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Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Truncation of ADAMTS13 by Plasmin Enhances Its Activity in Plasma
Chantal C Clark1, Mirjam M Mebius1,2, Steven de Maat1
1Department for Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Abstract:
ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) cleaves von Willebrand Factor (VWF) multimers to control their thrombogenicity. The fibrinolytic enzyme plasmin can cleave VWF in a similar manner. However, plasmin can also cleave ADAMTS13, which ultimately inactivates it. This leaves the overall role of plasmin in primary haemostasis uncertain.We investigated the combined molecular effects of plasmin on VWF and ADAMTS13. We first identified that plasmin destroys FRETS-VWF73 substrate by cleaving the ADAMTS13 binding region in a buffered system. We next investigated how plasmin affects both VWF and ADAMTS13 under static conditions in plasma by western blotting. We found that globular VWF is largely protected from plasmin cleavage. However, ADAMTS13 is rapidly cleaved under these conditions, suggesting inactivation. Surprisingly, we observed that plasmin enhances ADAMTS13 activity in a modified two-stage FRETS-VWF73 assay that protects FRETS-VWF73 substrate from degradation. In direct binding studies under the same conditions, we found that plasmin generates multiple C-terminally truncated forms of ADAMTS13 with VWF-binding capacity. In an effort to seek evidence for this mechanism in vivo, we analysed plasma from patients with systemic amyloidosis, which is hallmarked by a hyperfibrinolytic state. We found that their plasma contained increased levels of C-terminally truncated forms of ADAMTS13, which correlated with their hyperfibrinolytic state.We propose that truncation of ADAMTS13 by plasmin abolishes intramolecular self-association, which improves interaction with unfolded VWF.
Insights
Plasmin cleaves ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), but surprisingly enhances its activity on von Willebrand Factor (VWF). This interaction generates truncated ADAMTS13 forms, impacting primary hemostasis.
Area of Science:
- Hematology
- Biochemistry
- Molecular Biology
Background:
- ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) regulates von Willebrand Factor (VWF) thrombogenicity.
- Plasmin, a fibrinolytic enzyme, can cleave both VWF and ADAMTS13, creating uncertainty about plasmin's role in primary hemostasis.
- Plasmin's cleavage of ADAMTS13 can lead to its inactivation.
Purpose of the Study:
- To investigate the combined molecular effects of plasmin on VWF and ADAMTS13.
- To elucidate the mechanism by which plasmin influences ADAMTS13 activity and VWF cleavage.
- To explore the in vivo relevance of these interactions in a hyperfibrinolytic state.
Main Methods:
- Utilized a FRETS-VWF73 substrate assay to assess plasmin's effect on VWF cleavage.
- Employed western blotting to analyze VWF and ADAMTS13 cleavage in plasma under static conditions.
- Conducted modified two-stage FRETS-VWF73 assays and direct binding studies to evaluate ADAMTS13 activity and VWF-binding capacity.
- Analyzed plasma samples from patients with systemic amyloidosis.
Main Results:
- Plasmin cleaves the ADAMTS13 binding region of the FRETS-VWF73 substrate.
- While globular VWF is protected, ADAMTS13 is rapidly cleaved and inactivated by plasmin in plasma.
- Surprisingly, plasmin enhances ADAMTS13 activity in a modified assay, generating C-terminally truncated ADAMTS13 forms with VWF-binding capacity.
- Elevated levels of truncated ADAMTS13 were observed in patients with systemic amyloidosis, correlating with their hyperfibrinolytic state.
Conclusions:
- Plasmin-mediated truncation of ADAMTS13 enhances its interaction with unfolded VWF, potentially by abolishing intramolecular self-association.
- These findings suggest a complex regulatory role for plasmin in primary hemostasis involving modulation of ADAMTS13 activity.
- The presence of truncated ADAMTS13 in hyperfibrinolytic states like systemic amyloidosis highlights the in vivo significance of this interaction.
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