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1.
ACS Chem Neurosci ; 12(21): 4144-4152, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34669381

RESUMO

Cerebrovascular dysfunction is a common phenomenon in Alzheimer's patients, where fibrinogen is a major player. With the blood-brain barrier compromised, fibrinogen gains access to the brain, where its interaction with Aß42 results in plasmin-resistant abnormal blood clots that are deposited in the cerebral blood vessels, a condition commonly encountered in Alzheimer's disease (AD) patients called cerebral amyloid angiopathy (CAA). So far, there have been no effective therapeutics available to combat AD-associated CAA. This study reports a 13-amino acid peptide (Pα-NPGRPEPGSAGTW) as a potential inhibitor of the fibrin-Aß42 interaction along with the property to dissolve pre-existing plasmin-resistant abnormal clots. Strikingly, the identified sequence was found to be partially similar to a fragment of the fibrinogen α-chain reported to bind Aß42, the plasmin-resistant fibrinogen fragment (PRFF). Mechanistically, Pα interacts with Aß42 in place of fibrinogen, thus inhibiting the toxic fibrin-Aß42 interaction. However, it does not interfere with normal fibrin polymerization.


Assuntos
Doença de Alzheimer , Fibrinolisina , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Fibrina , Fibrinogênio , Humanos , Fragmentos de Peptídeos
2.
Int J Mol Sci ; 22(17)2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34502397

RESUMO

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.


Assuntos
Pré-Albumina/metabolismo , alfa 1-Antitripsina/metabolismo , Fatores Etários , Amiloide/metabolismo , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/fisiopatologia , Amiloidose/genética , Amiloidose/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Fibrinolisina , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pré-Albumina/genética , Pré-Albumina/fisiologia , Proteólise , alfa 1-Antitripsina/fisiologia
3.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445684

RESUMO

The shape and transparency of the cornea are essential for clear vision. However, its location at the ocular surface renders the cornea vulnerable to pathogenic microorganisms in the external environment. Pseudomonas aeruginosa and Staphylococcus aureus are two such microorganisms and are responsible for most cases of bacterial keratitis. The development of antimicrobial agents has allowed the successful treatment of bacterial keratitis if the infection is diagnosed promptly. However, no effective medical treatment is available after progression to corneal ulcer, which is characterized by excessive degradation of collagen in the corneal stroma and can lead to corneal perforation and corneal blindness. This collagen degradation is mediated by both infecting bacteria and corneal fibroblasts themselves, with a urokinase-type plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP) cascade playing a central role in collagen destruction by the host cells. Bacterial factors stimulate the production by corneal fibroblasts of both uPA and pro-MMPs, released uPA mediates the conversion of plasminogen in the extracellular environment to plasmin, and plasmin mediates the conversion of secreted pro-MMPs to the active form of these enzymes, which then degrade stromal collagen. Bacterial factors also stimulate expression by corneal fibroblasts of the chemokine interleukin-8 and the adhesion molecule ICAM-1, both of which contribute to recruitment and activation of polymorphonuclear neutrophils, and these cells then further stimulate corneal fibroblasts via the secretion of interleukin-1. At this stage of the disease, bacteria are no longer necessary for collagen degradation. In this review, we discuss the pivotal role of corneal fibroblasts in corneal ulcer associated with infection by P. aeruginosa or S. aureus as well as the development of potential new modes of treatment for this condition.


Assuntos
Úlcera da Córnea/metabolismo , Fibroblastos/metabolismo , Ceratite/microbiologia , Animais , Colágeno/metabolismo , Córnea/metabolismo , Córnea/fisiologia , Substância Própria/metabolismo , Úlcera da Córnea/etiologia , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/fisiopatologia , Fibrinolisina/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Plasminogênio/metabolismo , Ativadores de Plasminogênio/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
5.
Blood ; 138(3): 208-209, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34292330

Assuntos
Fibrinolisina , Fígado
6.
Eur J Med Chem ; 223: 113653, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34161866

RESUMO

Si113, a pyrazolo[3,4-d]pyrimidine derivative, gained more attention as an anticancer agent due to its potent anticancer activity on both in vitro and in vivo hepatocellular carcinomas (HCC) and ovarian carcinoma models. But the drawback is the low water solubility which prevents its further development. In this context, we successfully overcame this limitation by synthesizing two novel prodrugs introducing the amino acid sequence D-Ala-Leu-Lys (TP). Moreover, TP sequence has a high affinity with plasmin, a protease recognized as overexpressed in many solid cancers, including HCC and ovarian carcinoma. The prodrugs were synthesized and fully characterized in terms of in vitro ADME properties, plasma stability and plasmin-induced release of the parent drug. The inhibitory activity against Sgk1 was evaluated and in vitro growth inhibition was evaluated on ovarian carcinoma and HCC cell lines in the presence and absence of human plasmin. In vivo pharmacokinetic properties and preliminary tissue distribution confirmed a better profile highlighting the importance of the prodrug approach. Finally, the prodrug antitumor efficacy was evaluated in an HCC xenografted murine model, where a significant reduction (around 90%) in tumor growth was observed. Treatment with ProSi113-TP in combination with paclitaxel in a paclitaxel-resistant ovarian carcinoma xenografted murine model, resulted in an impressive reduction of tumor volume greater than 95%. Our results revealed a promising activity of Si113 prodrugs and pave the way for their further development against resistant cancer.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Fibrinolisina/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Pró-Fármacos/metabolismo , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estabilidade de Medicamentos , Feminino , Meia-Vida , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Transplante Heterólogo
7.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069309

RESUMO

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients' plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and "D:D" interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.


Assuntos
Afibrinogenemia/genética , Fibrinogênios Anormais/genética , Fibrinogênios Anormais/metabolismo , Mutação , Adulto , Afibrinogenemia/sangue , Animais , Testes de Coagulação Sanguínea , Células CHO , Cricetulus , Fator XIIIa/química , Fator XIIIa/metabolismo , Feminino , Fibrina/metabolismo , Fibrinogênios Anormais/química , Fibrinolisina/metabolismo , Heterozigoto , Humanos , Immunoblotting , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/metabolismo
8.
J Med Chem ; 64(9): 5667-5688, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33949859

RESUMO

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) that causes severe motor, sensory, and cognitive impairments. Kallikrein-related peptidase (KLK)6 is the most abundant serine protease secreted in the CNS, mainly by oligodendrocytes, the myelin-producing cells of the CNS, and KLK6 is assumed to be a robust biomarker of MS, since it is highly increased in the cerebrospinal fluid (CSF) of MS patients. Here, we report the design and biological evaluation of KLK6's low-molecular-weight inhibitors, para-aminobenzyl derivatives. Interestingly, selected hit compounds were selective of the KLK6 proteolytic network encompassing KLK1 and plasmin that also participate in the development of MS physiopathology. Moreover, hits were found noncytotoxic on primary cultures of murine neurons and oligodendrocyte precursor cells (OPCs). Among them, two compounds (32 and 42) were shown to promote the differentiation of OPCs into mature oligodendrocytes in vitro constituting thus emerging leads for the development of regenerative therapies.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Calicreínas/antagonistas & inibidores , Inibidores de Serino Proteinase/farmacologia , Animais , Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Derivados de Benzeno/farmacologia , Sítios de Ligação , Domínio Catalítico , Células Cultivadas , Desenho de Fármacos , Fibrinolisina/antagonistas & inibidores , Fibrinolisina/metabolismo , Humanos , Calicreínas/metabolismo , Cinética , Camundongos , Simulação de Acoplamento Molecular , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Relação Estrutura-Atividade
9.
Artigo em Inglês | MEDLINE | ID: mdl-33838314

RESUMO

Bee venom is a mixture of bioactive components that include proteases and protease inhibitors. A metalloprotease inhibitor has been predicted to be a bumblebee-specific toxin in the venom proteome of Bombus terrestris; however, the identification and functional roles of bee venom metalloprotease inhibitors have not been previously determined. In this study, we identified a bumblebee (B. ignitus) venom metalloprotease inhibitor (BiVMPI) that exhibits anti-fibrinolytic activity. BiVMPI contains a trypsin inhibitor-like cysteine-rich domain that exhibits similarity to inducible metalloprotease inhibitor. Using an anti-BiVMPI antibody raised against a recombinant BiVMPI protein produced in baculovirus-infected insect cells, the presence of BiVMPI in the venom gland and secreted venom of B. ignitus worker bees was confirmed. The recombinant BiVMPI protein demonstrated inhibitory activity against a metalloprotease, trypsin, chymotrypsin, protease K, and plasmin, but not subtilisin A, elastase, or thrombin. Additionally, the recombinant BiVMPI bound to plasmin and inhibited the plasmin-mediated degradation of fibrin, demonstrating an anti-fibrinolytic role for BiVMPI as a bee venom metalloprotease inhibitor. Our results provide the first evidence for the identification and anti-fibrinolytic activity of a metalloprotease inhibitor from bee venom.


Assuntos
Venenos de Abelha/química , Fibrinogênio/química , Proteínas de Insetos/química , Inibidores de Metaloproteinases de Matriz/química , Proteínas Recombinantes/química , Animais , Abelhas , Fibrinolisina/química , Humanos
10.
Molecules ; 26(8)2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33920584

RESUMO

Age gelation is a major quality defect in ultra-high-temperature (UHT) pasteurized milk during extended storage. Changes in plasmin (PL)-induced sedimentation were investigated during storage (23 °C and 37 °C, four weeks) of UHT skim milk treated with PL (2.5, 10, and 15 U/L). The increase in particle size and broadening of the particle size distribution of samples during storage were dependent on the PL concentration, storage period, and storage temperature. Sediment analysis indicated that elevated storage temperature accelerated protein sedimentation. The initial PL concentration was positively correlated with the amount of protein sediment in samples stored at 23 °C for four weeks (r = 0.615; p < 0.01), whereas this correlation was negative in samples stored at 37 °C for the same time (r = -0.358; p < 0.01) due to extensive proteolysis. SDS-PAGE revealed that whey proteins remained soluble over storage at 23 °C for four weeks, but they mostly disappeared from the soluble phase of PL-added samples after two weeks' storage at 37 °C. Transmission electron micrographs of PL-containing UHT skim milk during storage at different temperatures supported the trend of sediment analysis well. Based on the Fourier transform infrared spectra of UHT skim milk stored at 23 °C for three weeks, PL-induced particle size enlargement was due to protein aggregation and the formation of intermolecular ß-sheet structures, which contributed to casein destabilization, leading to sediment formation.


Assuntos
Fibrinolisina/química , Conservação de Alimentos , Proteínas do Leite/química , Leite/química , Animais , Caseínas/química , Bovinos , Fibrinolisina/isolamento & purificação , Fibrinolisina/ultraestrutura , Manipulação de Alimentos , Temperatura Alta/efeitos adversos , Humanos , Proteínas do Leite/isolamento & purificação , Proteínas do Leite/ultraestrutura , Tamanho da Partícula , Proteínas do Soro do Leite
11.
Clin Lab ; 67(4)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33865270

RESUMO

BACKGROUND: Venous thromboembolism (VTE) is a common complication in orthopedic trauma patients. Accurate prediction of individual thrombosis risk is important in determining whether prophylactic treatment with anticoagulants is necessary. In this study, we screened for biomarkers that could be used as predictors of VTE risk and evaluated their efficacy and benefit in treating orthopedic traumatic patients. METHODS: A total of 683 patients with orthopedic trauma were consecutively enrolled between January 2017 and June 2018 at Renmin Hospital of Wuhan University. Demographic and clinical information was collected, and VTE risk was assessed using the Caprini risk assessment score. The concentrations of PIC, coagulation parameters and other routine biochemical markers were quantified. The Mann-Whitney U test was used to identify potential biomarkers which were significantly different between patients who developed VTE and those who did not. Correlation between individual parameters was assessed using Pearson's correlation. A nomogram model was constructed to predict VTE risk using a combination of biomarkers, and a decision curve analysis was performed to assess the net benefit of using each biomarker. RESULTS: Patients with VTE had significantly higher levels of PIC (p = 0.037) and DD (p = 0.042) than those without, even after adjusting for confounding factors. PIC and DD levels increased in a stepwise fashion with increasing VTE risk and were the markers most strongly associated with Caprini score (PIC, r = 0.408; DD, r = 0.474; p < 0.001). In decision curve analysis, PIC levels provided greater net benefit than the Caprini score or DD level across patients with various bleeding risks. CONCLUSIONS: Plasma PIC levels are a useful biomarker of VTE risk and can be used to determine whether pharmaco-prophylaxis is needed in orthopedic trauma patients.


Assuntos
Tromboembolia Venosa , Anticoagulantes/uso terapêutico , Biomarcadores , Fibrinolisina , Humanos , Medição de Risco , Fatores de Risco , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/etiologia , alfa 2-Antiplasmina
12.
Sci Transl Med ; 13(588)2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827973

RESUMO

Neuroprotection for acute ischemic stroke is achievable with the eicosapeptide nerinetide, an inhibitor of the protein-protein interactions of the synaptic scaffolding protein PSD-95. However, nerinetide is subject to proteolytic cleavage if administered after alteplase, a standard-of-care thrombolytic agent that nullifies nerinetide's beneficial effects. Here, we showed, on the basis of pharmacokinetic data consistent between rats, primates, and humans, that in a rat model of embolic middle cerebral artery occlusion (eMCAO), nerinetide maintained its effectiveness when administered before alteplase. Because of its short plasma half-life, it can be followed by alteplase within minutes without reducing its neuroprotective effectiveness. In addition, the problem of protease sensitivity is solved by substituting cleavage-prone amino acids from their l- to their d-enantiomeric form. Treatment of rats subjected to eMCAO with such an agent, termed d-Tat-l-2B9c, eliminated protease sensitivity and maintained neuroprotective effectiveness. Our data suggest that both the clinical-stage PSD-95 inhibitor nerinetide and protease-resistant agents such as d-Tat-l-2B9c may be practically integrated into existing stroke care workflows and standards of care.


Assuntos
Antifibrinolíticos , Isquemia Encefálica , Proteína 4 Homóloga a Disks-Large/antagonistas & inibidores , Fibrinolisina/farmacologia , Acidente Vascular Cerebral , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Antifibrinolíticos/farmacologia , Interações Medicamentosas , Ratos , Acidente Vascular Cerebral/tratamento farmacológico
13.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803235

RESUMO

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID-19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired fibrinolytic disorders.


Assuntos
Análise Química do Sangue/métodos , Doença , Fibrinolisina/análise , Fibrinolisina/metabolismo , Animais , Antifibrinolíticos/sangue , Fibrina/análise , Fibrina/química , Fibrinolíticos/sangue , Humanos , Plasminogênio/análise , Plasminogênio/química , Plasminogênio/metabolismo
14.
PLoS One ; 16(3): e0248431, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33720950

RESUMO

BACKGROUND: Ischemic stroke is a common and debilitating disease with limited treatment options. Protease activated receptor 1 (PAR1) is a fundamental cell signaling mediator in the central nervous system (CNS). It can be activated by many proteases including thrombin and plasmin, with various down-stream effects, following brain ischemia. METHODS: A permanent middle cerebral artery occlusion (PMCAo) model was used in PAR1 KO and WT C57BL/6J male mice. Mice were evaluated for neurological deficits (neurological severity score, NSS), infarct volume (Tetrazolium Chloride, TTC), and for plasmin and thrombin activity in brain slices. RESULTS: Significantly low levels of plasmin and thrombin activities were found in PAR1 KO compared to WT (1.6±0.4 vs. 3.2±0.6 ng/µl, p<0.05 and 17.2±1.0 vs. 21.2±1.0 mu/ml, p<0.01, respectively) along with a decreased infarct volume (178.9±14.3, 134.4±13.3 mm3, p<0.05). CONCLUSIONS: PAR1 KO mice have smaller infarcts, with lower thrombin and plasmin activity levels. These findings may suggest that modulation of PAR1 is a potential target for future pharmacological treatment of ischemic stroke.


Assuntos
Encéfalo , Fibrinolisina/metabolismo , AVC Isquêmico , Receptor PAR-1/deficiência , Trombina/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Fibrinolisina/genética , AVC Isquêmico/genética , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Masculino , Camundongos , Camundongos Knockout , Receptor PAR-1/metabolismo , Trombina/genética
15.
J Biotechnol ; 330: 61-69, 2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33689867

RESUMO

The 57-amino acid Kunitz Protease Inhibitor (KPI) domain of Protease Nexin 2 inhibits Factor XIa (FXIa) and other proteases. We previously fused KPI to human serum albumin (KPIHSA). KPIHSA inhibits coagulation Factor XIa (FXIa) 6-fold more rapidly than plasmin. We screened a bacterial expression library of KPI variants randomized at M17, and selected M17D as having the highest anti-FXIa: antiplasmin activity ratio. Expressed as HSA fusion proteins in Pichia pastoris, KPIHSA and KPI(M17D)HSA inhibited FXIa indistinguishably (Ki 9 nM) but KPI(M17D)HSA lacked detectable antiplasmin activity. Purified variant and wild-type KPIHSA were expressed and injected into mice with ferric chloride-treated carotid arteries, with or without systemic administration of tissue plasminogen activator (Tenecteplase, TNKase). The time to arterial occlusion (TTO) or reperfusion (TTR) was assessed by Doppler ultrasound. TTR did not differ between mice treated with TNKase alone or with TNKase supplemented with 38 mg/kg KPI(M17D)HSA but was significantly prolonged to >60 min in all mice treated with TNKase and 38 mg/kg KPIHSA. TTO was significantly but equally prolonged by either 38 mg/kg KPIHSA or KPI(M17D)HSA versus vehicle controls. The antiplasmin activity of KPI is relevant in vivo but its elimination did not enhance counter-thrombosis by KPI.


Assuntos
Precursor de Proteína beta-Amiloide , Fator XIa , Animais , Fibrinolisina , Camundongos , Inibidores de Proteases , Saccharomycetales , Ativador de Plasminogênio Tecidual/genética
16.
J Ethnopharmacol ; 273: 114000, 2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-33705919

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: In folk medicine, parts of Plumeria alba L. are used for the treatment of many diseases, with its latex being used for curing skin diseases and promoting wound healing. AIM OF THE STUDY: This study aimed to study the role of P. alba L. latex in hemostasis and platelet aggregation. MATERIALS AND METHODS: The latex of P. alba L. was processed to remove waxes and enrich protein content, and the final extract was named Plumeria alba L. natant latex (PaNL). PaNL was analyzed for protease activity against casein. The type of protease in PaNL was identified by using protease inhibitors such as E-64, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, and pepstatin A. Human fibrinogen, fibrin, and collagen types I and IV were subjected to hydrolysis with different concentrations of PaNL. The thrombin-like activity of PaNL was determined by analyzing its fibrinogen-clotting and procoagulant activities. The role of PaNL in platelet aggregation was also investigated. Its hemorrhagic and edema-inducing activities were evaluated in a mouse model. Phytochemical compounds were identified by gas chromatography-mass spectroscopy. RESULTS: The findings of casein/gelatin zymography confirmed that PaNL possesses protease activity. The results of the protease inhibition study indicated the presence of a cysteine-type protease(s) in PaNL. PaNL hydrolyzed the subunits of fibrinogen, fibrin, and collagen types I and IV. Its fibrin-degradation activity indicated that PaNL possesses plasmin-like activity. PaNL induced clotting of citrated human plasma within 3 min of incubation in the absence of CaCl2, indicating the presence of thrombin-like activity, which was further confirmed by the results of the fibrinogen-clotting assay. PaNL induced platelet aggregation in the absence of agonists. There was no hemolytic activity. Mice injected with PaNL did not show edema/ hemorrhagic activity. CONCLUSION: PaNL possesses procoagulant, fibrino(geno)lytic, thrombin- and plasmin-like activities and induces platelet aggregation, which could explain its usage for wound treatment in folk medicine.


Assuntos
Apocynaceae/química , Cisteína Proteases/metabolismo , Fibrinolisina , Látex/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Trombina , Animais , Coagulação Sanguínea/efeitos dos fármacos , Cisteína Proteases/genética , Edema/induzido quimicamente , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Hemorragia/induzido quimicamente , Látex/efeitos adversos , Látex/química , Masculino , Camundongos , Compostos Fitoquímicos , Fitoterapia
17.
Food Chem ; 353: 129373, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-33730667

RESUMO

High-resolution ultrasonic spectroscopy (HR-US) was applied for precise detection of plasmin activity towards ß-casein in buffer at pH 7.8 and 37 °C. The evolution of ultrasonic velocity and ultrasonic attenuation measured at 15.5 MHz is related to the concentration of peptide bonds hydrolyzed and loss of ß-casein aggregates, respectively. The ultrasonic assay presents sensitive and direct activity-based quantification of plasmin levels in milk. The variation in plasmin concentration between HR-US and ELISA method owed to the differing detection principles. The real-time ultrasonic profiles of hydrolysis were utilized to describe the kinetic aspect of plasmin activity. The non-linear activity curve was fitted with classic and inverse Michaelis-Menten type models. Within 1-8.6 mg·mL-1 ß-casein, the Vmax and KM obtained were (6.30 ± 2.21) × 10-5 mol.kg-1·min-1 and 10.33 ± 3.50 mg·mL-1, respectively. The maximum peptide bond cleaved was 5-6 (2.7% degree of hydrolysis) achieved at 1 mg·mL-1 ß-casein.


Assuntos
Caseínas/análise , Fibrinolisina/análise , Análise Espectral/métodos , Ultrassom/métodos , Animais , Ensaio de Imunoadsorção Enzimática , Limite de Detecção , Leite/química , Proteólise , Reprodutibilidade dos Testes
18.
Neurochem Res ; 46(5): 1166-1176, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33523394

RESUMO

Poly-arginine peptides R18 and R18D have previously been demonstrated to be neuroprotective in ischaemic stroke models. Here we examined the proteolytic stability and efficacy of R18 and R18D in reducing infarct core growth and preserving the ischaemic penumbra following middle cerebral artery occlusion (MCAO) in the Sprague Dawley rat. R18 (300 or 1000 nmol/kg), R18D (300 nmol/kg) or saline were administered intravenously 10 min after MCAO induced using a filament. Serial perfusion and diffusion-weighted MRI imaging was performed to measure changes in the infarct core and penumbra from time points between 45- and 225-min post-occlusion. Repeated measures analyses of infarct growth and penumbral tissue size were evaluated using generalised linear mixed models (GLMMs). R18D (300 nmol/kg) was most effective in slowing infarct core growth (46.8 mm3 reduction; p < 0.001) and preserving penumbral tissue (21.6% increase; p < 0.001), followed by R18 at the 300 nmol/kg dose (core: 29.5 mm3 reduction; p < 0.001, penumbra: 12.5% increase; p < 0.001). R18 at the 1000 nmol/kg dose had a significant impact in slowing core growth (19.5 mm3 reduction; p = 0.026), but only a modest impact on penumbral preservation (6.9% increase; p = 0.062). The in vitro anti-excitotoxic neuroprotective efficacy of R18D was also demonstrated to be unaffected when preincubated for 1-3 h or overnight, in a cell lysate prepared from dying neurons or with the proteolytic enzyme, plasmin, whereas the neuroprotective efficacy of R18 was significantly reduced after a 2-h incubation. These findings highlight the capacity of poly-arginine peptides to reduce infarct growth and preserve the ischaemic penumbra, and confirm the superior efficacy and proteolytic stability of R18D, which indicates that this peptide is likely to retain its neuroprotective properties when co-administered with alteplase during thrombolysis for acute ischaemic stroke.


Assuntos
Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Peptídeos/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Células Cultivadas , Fibrinolisina/metabolismo , Masculino , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Estabilidade Proteica , Ratos Sprague-Dawley , Estereoisomerismo
19.
Exp Eye Res ; 204: 108459, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33493476

RESUMO

The cornea is a relatively unique tissue in the body in that it possesses specific features such as a lack of blood vessels that contribute to its transparency. The cornea is supplied with soluble blood components such as albumin, globulin, and fibrinogen as well as with nutrients, oxygen, and bioactive substances by diffusion from aqueous humor and limbal vessels as well as a result of its exposure to tear fluid. The healthy cornea is largely devoid of cellular components of blood such as polymorphonuclear leukocytes, monocytes-macrophages, and platelets. The location of the cornea at the ocular surface renders it susceptible to external insults, and its avascular nature necessitates the operation of healing and defense mechanisms in a manner independent of a direct blood supply. The fibrinolytic system, which was first recognized for its role in the degradation of fibrin clots in the vasculature, has also been found to contribute to various biological processes outside of blood vessels. Fibrinolytic factors thus play an important role in biological defense of the cornea. In this review, we address the function of the fibrinolytic system in corneal defense including wound healing and the inflammatory response.


Assuntos
Córnea/metabolismo , Lesões da Córnea/metabolismo , Fibrinolisina/fisiologia , Cicatrização/fisiologia , Animais , Antifibrinolíticos/uso terapêutico , Fibrinólise/fisiologia , Humanos
20.
PLoS Negl Trop Dis ; 15(1): e0008997, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33406071

RESUMO

BACKGROUND: Scabies, a highly contagious skin disease affecting more than 200 million people worldwide at any time, is caused by the parasitic mite Sarcoptes scabiei. In the absence of molecular markers, diagnosis requires experience making surveillance and control challenging. Superficial microthrombi in the absence of vasculitis in scabies-affected skin are a recognised, yet unexplained histopathological differential of scabies infection. This study demonstrates that a family of Scabies Mite Inactivated Cysteine Protease Paralogues (SMIPP-Cs) excreted by the mites plays a role in formation of scabies-induced superficial microthrombi. METHODOLOGY/PRINCIPAL FINDINGS: A series of in vitro and ex vivo experiments involving two representative recombinant SMIPP-Cs was carried out. In the presence of SMIPP-Cs, the thrombin clotting time (TCT), fibrin formation and plasmin induced fibrinolysis were monitored in vitro. The ultrastructure of the SMIPP-C-modulated fibrin was analysed by Scanning Electron Microscopy (SEM). Immuno-histological analyses were performed ex vivo, to localise the SMIPP-C proteins within scabies infected skin biopsies. SMIPP-Cs displayed pro-coagulant properties. They bound calcium ions, reduced the thrombin clotting time, enhanced the fibrin formation rate and delayed plasmin-induced fibrinolysis. The SMIPP-Cs associated with fibrin clots during fibrinogen polymerisation and did not bind to preformed fibrin. Scanning electron microscopy revealed that the fibrin clots formed in the presence of SMIPP-Cs were aberrant and denser than normal fibrin clots. SMIPP-Cs were detected in microthrombi which are commonly seen in scabietic skin. CONCLUSIONS/SIGNIFICANCE: The SMIPP-Cs are the first scabies mite proteins found in sub-epidermal skin layers and their pro-coagulant properties promote superficial microthrombi formation in scabetic skin. Further research is needed to evaluate their potential as diagnostic or therapeutic target.


Assuntos
Coagulação Sanguínea , Cisteína Proteases/fisiologia , Fibrinolisina/farmacologia , Fibrinólise , Sarcoptes scabiei/enzimologia , Escabiose/parasitologia , Pele/irrigação sanguínea , Animais , Cálcio/metabolismo , Cisteína Proteases/análise , Fibrina/biossíntese , Humanos
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