{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T00:56:38Z","timestamp":1768524998972,"version":"3.49.0"},"reference-count":38,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T00:00:00Z","timestamp":1763683200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012190","name":"Ministry of Science and Higher Education of the Russian Federation","doi-asserted-by":"crossref","award":["075-15-2025-518"],"award-info":[{"award-number":["075-15-2025-518"]}],"id":[{"id":"10.13039\/501100012190","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>The saliva of the medicinal leech Hirudo medicinalis contains a wide range of biologically active compounds, including multiple anticoagulants. Previously, we identified a novel cysteine-rich anticoagulant protein (CRA) from leech saliva and produced it recombinantly in Escherichia coli, demonstrating its potential as a basis for new anticoagulant drugs. In this study, we developed an optimized procedure for scalable production and purification of recombinant CRA. The purified protein was analyzed for common contaminants originating from E. coli, such as endotoxins, bacterial proteins, and DNA, and its anticoagulant properties were evaluated using standard clotting assays. Across three independent experiments, the yield of purified CRA ranged from 3.7 to 5.5 mg per liter of bacterial culture, with impurity levels per milligram of protein ranging from 7.1\u201331.2 ng of bacterial proteins, 1.2\u201315.1 ng of DNA, and 60\u20131445 EU of endotoxins. The purified CRA displayed electrophoretic and chromatographic homogeneity and retained strong anticoagulant activity. Additionally, a truncated form of CRA lacking the C-terminal region was produced and characterized. This variant lost membrane affinity and showed altered activity profiles, with higher thrombin time activity but reduced prothrombin time and activated partial thromboplastin time activities compared with the full-length protein.<\/jats:p>","DOI":"10.3390\/biom15121633","type":"journal-article","created":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T08:59:46Z","timestamp":1763715586000},"page":"1633","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Production, Purification, and Characterization of a Novel Cysteine-Rich Anticoagulant from the Medicinal Leech and the Functional Role of Its C-Terminal Motif"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2471-0563","authenticated-orcid":false,"given":"Valentin A.","family":"Manuvera","sequence":"first","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Center for Genetic Reprogramming and Gene Therapy, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Moscow Center for Advanced Studies, 20 Kulakova Str., 123592 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-6309-2817","authenticated-orcid":false,"given":"Ksenia A.","family":"Brovina","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Center for Genetic Reprogramming and Gene Therapy, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Moscow Center for Advanced Studies, 20 Kulakova Str., 123592 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2242-2127","authenticated-orcid":false,"given":"Vladislav V.","family":"Babenko","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5735-0042","authenticated-orcid":false,"given":"Pavel A.","family":"Bobrovsky","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Center for Genetic Reprogramming and Gene Therapy, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Moscow Center for Advanced Studies, 20 Kulakova Str., 123592 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3514-2303","authenticated-orcid":false,"given":"Daria D.","family":"Kharlampieva","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8957-6142","authenticated-orcid":false,"given":"Ekaterina N.","family":"Grafskaia","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Center for Genetic Reprogramming and Gene Therapy, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Moscow Center for Advanced Studies, 20 Kulakova Str., 123592 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-1663-9311","authenticated-orcid":false,"given":"Maria Y.","family":"Serebrennikova","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Center for Genetic Reprogramming and Gene Therapy, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Moscow Center for Advanced Studies, 20 Kulakova Str., 123592 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nikita R.","family":"Rusavskiy","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Moscow Center for Advanced Studies, 20 Kulakova Str., 123592 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0359-0785","authenticated-orcid":false,"given":"Nadezhda F.","family":"Polina","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vassili N.","family":"Lazarev","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Center for Genetic Reprogramming and Gene Therapy, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia"},{"name":"Moscow Center for Advanced Studies, 20 Kulakova Str., 123592 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2350","DOI":"10.1016\/j.jacc.2023.11.007","article-title":"Global Burden of Cardiovascular Diseases and Risks Collaborators Global Burden of Cardiovascular Diseases and Risks, 1990-2022","volume":"82","author":"Mensah","year":"2023","journal-title":"J. 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