Carboxyl terminus of ADAMTS13 directly inhibits platelet aggregation and ultra large von Willebrand factor string formation under flow in a free-thiol-dependent manner

Abstract

Objective: ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, 13) cleaves von Willebrand factor (VWF), thereby inhibiting thrombus formation. Proteolytic cleavage relies on the amino-terminal (MDTCS) domains, but the role of the more distal carboxyl-terminal domains of ADAMTS13 is not fully understood. A previous study demonstrated the presence of multiple surface-exposed free sulfhydryls on ADAMTS13 that seemed to interact with those on VWF under shear. Here, we determined the physiological relevance of such an interaction in antithrombotic responses under flow.

Approach and results: A microfluidic assay demonstrated that a carboxyl-terminal fragment of ADAMTS13, comprising either 2 to 8 thrombospondin type 1 (TSP1) repeats and CUB domains (T2C) or 5 to 8 Thrombospondin type 1 (TSP1) repeats and CUB domains (T5C), directly inhibited platelet adhesion/aggregation on a collagen surface under arterial shear. In addition, an intravital microscopic imaging analysis showed that the carboxyl-terminal fragment of ADAMTS13 (T2C or T5C) was capable of inhibiting the formation and elongation of platelet-decorated ultra large (UL) VWF strings and the adhesion of platelets/leukocytes on endothelium in mesenteric venules after oxidative injury. The inhibitory activity of T2C and T5C on platelet aggregation and ULVWF string formation were dependent on the presence of their surface free thiols; pretreatment of T2C and T5C or full-length ADAMTS13 with N-ethylmaleimide that reacts with free sulfhydryls abolished or significantly reduced its antithrombotic activity.

Conclusions: Our results demonstrate for the first time that the carboxyl terminus of ADAMTS13 has direct antithrombotic activity in a free-thiol-dependent manner. The free thiols in the carboxyl-terminal domains of ADAMTS13 may also contribute to the overall antithrombotic function of ADAMTS13 under pathophysiological conditions.

Keywords: ADAMTS13; animal model; arterial thrombosis; thrombotic thrombocytopenic purpura; von Willeband factor.

Figure 1

Constructs and purified recombinant ADAMTS13 and its C-terminal fragments. A. Full-length human ADAMTS13 (rA13) comprises a signal peptide (S), a propeptide (P), a metalloprotease (M), a disintegrin domain (D), a cysteine rich (C), the first thrombospondin type 1 repeats (TSP1), and a spacer domain (S). The more distal C-terminus contains 7 more TSP1 repeats (2-8) and two CUB domains (C1-C2). The signal peptide and propeptide are cleaved ( ) upon secretion and a V5-His epitope is attached to the C-terminal end in each construct. In addition, a 3xFlag tag was added to the C-terminal fragments of ADAMTS13 after the leader sequence (Igk) was removed ( ) during secretion. T2C or T5C refers the construct consisting of 2-8 TSP1 repeats plus CUB domains or 5-8 TSP1 repeats plus CUB domains. B. SDS-polyacrylamide (10%) gel electrophoresis and coomassie blue staining determined the purity of recombinant ADAMTS13 (rA13), T2C, and T5C (~5-10 μg of total protein/lane). M in lane 1 is a pre-stained and broad range protein marker. C and D are the relative activity of recombinant ADAMTS13 (rA13) with or without NEM treatment in the proteolytic cleavage of FRETS-VWF73 peptide and multimeric VWF, respectively.

Figure 2

A microfluidic flow system determines antithrombotic activity of rA13 and its C-terminal fragments. A. Representative images depicting the surface coverage of platelet aggregates on a fibrillar collagen-coated surface under various arterial shears (20, 50, 100, and 200 dyne/cm²) after perfusion of PPACK-anticoagulated whole blood from wild-type mice (WT) and Adamts13^−/− (KO) mice. B. Quantitation of relative surface platelet coverage (or relative fluorescent intensity) from images obtained in panel A as a function of fluid shear stress from three individual experiments (n=3). The maximal fluorescent intensity in the KO mice was defined as 100% at the end of 120 seconds. The data shown are the means ± one standard deviation. C. The percentage of surface coverage of WT and KO murine platelets on fibrillar collagen under high shear (100 dyne/cm²) in the presence of rA13 (0, 12.5, 25, 50, and 100 nM) (mean ± standard deviation, N=3). D. The percentage of murine (KO) platelet coverage on a fibrillar collagen surface in the presence of active human rA13 (100 nM) and heat-inactivated rA13 (iA13) (100 and 500 nM) under 100 dyne/cm². The data shown are the mean+ standard error) (n=3). E. The relative proteolytic activity of rA13 and heat-inactivated rA13 (iA13) at various concentrations (10, 100, and 1,000 nM) in cleaving the FRETS-VWF73 peptide.

Figure 3

The C-terminal ADAMTS13 fragments inhibit platelet aggregation on a collagen surface under flow. A to F represent dynamic changes in the mean percentage of coverage or aggregation of human platelets from a PPACK anti-coagulated whole blood on type 1 soluble collagen surface under shear (~20 dyne/cm²) over time (180 seconds) in the absence (0 nM) and presence of various concentrations (100, 500, and 1000 nM) of untreated (A, D) or NEM-treated (B, E) T2C or T5C as indicated in each panel. G to L indicate the dynamic changes in the mean percentage of coverage of murine (Adamts13^−/−) platelets on type 1 fibrillar collagen surface over time under high shear (~100 dyne/cm²) in the absence (0 nM) and presence of various concentrations (100, 500, and 1000 nM) of untreated (G, J) or NEM-treated (H, K) T2C or T5C as indicated in each panel. C, F, I, and L are the means and standard errors (SEM) of the mean platelet coverage or aggregation on collagen surfaces at the end of perfusion. Each experiment was repeated for 3-5 times as indicated in each panel. Statistical analysis was performed by ANOVA analysis. The p values<0.05 and 0.01 were considered statistically significant and highly significant, respectively.

Figure 4

Representative snapshots of platelet/leukocyte-decorated ULVWF strings on the endothelial cells in mesenteric venules. A, C, E, G, I, K, and M represent the ULVWF string formation prior to infusion of rA13 or a C-terminal fragment; B, D, F, H, J, L, and N show the ULVWF strings one minute after infusion of PBS, rA13 (10 nM), NEM-treated rA13 (10 nM), T2C (500 nM), NEM-treated T2C (500 nM), T5C (500 nM), and NEM-treated T5C (500 nM), respectively. Asterisks indicate the presence of long and short platelet-decorated ULVWF strings; arrows indicate the blood flow direction.

Figure 5

Dynamic changes in the mean length of platelet-decorated ULVWF strings on endothelial cells before and after infusion of rA13 and its C-terminal fragments. A to I show the dynamic changes in the mean length of platelet-decorated ULVWF strings on the endothelial cells in mesenteric venules after oxidative injury prior to and after infusion (↓) of PBS, rA13 (10 nM), T2C (10 and 500 nM), and T5C (10 and 500 nM) which were treated with or without NEM as indicated in each panel. The data represent the mean ± standard errors (SEM) from three mice (N=3) for each treatment. J summarizes the percentage changes (mean ± SEM, n=3) in the average length of platelet-decorated ULVWF strings on endothelial cells after infusion of PBS, rA13 (10 nM), T2C (10 and 500 nM), and T5C (10 and 500 nM) with (red bars) or without (blue bars) NEM treatment. Statistical analysis was performed using ANOVA analysis. Stars in panel J indicate the differences were statistically highly significant compared with PBS-control.

Figure 6

Dynamic changes in the surface coverage of platelet/leukocyte aggregates on endothelium before and after infusion of rA13 and its C-terminal fragments. A to I show the dynamic changes in the percentage of surface coverage of platelet/leukocyte aggregates on endothelial cells after oxidative injury prior to and after infusion (↓) of PBS, rA13 (10 nM), T2C (10 and 500 nM), and T5C (10 and 500 nM) that were untreated or treated with NEM as indicated in each panel. The data are the mean ± standard errors (SEM) from three mice (N=3) for each treatment. J summarizes the percent changes (mean ± SEM, n=3) in the surface coverage of platelet/leukocyte aggregates on endothelium after infusion of PBS, rA13 (10 nM), T2C (10 and 500 nM), and T5C (10 and 500 nM) untreated (blue bars) or treated (red bars) with NEM. Statistical analysis was performed using ANOVA analysis. Stars in panel J indicate that the differences were statistically highly significant compared with PBS-control.