/metabolism , Humans , Hydrolysis , Insect Proteins/classification , Insect Proteins/genetics , Lactoglobulins/chemistry , Lepidoptera , Models, Molecular , Molecular Sequence Data , Protein Conformation , Prothrombin/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Serine Endopeptidases/classification , Serine Endopeptidases/genetics , Structure-Activity RelationshipABSTRACT
BuXI, o inibidor do tipo Kunitz isolado das sementes de B. ungulata inativa fator Xa, calicreína plasmática humana, tripsina e o LOPAP, ativador de protrombina isolado das cerdas da lagarta Lonomia obliqua. BvTI, isolado das sementes de B. variegata,l extensamente (70 por cento) homólogo a BuXI, inibe tripsina mas não inibe fator Xa e LOPAP. A diferença mais marcante entre os sítios reativos de BuXI e BvTI encontra-se nos resíduos Met59, Met66 e Thrs7. As constantes de inibição medidas foram confirmadas para tripsina e fator Xa,1 em sistema Biacore. BuXI também inibe o fator Xa no complexo protrombinase. A hidrólise de substratos peptídicos de fluorescência apagada, sintetizados com base nos sítios reativos desses inibidores, foi estudada com fator Xa, LOPAP,1 calicreína plasmática, calicreína pancreática de porco, trombina e tripsina. Abz-VMIAALPRTMFIQ-EDDnp,l peptídeo modelo baseado em BuXI, foi eficientemente hidrolisado fator Xa, mas não Abz-WISALPRSLFIQ-EDDnp baseado em BvTI. A influência do número de resíduos de aminoácidos, para a interação com o tor Xá, foi demonstrada pelo decréscimo de eficiência catalítica da hidrólise de bstratos de cadeia mais curta. Contudo, para trombina, tais mudanças foram de eito menor. Devido à constatação de que BuXI deixa de inibir o fator Xa após ser oxidado, a articipação dos resíduos de metionina para a interação enzima-substrato foi estudada. ais resíduos efetivamente estão envolvidos nas reações catalisadas por fator Xa e OPAP como demonstrado na série de substratos peptídicos baseados nos sítios ativos de BuXI e BvTI, que é desprovido de metionina...(au)
