Truncation of ADAMTS13 by Plasmin Enhances Its Activity in Plasma

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.

Keywords

ADAMTS13 - von willebrand factor - plasmin

Author Contributions

C.C. Clark, M.M. Mebius, S.d. Maat and C. Maas designed and performed research, analysed data and wrote the manuscript. B.P.C. Hazenberg, A.G.M. Tielens, P.G.d. Groot, R.T. Urbanus and J.J.v. Hellemond contributed vital new reagents and analytical tools. C.C. Clark and Mirjam M. Mebius contributed equally.

Financial Support

CM gratefully acknowledges financial support from the Landsteiner Foundation for Blood Transfusion Research and the Netherlands Thrombosis Foundation.

^* Chantal C. Clark and Mirjam M. Mebius contributed equally.

• References

• 1 Kremer Hovinga JA, Coppo P, Lämmle B, Moake JL, Miyata T, Vanhoorelbeke K. Thrombotic thrombocytopenic purpura. Nat Rev Dis Primers 2017; 3: 17020
• 2 Tersteeg C, de Maat S, De Meyer SF. , et al. Plasmin cleavage of von Willebrand factor as an emergency bypass for ADAMTS13 deficiency in thrombotic microangiopathy. Circulation 2014; 129 (12) 1320-1331
• 3 Crawley JTB, Lam JK, Rance JB, Mollica LR, O'Donnell JS, Lane DA. Proteolytic inactivation of ADAMTS13 by thrombin and plasmin. Blood 2005; 105 (03) 1085-1093
• 4 Berkowitz SD, Dent J, Roberts J. , et al. Epitope mapping of the von Willebrand factor subunit distinguishes fragments present in normal and type IIA von Willebrand disease from those generated by plasmin. J Clin Invest 1987; 79 (02) 524-531
• 5 Kokame K, Nobe Y, Kokubo Y, Okayama A, Miyata T. FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br J Haematol 2005; 129 (01) 93-100
• 6 de Maat S, Björkqvist J, Suffritti C. , et al. Plasmin is a natural trigger for bradykinin production in patients with hereditary angioedema with factor XII mutations. J Allergy Clin Immunol 2016; 138 (05) 1414-1423.e9
• 7 Wilkins MR, Gasteiger E, Bairoch A. , et al. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 1999; 112: 531-552
• 8 Majerus EM, Anderson PJ, Sadler JE. Binding of ADAMTS13 to von Willebrand factor. J Biol Chem 2005; 280 (23) 21773-21778
• 9 Maas C, Govers-Riemslag JW, Bouma B. , et al. Misfolded proteins activate factor XII in humans, leading to kallikrein formation without initiating coagulation. J Clin Invest 2008; 118 (09) 3208-3218
• 10 Hazenberg BP, van Rijswijk MH, Piers DA. , et al. Diagnostic performance of ¹²³I-labeled serum amyloid P component scintigraphy in patients with amyloidosis. Am J Med 2006; 119 (04) 355.e15-355.e24
• 11 Bouma B, Maas C, Hazenberg BPC, Lokhorst HM, Gebbink MF. Increased plasmin-α2-antiplasmin levels indicate activation of the fibrinolytic system in systemic amyloidoses. J Thromb Haemost 2007; 5 (06) 1139-1142
• 12 Brophy TM, Ward SE, McGimsey TR. , et al. Plasmin cleaves von Willebrand factor at K1491-R1492 in the A1-A2 linker region in a shear- and glycan-dependent manner in vitro. Arterioscler Thromb Vasc Biol 2017; 37 (05) 845-855
• 13 Feys HB, Vandeputte N, Palla R. , et al. Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura. J Thromb Haemost 2010; 8 (09) 2053-2062
• 14 South K, Luken BM, Crawley JT. , et al. Conformational activation of ADAMTS13. Proc Natl Acad Sci U S A 2014; 111 (52) 18578-18583
• 15 Feys HB, Anderson PJ, Vanhoorelbeke K, Majerus EM, Sadler JE. Multi-step binding of ADAMTS-13 to von Willebrand factor. J Thromb Haemost 2009; 7 (12) 2088-2095
• 16 Hammarström P. The bloody path of amyloids and prions. J Thromb Haemost 2007; 5 (06) 1136-1138
• 17 Hiura H, Matsui T, Matsumoto M. , et al. Proteolytic fragmentation and sugar chains of plasma ADAMTS13 purified by a conformation-dependent monoclonal antibody. J Biochem 2010; 148 (04) 403-411
• 18 Lam JK, Chion CK, Zanardelli S, Lane DA, Crawley JT. Further characterization of ADAMTS-13 inactivation by thrombin. J Thromb Haemost 2007; 5 (05) 1010-1018
• 19 Crawley JT, de Groot R, Xiang Y, Luken BM, Lane DA. Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor. Blood 2011; 118 (12) 3212-3221
• 20 Wohner N, Kovács A, Machovich R, Kolev K. Modulation of the von Willebrand factor-dependent platelet adhesion through alternative proteolytic pathways. Thromb Res 2012; 129 (04) e41-e46