Activated protein C (APC) resistance is usually associated with a single DNA mutation predicting replacement of Arg506 by Gln in factor V (FV). Studies using synthetic peptides suggest that FV residues 493-506 provide factor Xa (FXa) and protein S binding sites. Biochemical studies were performed to test the hypothesis that the Arg506Gln FV mutation causes APC resistance and to define the nature of the resistance of Gln506-FVa to APC. Purified Gln506-FV conveyed APC resistance to FV-deficient plasma in APTT and FXa-1-stage assays. Purified Gln506-FVa, generated either by thrombin or by FXa, was resistant to APC. Nonetheless, Gln506-FVa was not completely resistant to APC since it was inactivated by APC approximately 10-fold slower than normal Arg506-FVa, probably due to cleavage at Arg306. This reduced but significant susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance, especially for heterozygotes, is a relatively moderate risk factor for venous thrombosis. Cardiolipin promotes APC anticoagulant activity better than FXa coagulant activity, and antibodies from some antiphospholipid antibody syndrome patients downregulate APC activity. Thus, acquired APC resistance may contribute to pathogenesis of thrombosis in the antiphospholipid antibody syndrome.
Purpose: To evaluate the long-term safety of vascular endothelial growth factor (VEGF) suppression with sustained aflibercept expression after a single intravitreal injection (IVI) of ADVM-022, an anti-VEGF gene therapy, in non-human primates (NHPs). Methods: Non-human primates received bilateral IVI of ADVM-022, a gene therapy vector encoding aflibercept, a standard of care for the treatment of VEGF-based retinal disease. Aflibercept levels from ocular fluids and tissues were measured. Ocular inflammation was assessed by slit lamp biomicroscopy and fundoscopy. The integrity of the retinal structure was analyzed by optical coherence tomography and blue light fundus autofluorescence and electroretinography was performed to determine retinal function. Histologic evaluation of the retina was performed at the longest time point measured (2.5 years after injection). Results: Sustained expression of aflibercept was noted out to the last time point evaluated. Mild to moderate inflammatory responses were observed, which trended toward spontaneous resolution without anti-inflammatory treatment. No abnormalities in retinal structure or function were observed, as measured by optical coherence tomography and electroretinography, respectively. RPE integrity was maintained throughout the study; no histologic abnormalities were observed 2.5 years after ADVM-022 IVI. Conclusions: In non-human primates, long-term, sustained aflibercept expression and the resulting continuous VEGF suppression by a single IVI of ADVM-022, appears to be safe, with no measurable adverse effects on normal retinal structure and function evaluated out to 2.5 years. Translational Relevance: Together with the results from previous ADVM-022 preclinical studies, these data support the evaluation of this gene therapy candidate in clinical trials as a potential durable treatment for various VEGF-mediated ophthalmic disorders.
Abstract Three‐dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site‐directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3‐dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.
Inhibition of vascular endothelial growth factor, a key contributor to the choroidal neovascularization associated with wet age-related macular degeneration, is the mode of action of several approved therapies, including aflibercept, which requires frequent intravitreal injections to provide clinical benefit. Lack of compliance with the dosing schedule may result in recurrence of active wet macular degeneration, leading to irreversible vision impairment. Gene therapy providing sustained anti-vascular endothelial growth factor levels in the retina following a single injection could drastically reduce the treatment burden and improve visual outcomes. ADVM-022, an adeno-associated virus vector encoding aflibercept, is optimized for intravitreal delivery and strong protein expression. Here, we report the long-term expression and efficacy of ADVM-022-derived aflibercept in a laser-induced choroidal neovascularization model in non-human primates. Intravitreal administration of ADVM-022 was well tolerated and resulted in sustained aflibercept levels. In addition, ADVM-022 administration 13 months before lasering prevented the occurrence of clinically relevant choroidal neovascularization lesions, similar to animals that received a bolus of intravitreal aflibercept (standard of care) at the time of lesioning. These results demonstrate that a single intravitreal administration of ADVM-022 may provide a safe and effective long-term treatment option for wet macular degeneration and may ultimately improve patients' visual outcomes.
Certain activators of the contact system of coagulation have been reported to induce full activity in the singlechain form (80,000 MW) of Factor XII (FXII). The effects of soluble ellagic acid (EA) and dextran sulfate (DXS) (∼500,000 MW) on purified components of the contact system were studied. Soluble EA (<40 yM) was found to exert a dose-dependent procoagulant effect on purified FXII added to FXII-deficient plasma although the maximal activity observed was much less than that elicited by kaolin in the same mixtures. 5 μM soluble EA increased the amidolytic activity of FXII against H-D-Pro-Phe-Arg-pNA from 2.5 to 5.0 mol substrate/mol enzyme/min. Purified β-FXIIa (28,000 MW) or FXII that had been preincubated with trypsin hydrolyzed 900 mol substrate/mol enzyme/min in the presence or absence of soluble EA. Thus, soluble EA induces minimal (≤0.3%) amidolytic activity in FXII. We also examined the generation of FXIa in the presence of 5 μM soluble EA, FXI, high MW kininogen, and FXII or α-FXIIa (two-chain form, 80.000 MW). The rate of cleavage of 125I-FXI in the presence of 10 μg/ml DXS, FXI, high MW kininogen, and FXII or α-FXIIa was also measured. In the presence of either DXS or soluble EA under the concentrations and conditions employed, α-FXIIa exhibited an initial rate of FXI activation that was 20 times higher than that achieved by single-chain FXII. In addition, the presence of both high MW kininogen and either EA or DXS enhanced the activity of α-FXIIa 20 fold in the activation of FXI. The results show that single chain FXII in the presence of either DXS or soluble EA expresses less than 5% of the enzymatic activities of its proteolytically derived forms whereas either “activator” greatly enhances the action of α-FXIIa on FXI in the presence of high MW kininogen.
Summary Analysis of naturally occurring protein mutations yields valuable insights into functionally important sequences. Characterizing mutations responsible for protein C deficiency at the molecular level has been the subject of intensive investigation. In a previous study, a three-dimensional model of the serine protease domain of protein C was used to analyze the set of protease domain mutations previously available. The mutations were largely found to fall into a limited number of categories. A recently updated protein C mutation data base has provided a number of new mutations which have been analyzed for structural predictions.
Abstract: Protein S, an anticoagulant factor in the protein C antithrombotic pathway, was found to be synthesized and released by six tumor cell lines of neural origin by western blotting and ELISA. The rate of synthesis ranged from three‐to 11‐fold higher than that of a microvascular endothelial cell line and 36–144% that of a hepatoma cell line. The secreted protein S displayed specific anticoagulant activity similar to that of purified plasma protein S, implying that it was fully γ‐carboxylated. Ten primary brain tumor tissues also expressed protein S antigen, as shown by western blot analysis. Expression of anticoagulantly active protein S by neural cells raises important questions concerning possible physiologic roles for this multidomain protein beyond its function in control of thrombosis.
Several standard-of-care therapies for the treatment of retinal disease, including aflibercept, inhibit vascular endothelial growth factor (VEGFA). The main shortcoming of these therapies is potential undertreatment due to a lack of compliance resulting from the need for repeated injections. Gene therapy may provide sustained levels of anti-VEGFA proteins in the retina following a single injection. In this nonhuman primate study, we explored whether ADVM-022, a recombinant adeno-associated virus (AAV) vector designed to express aflibercept, could induce anti-VEGFA protein levels comparable with those observed following a single-bolus intravitreal (IVT) injection of the standard-of-care aflibercept recombinant protein. The results demonstrated that intraocular levels of aflibercept measured at 56 days after a single IVT injection of ADVM-022 were equivalent to those in the aflibercept recombinant protein-injected animals measured 21-32 days post-administration. ADVM-022-injected animals exhibited signs of an initial self-limiting inflammatory response, but overall all doses were well tolerated. ADVM-022 administration did not result in systemic exposure to aflibercept at any dose evaluated. These results demonstrated that a single IVT injection of ADVM-022 resulted in safe and efficacious aflibercept levels in the therapeutic range, suggesting the potential of a gene therapy approach for long-term treatment of retinal disease with anti-VEGF therapy.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTReceptors for high molecular weight kininogen on stimulated washed human plateletsJudith S. Greengard and John H. GriffinCite this: Biochemistry 1984, 23, 26, 6863–6869Publication Date (Print):December 18, 1984Publication History Published online1 May 2002Published inissue 18 December 1984https://pubs.acs.org/doi/10.1021/bi00321a090https://doi.org/10.1021/bi00321a090research-articleACS PublicationsRequest reuse permissionsArticle Views49Altmetric-Citations81LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
