Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin
BACKGROUND A subset of trauma patients undergo fibrinolysis shutdown rather than pathologic hyperfibrinolysis, contributing to organ failure. The molecular basis for fibrinolysis shutdown in trauma is incompletely understood. Elastase released from primed/activated human neutrophils (HNE) has histor...
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| Format: | Article |
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Ovid Technologies (Wolters Kluwer Health)
2019
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| Online Access: | http://hdl.handle.net/1721.1/119876 https://orcid.org/0000-0001-9720-8155 https://orcid.org/0000-0002-9547-3251 |
| _version_ | 1826216280995135488 |
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| author | Moore, Hunter B. Banerjee, Anirban Silliman, Christopher C. Moore, Ernest E. Barrett, Christopher D Yaffe, Michael B |
| author2 | Koch Institute for Integrative Cancer Research at MIT |
| author_facet | Koch Institute for Integrative Cancer Research at MIT Moore, Hunter B. Banerjee, Anirban Silliman, Christopher C. Moore, Ernest E. Barrett, Christopher D Yaffe, Michael B |
| author_sort | Moore, Hunter B. |
| collection | MIT |
| description | BACKGROUND A subset of trauma patients undergo fibrinolysis shutdown rather than pathologic hyperfibrinolysis, contributing to organ failure. The molecular basis for fibrinolysis shutdown in trauma is incompletely understood. Elastase released from primed/activated human neutrophils (HNE) has historically been described as fibrin(ogen)olytic. However, HNE can also degrade plasminogen (PLG) to angiostatin (ANG), retaining the kringle domains but not the proteolytic function, and could thereby compete for generation of active plasmin by tissue plasminogen activator (tPA). We hypothesized that HNE can drive fibrinolysis shutdown rather than fibrinolysis. METHODS Turbidometry was performed using light scatter (λ = 620 nm) in a purified fibrinogen + PLG system and in healthy citrate plasma clotted with Ca2+/thrombin ± tPA, ±HNE, and ±ANG to evaluate HNE effects on fibrinolysis, quantified by time to transition midpoint (Tm). ΔTmfrom control is reported as percent of control ±95% CI. Purified HNE coincubated with PLG or tPA was analyzed by western blot to identify cleavage products. Exogenous HNE was mixed ex vivo with healthy volunteer blood (n = 7) and used in TEG ± tPA to evaluate effects on fibrinolysis. RESULTS HNE did not cause measurable fibrinolysis on fibrin clots, clotted plasma, or whole blood as assessed by turbidometry or TEG in the absence of tPA. Upon tPA treatment, all three methods of evaluating fibrinolysis showed delays and decreases in fibrinolysis caused by HNE relative to control: fibrin clot turbidometry ΔTm= 110.7% (CI 105.0-116.5%), clotted citrate plasma (n = 6 healthy volunteers) ΔTm= 126.1% (CI 110.4-141.8%), and whole blood native TEG (n = 7 healthy volunteers) with ΔLY30 = 28% (p = 0.043). Western blot analysis of HNE-PLG co-incubation confirmed that HNE generates angiostatin K1-3, and plasma turbidity assays treated with angiostatin K1-3 delayed fibrinolysis. CONCLUSION HNE degrades PLG and generates angiostatin K1-3, which predominates over HNE cleavage of fibrin(ogen). These findings suggest that neutrophil release of elastase may underlie trauma-induced fibrinolytic shutdown. |
| first_indexed | 2024-09-23T16:44:54Z |
| format | Article |
| id | mit-1721.1/119876 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T16:44:54Z |
| publishDate | 2019 |
| publisher | Ovid Technologies (Wolters Kluwer Health) |
| record_format | dspace |
| spelling | mit-1721.1/1198762024-06-27T18:54:30Z Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin Moore, Hunter B. Banerjee, Anirban Silliman, Christopher C. Moore, Ernest E. Barrett, Christopher D Yaffe, Michael B Koch Institute for Integrative Cancer Research at MIT Barrett, Christopher D Yaffe, Michael B BACKGROUND A subset of trauma patients undergo fibrinolysis shutdown rather than pathologic hyperfibrinolysis, contributing to organ failure. The molecular basis for fibrinolysis shutdown in trauma is incompletely understood. Elastase released from primed/activated human neutrophils (HNE) has historically been described as fibrin(ogen)olytic. However, HNE can also degrade plasminogen (PLG) to angiostatin (ANG), retaining the kringle domains but not the proteolytic function, and could thereby compete for generation of active plasmin by tissue plasminogen activator (tPA). We hypothesized that HNE can drive fibrinolysis shutdown rather than fibrinolysis. METHODS Turbidometry was performed using light scatter (λ = 620 nm) in a purified fibrinogen + PLG system and in healthy citrate plasma clotted with Ca2+/thrombin ± tPA, ±HNE, and ±ANG to evaluate HNE effects on fibrinolysis, quantified by time to transition midpoint (Tm). ΔTmfrom control is reported as percent of control ±95% CI. Purified HNE coincubated with PLG or tPA was analyzed by western blot to identify cleavage products. Exogenous HNE was mixed ex vivo with healthy volunteer blood (n = 7) and used in TEG ± tPA to evaluate effects on fibrinolysis. RESULTS HNE did not cause measurable fibrinolysis on fibrin clots, clotted plasma, or whole blood as assessed by turbidometry or TEG in the absence of tPA. Upon tPA treatment, all three methods of evaluating fibrinolysis showed delays and decreases in fibrinolysis caused by HNE relative to control: fibrin clot turbidometry ΔTm= 110.7% (CI 105.0-116.5%), clotted citrate plasma (n = 6 healthy volunteers) ΔTm= 126.1% (CI 110.4-141.8%), and whole blood native TEG (n = 7 healthy volunteers) with ΔLY30 = 28% (p = 0.043). Western blot analysis of HNE-PLG co-incubation confirmed that HNE generates angiostatin K1-3, and plasma turbidity assays treated with angiostatin K1-3 delayed fibrinolysis. CONCLUSION HNE degrades PLG and generates angiostatin K1-3, which predominates over HNE cleavage of fibrin(ogen). These findings suggest that neutrophil release of elastase may underlie trauma-induced fibrinolytic shutdown. National Institutes of Health (U.S.) (Grant F32-HL134244) National Institutes of Health (U.S.) (Grant L30-GM120751 United States. Department of Defense (Grant 151953) 2019-01-08T18:06:39Z 2019-01-08T18:06:39Z 2017-12 2017-07 2018-12-19T15:46:57Z Article http://purl.org/eprint/type/JournalArticle 2163-0755 http://hdl.handle.net/1721.1/119876 Barrett, Christopher D. et al. “Human Neutrophil Elastase Mediates Fibrinolysis Shutdown through Competitive Degradation of Plasminogen and Generation of Angiostatin.” Journal of Trauma and Acute Care Surgery 83, 6 (December 2017): 1053–1061 © 2017 Wolters Kluwer Health, Inc https://orcid.org/0000-0001-9720-8155 https://orcid.org/0000-0002-9547-3251 http://dx.doi.org/10.1097/TA.0000000000001685 Journal of Trauma and Acute Care Surgery Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Ovid Technologies (Wolters Kluwer Health) PMC |
| spellingShingle | Moore, Hunter B. Banerjee, Anirban Silliman, Christopher C. Moore, Ernest E. Barrett, Christopher D Yaffe, Michael B Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin |
| title | Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin |
| title_full | Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin |
| title_fullStr | Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin |
| title_full_unstemmed | Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin |
| title_short | Human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin |
| title_sort | human neutrophil elastase mediates fibrinolysis shutdown through competitive degradation of plasminogen and generation of angiostatin |
| url | http://hdl.handle.net/1721.1/119876 https://orcid.org/0000-0001-9720-8155 https://orcid.org/0000-0002-9547-3251 |
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