Removal of the C-Terminal Domains of ADAMTS13 by Activated Coagulation Factor XI induces Platelet Adhesion on Endothelial Cells under Flow Conditions

Abstract

Platelet recruitment to sites of vascular injury is mediated by von Willebrand factor (VWF). The shear-induced unraveling of ultra-large VWF multimers causes the formation of a "stringlike" conformation, which rapidly recruits platelets from the bloodstream. A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) regulates this process by cleaving VWF to prevent aberrant platelet adhesion; it is unclear whether the activity of ADAMTS13 itself is regulated. The serine proteases α-thrombin and plasmin have been shown to cleave ADAMTS13. Based on sequence homology, we hypothesized that activated coagulation factor XI (FXIa) would likewise cleave ADAMTS13. Our results show that FXIa cleaves ADAMTS13 at the C-terminal domains, generating a truncated ADAMTS13 with a deletion of part of the thrombospondin type-1 domain and the CUB1-2 domains, while α-thrombin cleaves ADAMTS13 near the CUB1-2 domains and plasmin cleaves ADAMTS13 at the metalloprotease domain and at the C-terminal domain. Using a cell surface immunoassay, we observed that FXIa induced the deletion of the CUB1-2 domains from ADAMTS13 on the surface of endothelial cells. Removal of the C-terminal domain of ADAMTS13 by FXIa or α-thrombin caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS) and blocked the ability of ADAMTS13 to cleave VWF on the endothelial cell surface, resulting in persistence of VWF strands and causing an increase in platelet adhesion under flow conditions. We have demonstrated a novel mechanism for coagulation proteinases including FXIa in regulating ADAMTS13 activity and function. This may represent an additional hemostatic function by which FXIa promotes local platelet deposition at sites of vessel injury.

Keywords: a disintegrin and metalloproteinase with a thrombospondin type 1 motif; coagulation; factor XI; member 13; platelets; von Willebrand factor.

Figure 1

Proteolysis of ADAMTS13 by FXIa. (A) rADAMTS13 (250 nM) was incubated with FXIa (50 nM), in the absence or presence of aprotinin (50 µM) for selected times (0–120 min) at 37°C before being separated by SDS-PAGE under reduced conditions and analyzed by Coomassie blue staining. rADAMTS13 fragment size (kDa) is shown following proteolysis by FXIa (B) rADAMTS13 (250 nM) was incubated with FXIa (100–15 nM), plasmin (50–0.5 nM), or α-thrombin (100–15 nM) for selected times (0–4 h) at 37°C. rADAMTS13 was analyzed by western blotting using an anti-ADAMTS13 MET domain antibody (C) rADAMTS13 (250 nM) was incubated with FXa (50 nM), FXIIa (50 nM), and kallikrein (50 nM) for selected times (0–4 h) at 37°C. rADAMTS13 was analyzed by western blotting using an anti-ADAMTS13 MET domain antibody (D) rADAMTS13 (250 nM) was incubated with FXIa (50 nM) for selected times (0–4 h) at 37°C before being analyzed by western blotting using an anti-ADAMTS13 MET domain antibody in the absence or presence of CaCl₂ or ZnCl₂ (n = 3).

Figure 2

Characterization of ADAMTS13 proteolysis by FXIa, α-thrombin, and plasmin. rADAMTS13 (250 nM) was incubated with (A) FXIa (50 nM), (B) α-thrombin (50 nM), or (C) plasmin (5 nM) for selected times (0–4 h) at 37°C before being analyzed with three different antibodies: anti-ADAMTS13 MET domain antibody, anti-ADAMTS13 CUB1-2 domain antibody, and anti-TSP4 domain antibody. The predicted identities of the formed fragments upon cleavage of rADAMTS13 by FXIa in (A), or by α-thrombin in (B) or by plasmin in (C) are depicted in a schematic overview (n = 3).

Figure 3

FXIa cleaves the CUB1-2 domain of ADAMTS13 on the endothelial surface. (A) HUVECs were incubated with or without rADAMTS13 (50 nM) for 1 h at 37°C, washed, and treated with or without FXIa (30 nM) for 2 h. Reactions were stopped with aprotinin (50 µM), followed by cell surface detection of ADAMTS13 by using either an anti-ADAMTS13 CUB1-2 domain antibody or an anti-ADAMTS13 TSP4 domain antibody. (B) HUVECs incubated with rADAMTS13 (50 nM) treated with FXIa (30 nM), (C) α-thrombin (30 nM), or (D) plasmin (30 nM) for 0–2 h at 37°C. Reactions were stopped with aprotinin (50 µM) and hirudin (10 µg/mL), followed by cell surface detection of ADAMTS13 by using either an anti-ADAMTS13 CUB1-2 domain antibody (○) or an anti-ADAMTS13 TSP4 domain antibody (●). Data are mean ± SE (n = 3).

Figure 4

ADAMTS13 activity following proteolysis by FXIa. (A) rADAMTS13 (30 nM) was incubated at 37°C for 4 h with the following: FXIa (5 nM), plasmin (5 nM), or α-thrombin (5 nM) in HBS with 5 mM CaCl₂. Reactions were stopped with aprotinin (50 µM) and hirudin (10 µg/mL). rADAMTS13 was diluted to 4 nM in reaction buffer (5 mM Bis-Tris pH 6.0, 25 mM CaCl2, and 0.005% Tween-20) and the reaction was initiated by the addition of an equal volume of FRETS-VWF73 substrate (4 µM). Data are mean ± SE (n = 3). (B) Western blot of the samples using an anti-ADAMTS13 MET domain antibody to confirm proteolytic cleavage of ADAMTS13 by the proteases.

Figure 5

FXIa inhibits ADAMTS13 cleavage of VWF. rADAMTS13 (250 nM) was incubated at 37°C for 4 h with the following: FXIa (50 nM), α-thrombin (50 nM), and plasmin (50 nM) in HBS with 5 mM CaCl₂. Reactions were stopped with aprotinin (50 µM) and hirudin (10 µg/mL). Endothelialized parallel-plate flow chambers were prepared and EC’s were stimulated with TNFα to release VWF as described above. (A) VWF string formation and platelet adhesion depicted with fluorescence following perfusion of washed platelets at a venous flow rate of 2.5 dyne/cm² in the absence or presence of rADAMTS13 (2.5 nM) incubated with either vehicle, FXIa, α-thrombin, plasmin, or an anti-ADAMTS13 CUB domain antibody (20 ng/mL). (B) Quantification of platelet string formation, total VWF number, and VWF length compared between noted substrates. Using Dunnett’s multiple comparison test, * and ^# indicate statistical significance (p < 0.05). Data are mean ± SE (n = 3).

Figure 6

Schematic representation of the proposed model for ADAMTS13 substrate cleavage. (A) ADAMTS13 circulates in plasma in a closed conformation due to an interaction between its CUB-1 domain and the spacer domain. Following the binding of the ADAMTS13 TSP7-CUB2 domain to the VWF D4CK domains under shear flow, ADAMTS13 unfolds and the spacer domain of ADAMTS13 becomes available for binding to the A2 domain of VWF, leading to the efficient cleavage of VWF. (B) However, deletion of the C-terminal domains of ADAMTS13 induce a conformational change, increasing the cleavage of the FRETS-VWF73 peptide substrate under static conditions. In contrast, the removal of the C-terminal TSP1 and CUB domains from ADAMTS13 decreases the binding of ADAMTS13 to VWF and also decreases its capacity to cleave VWF when subjected to shear flow. In this study, we show that the removal of the C-terminal domains of ADAMTS13 by the serine proteases FXIa and α-thrombin enhance the capacity of ADAMTS13 to cleave the FRETS peptide substrate under static conditions and abolished the ability to cleave VWF strings on endothelial cells under flow conditions.