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Journal of Clinical Medicine Feb 2023Several plant protein inhibitors with anticoagulant properties have been studied and characterized, including the trypsin inhibitor (DrTI). This protein inhibits serine...
Peptides Derived from a Plant Protease Inhibitor of the Coagulation Contact System Decrease Arterial Thrombus Formation in a Murine Model, without Impairing Hemostatic Parameters.
Several plant protein inhibitors with anticoagulant properties have been studied and characterized, including the trypsin inhibitor (DrTI). This protein inhibits serine proteases (trypsin) and enzymes directly involved in coagulation, such as plasma kallikrein, factor XIIa, and factor XIa. In this study, we evaluated the effects of two new synthetic peptides derived from the primary sequence of DrTI in coagulation and thrombosis models to understand the mechanisms involved in the pathophysiology of thrombus formation as well as in the development of new antithrombotic therapies. Both peptides acted on in vitro hemostasis-related parameters, showing promising results, prolonging the Partially Activated Thromboplastin Time (aPTT) and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid. In murine models, for arterial thrombosis induced by photochemical injury, and platelet-endothelial interactions monitored by intravital microscopy, both peptides at doses of 0.5 mg/kg significantly extended the time of artery occlusion and modified the platelet adhesion and aggregation pattern with no changes in bleeding time, demonstrating the high biotechnological potential of both molecules.
PubMed: 36902597
DOI: 10.3390/jcm12051810 -
Journal of Thrombosis and Haemostasis :... Jun 2023During plasma contact activation, factor XII (FXII) binds to surfaces through its heavy chain and undergoes conversion to the protease FXIIa. FXIIa activates...
BACKGROUND
During plasma contact activation, factor XII (FXII) binds to surfaces through its heavy chain and undergoes conversion to the protease FXIIa. FXIIa activates prekallikrein and factor XI (FXI). Recently, we showed that the FXII first epidermal growth factor-1 (EGF1) domain is required for normal activity when polyphosphate is used as a surface.
OBJECTIVES
The aim of this study was to identify amino acids in the FXII EGF1 domain required for polyphosphate-dependent FXII functions.
METHODS
FXII with alanine substitutions for basic residues in the EGF1 domain were expressed in HEK293 fibroblasts. Wild-type FXII (FXII-WT) and FXII containing the EGF1 domain from the related protein Pro-HGFA (FXII-EGF1) were positive and negative controls. Proteins were tested for their capacity to be activated, and to activate prekallikrein and FXI, with or without polyphosphate, and to replace FXII-WT in plasma clotting assays and a mouse thrombosis model.
RESULTS
FXII and all FXII variants were activated similarly by kallikrein in the absence of polyphosphate. However, FXII with alanine replacing Lys, Lys, and Lys (FXII-Ala) or Lys, His, and Lys (FXII-Ala) were activated poorly in the presence of polyphosphate. Both have <5% of normal FXII activity in silica-triggered plasma clotting assays and have reduced binding affinity for polyphosphate. Activated FXIIa-Ala displayed profound defects in surface-dependent FXI activation in purified and plasma systems. FXIIa-Ala reconstituted FXII-deficient mice poorly in an arterial thrombosis model.
CONCLUSION
FXII Lys, Lys, Lys, and Lys form a binding site for polyanionic substances such as polyphosphate that is required for surface-dependent FXII function.
Topics: Humans; Animals; Mice; Factor XII; Prekallikrein; Polyphosphates; HEK293 Cells; Factor XI; Thrombosis; Factor XIIa
PubMed: 36863563
DOI: 10.1016/j.jtha.2023.02.014 -
ACS Medicinal Chemistry Letters Feb 2023The invention in this patent application relates to ()-spiro[benzo[][1,3]oxazine-4,3'-pyrrolidin]-2(1)-one derivatives, represented generally by formula 1. These...
The invention in this patent application relates to ()-spiro[benzo[][1,3]oxazine-4,3'-pyrrolidin]-2(1)-one derivatives, represented generally by formula 1. These compounds are selective plasma kallikrein inhibitors and may potentially be beneficial in the treatment of several diseases and disorders, including hereditary angioedema, uveitis, posterior uveitis, wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy, and retinal vein occlusion.
PubMed: 36793429
DOI: 10.1021/acsmedchemlett.2c00526 -
Journal of Thrombosis and Haemostasis :... May 2023Titanium (Ti) and its alloys are widely used in manufacturing medical devices because of their strength and resistance to corrosion. Although Ti compounds are considered...
BACKGROUND
Titanium (Ti) and its alloys are widely used in manufacturing medical devices because of their strength and resistance to corrosion. Although Ti compounds are considered compatible with blood, they appear to support plasma contact activation and may be thrombogenic.
OBJECTIVES
The objective of this study was to compare Ti and titanium nitride (TiN) with known activators of contact activation (kaolin and silica) in plasma-clotting assays and to assess binding and activation of factor XII, (FXII), factor XI (FXI), prekallikrein, and high-molecular-weight kininogen (HK) with Ti/TiN.
METHODS
Ti-based nanospheres and foils were compared with kaolin, silica, and aluminum in plasma-clotting assays. Binding and activation of FXII, prekallikrein, HK, and FXI to surfaces was assessed with western blots and chromogenic assays.
RESULTS
Using equivalent surface amounts, Ti and TiN were comparable with kaolin and superior to silica, for inducing coagulation and FXII autoactivation. Similar to many inducers of contact activation, Ti and TiN are negatively charged; however, their effects on FXII are not neutralized by the polycation polybrene. Antibodies to FXII, prekallikrein, or FXI or coating Ti with poly-L-arginine blocked Ti-induced coagulation. An antibody to FXII reduced FXII and PK binding to Ti, kallikrein generation, and HK cleavage.
CONCLUSION
Titanium compounds induce contact activation with a potency comparable with that of kaolin. Binding of FXII with Ti shares some features with FXII binding to soluble polyanions but may have unique features. Inhibitors targeting FXII or FXI may be useful in mitigating Ti-induced contact activation in patients with titanium-based implants that are exposed to blood.
Topics: Humans; Factor XI; Factor XII; Kaolin; Prekallikrein; Titanium
PubMed: 36696212
DOI: 10.1016/j.jtha.2022.12.014 -
Frontiers in Physiology 2022Patients infected by the SARS-CoV-2 virus are commonly diagnosed with threatening liver conditions associated with drug-induced therapies and systemic viral action....
Patients infected by the SARS-CoV-2 virus are commonly diagnosed with threatening liver conditions associated with drug-induced therapies and systemic viral action. RNA-Seq data from cells in bronchoalveolar lavage fluid from COVID-19 patients have pointed out dysregulation of kallikrein-kinin and renin-angiotensin systems as a possible mechanism that triggers multi-organ damage away from the leading site of virus infection. Therefore, we measured the plasma concentration of biologically active peptides from the kallikrein-kinin system, bradykinin and des-Arg-bradykinin, and liver expression of its proinflammatory axis, bradykinin 1 receptor (B1R). We measured the plasma concentration of bradykinin and des-Arg-bradykinin of 20 virologically confirmed COVID-19 patients using a liquid chromatography-tandem mass spectrometry-based methodology. The expression of B1R was evaluated by immunohistochemistry from post-mortem liver specimens of 27 COVID-19 individuals. We found a significantly higher blood level of des-Arg-bradykinin and a lower bradykinin concentration in patients with COVID-19 compared to a healthy, uninfected control group. We also observed increased B1R expression levels in hepatic tissues of patients with COVID-19 under all hepatic injuries analyzed (liver congestion, portal vein dilation, steatosis, and ischemic necrosis). Our data indicate that des-Arg-bradykinin/B1R is associated with the acute hepatic dysfunction induced by the SARS-CoV-2 virus infection in the pathogenesis of COVID-19.
PubMed: 36601349
DOI: 10.3389/fphys.2022.1080837 -
Thrombosis Journal Jan 2023(p-BthTX-I) K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and...
A snake venom-analog peptide that inhibits SARS-CoV-2 and papain-like protease displays antithrombotic activity in mice arterial thrombosis model, without interfering with bleeding time.
BACKGROUND
(p-BthTX-I) K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains with weak cytotoxic effects. Complications of SARS-CoV-2 infection include vascular problems and increased risk of thrombosis; therefore, studies to identify new drugs for treating SARS-CoV-2 infections that also inhibit thrombosis and minimize the risk of bleeding are required.
OBJECTIVES
To determine whether (p-BthTX-I) K affects the hemostatic system.
METHODS
Platelet aggregation was induced by collagen, arachidonic acid, and adenosine diphosphate (ADP) in the Chronolog Lumi-aggregometer. The coagulation activity was evaluated by determining activated partial thromboplastin clotting time (aPTT) and prothrombin time (PT) with (p-BthTX-I) K (5.0-434.5 µg) or 0.9% NaCl. Arterial thrombosis was induced with a 540 nm laser and 3.5-20 mg kg Rose Bengal in the carotid artery of male C57BL/6J mice using (p-BthTX-I) K. Bleeding time was determined in mouse tails immersed in saline at 37 °C after (p-BthTX-I) K (4.0 mg/kg and 8.0 mg/kg) or saline administration.
RESULTS
(p-BthTX-I) K prolonged the aPTT and PT by blocking kallikrein and FXa-like activities. Moreover, (p-BthTX-I) K inhibited ADP-, collagen-, and arachidonic acid-induced platelet aggregation in a dose-dependent manner. Further, low concentrations of (p-BthTX-I) K extended the time to artery occlusion by the formed thrombus. However, (p-BthTX-I) K did not prolong the bleeding time in the mouse model of arterial thrombosis.
CONCLUSION
These results demonstrate the antithrombotic activity of the peptide (p-BthTX-I) K possibly by kallikrein inhibition, suggesting its strong biotechnological potential.
PubMed: 36593467
DOI: 10.1186/s12959-022-00436-5 -
Molecular and Cellular Endocrinology Feb 2023In this study, we investigated how platelets and aorta contribute to the creation and maintenance of a prothrombotic state in an experimental model of postmenopausal...
AIM
In this study, we investigated how platelets and aorta contribute to the creation and maintenance of a prothrombotic state in an experimental model of postmenopausal hypertension in ovariectomized rats.
METHODS
Bilateral ovariectomy was performed in both 14-week-old female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The animals were kept in phytoestrogen free diet. Vascular parameters, platelet, coagulation and aortic prothrombotic functions and mechanisms were assessed.
RESULTS
Exacerbated platelet aggregation was observed in both SHR and WKY animals after ovariectomy. The mechanism was related to aortic COX2 downregulation and reduction in AMP, ADP, and ATP hydrolysis in serum and platelets. A procoagulant potential was observed in plasma from ovariectomized rats and this was confirmed by kallikrein and factor Xa generation in aortic rings. Aortic rings derived from ovariectomized SHR presented a greater thrombin generation capacity compared to equivalent rings from WKY animals. The mechanism involved tissue factor and PAR-1 upregulation as well as an increase in extrinsic coagulation and fibrinolysis markers in aorta and platelets. Aortic smooth muscle cells pre-treated with a plasma pool derived from estrogen-depleted animals developed a procoagulant profile with tissue factor upregulation. This procoagulant profile was dependent on inflammatory signalling, since NFκB inhibition attenuated the procoagulant activity and tissue factor expression.
CONCLUSIONS
A prothrombotic phenotype was observed in both WKY and SHR ovariectomized rats being associated with platelet hyperreactivity and tissue factor upregulation in aorta and platelets. The mechanism involves proinflammatory signalling that supports greater thrombin generation in aorta and vascular smooth muscle cells.
Topics: Rats; Female; Animals; Rats, Inbred SHR; Rats, Inbred WKY; Thrombin; Thromboplastin; Hypertension; Aorta; Estrogens
PubMed: 36494014
DOI: 10.1016/j.mce.2022.111827 -
Materials Today. Bio Dec 2022Protein adsorption to biomaterial surfaces is considered a determining factor for the host response. Here we detail the protein adsorption profiles of alginate hydrogel...
Protein adsorption to biomaterial surfaces is considered a determining factor for the host response. Here we detail the protein adsorption profiles of alginate hydrogel microspheres relevant for cell therapy using mass spectrometry (MS)-based proteomics. The investigated microspheres include sulfated alginate (SA), high G alginate (HiG), and poly-l-lysine coated alginate (AP), which previously have been shown to exhibit different inflammatory and fibrotic responses. The biological significance was assessed in lepirudin-anticoagulated human whole blood (hWB) by functional analysis of the acute-phase responses (complement and coagulation). Proteomic profiling revealed distinct signatures for the microspheres, wherein Ingenuity Pathway Analysis identified complement and coagulation as the top enriched canonical pathways. The levels of complement and coagulation activators and inhibitors were distinctly different, which was reflected in the functional hWB analyses: SA was highly enriched with inhibitory factors of complement and coagulation (e.g. C1 inhibitor, factor H, antithrombin-III, heparin cofactor 2), other heparin-binding proteins and factors promoting fibrinolysis (factor XII, plasma kallikrein), conforming to an anti-inflammatory and anti-fibrotic profile. HiG enriched moderate levels of complement inhibitors, conforming to a low-inflammatory and pro-fibrotic profile. AP showed the most prominent enrichment of complement activators (e.g. C3, properdin, C-reactive protein) and low levels of inhibitors, conforming to a pro-inflammatory and highly pro-fibrotic profile. In conclusion, the extensive enrichment of inhibitory acute-phase proteins on SA could be a determining factor for its reduced host response. The interactions between the plasma proteins and hydrogel surfaces shown herein point to proteomics as an important supplement to existing and methods for designing biocompatible alginate-based hydrogels.
PubMed: 36420052
DOI: 10.1016/j.mtbio.2022.100490 -
Blood Advances Apr 2023A dysregulated plasma contact system is involved in various pathological conditions, such as hereditary angioedema, Alzheimer disease, and sepsis. We previously showed...
A dysregulated plasma contact system is involved in various pathological conditions, such as hereditary angioedema, Alzheimer disease, and sepsis. We previously showed that the 3E8 anti-high molecular weight kininogen (anti-HK) antibody blocks HK cleavage and bradykinin generation in human plasma ex vivo. Here, we show that 3E8 prevented not only HK cleavage but also factor XI (FXI) and prekallikrein (PK) activation by blocking their binding to HK in mouse plasma in vivo. 3E8 also inhibited contact system-induced bradykinin generation in vivo. Interestingly, FXII activation was also inhibited, likely because of the ability of 3E8 to block the positive feedback activation of FXII by kallikrein (PKa). In human plasma, 3E8 also blocked PK and FXI binding to HK and inhibited both thrombotic (FXI activation) and inflammatory pathways (PK activation and HK cleavage) of the plasma contact system activation ex vivo. Moreover, 3E8 blocked PKa binding to HK and dose-dependently inhibited PKa cleavage of HK. Our results reveal a novel strategy to inhibit contact system activation in vivo, which may provide an effective method to treat human diseases involving contact system dysregulation.
Topics: Humans; Animals; Mice; Prekallikrein; Factor XI; Bradykinin; Kininogen, High-Molecular-Weight; Thrombosis
PubMed: 36409609
DOI: 10.1182/bloodadvances.2021006485 -
Journal of Cardiovascular and Thoracic... 2022The tissue kallikrein-kinin system is an endogenous homeostatic pathway, which its stimulation is associated with cardioprotection. The present study aimed to determine...
The tissue kallikrein-kinin system is an endogenous homeostatic pathway, which its stimulation is associated with cardioprotection. The present study aimed to determine the effect of exercise training on plasma tissue kallikrein (TK) and bradykinin (BK) and their association with cardiac hypertrophy. 22 non-athlete and 22 athlete women were exposed to acute (Bruce test) and chronic (12-week swimming training) exercises. 2D echocardiography was used to evaluate morphological and functional features of the heart. Plasma concentrations of TK and BK were quantified by ELISA. Athletes had significantly higher values of left ventricle end-diastolic diameter index (LVEDDI) and left ventricle mass index (LVMI) than non-athletes. Exercise intervention affected echocardiographic features in neither of the study groups. Chronic exercise training notably increased plasma levels of TK and BK, which increase was more pronounced in the athletes. Plasma TK negatively correlated with LVEDDI (r=-0.64, =0.036 and r=-0.58, =0.027) and LVMI (r=-0.51, =0.032 and r=-0.63, =0.028) in the non-athlete and athlete groups. In opposition, there was a positive correlation between plasma TK and left ventricle ejection fraction in non-athletes (r=0.39, =0.049) and athletes (r=0.53, =0.019). The upregulation of the tissue kallikrein-kinin system may be a protective mechanism against excessive cardiac hypertrophy induced by chronic exercise training.
PubMed: 36398053
DOI: 10.34172/jcvtr.2022.28