Evidence for the Interaction Between VWF Carboxyl-Terminal Domains and ADAMTS13 Domains Distal to Its Spacer Domain

Abstract

ADAMTS13 plasma metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Y1605-M1606 bond in the A2 domain. Several studies have characterised high affinity binding sites in the A2 domain, probably interacting with the ADAMTS13 spacer and proximal domains. We have previously provided preliminary evidence for an additional ADAMTS13 binding site distal to the A domains, by measuring a ~7-fold increase in affinity for ADAMTS13 of plate-immobilised VWFA1CK (residues 1260-2813) (KD 11 nM) compared with VWFA1A2A3 (residues 1260-1874) (KD 78 nM) (Zanardelli et al, JTH, volume 5 Supplement I, XXI ISTH congress 2007, abstract number O-W-018). We subsequently expressed a series of VWF carboxyl-terminal fragments, VWFD4CK (residues 1874-2813), VWFA3CK (residues 1671-2813), VWFD4 (residues 1874-2299) and VWFB1CK (residues 2296-2813). Using an equilibrium binding assay, we assessed their binding to full-length ADAMTS13 and truncated ADAMTS13 proteins MDTCS (residues 34-685) and del(TSP5-CUB) (residues 34-894). KD values calculated for VWFD4CK and VWFA3CK binding to full-length ADAMTS13 were 134 and 108 nM, respectively. This confirmed the presence of an ADAMTS13 binding site outside VWF A2 domain, in the region D4CK. It also excluded a major role in the interaction for the VWF A3 domain. However, isolated VWFD4 and VWFB1CK did not bind significantly, suggesting that integrity of the VWF C-terminal region is also required for the binding. Furthermore, MDTCS bound to VWFA1CK and VWFA1A2A3 with similar affinity (KD 120 and 82 nM, respectively) and no significant interaction occurred with VWFD4CK. Similar results were found for the binding of del(TSP5-CUB) to the VWF fragments (VWFA1CK KD 85 nM, VWFA1A2A3 KD 65 nM and VWFD4CK no binding). Together these results suggested a multiple interaction between the carboxyl-terminal region of VWF (within D4CK) and the distal domains of ADAMTS13 (TSP5–8 and CUB domains). The VWF carboxyl-terminal binding site may be important for initial anchoring of ADAMTS13, allowing the subsequent binding between the spacer domain and VWF A2 domain.