Cleavage of von Willebrand factor by granzyme M destroys its factor VIII binding capacity

Abstract

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF.

Figure 1. GrM cleaves VWF.

(A) Purified plasma VWF (5 µg/mL) was incubated with various purified recombinant granzymes (300 nM) in TBS for 1 hour at 37°C and analyzed by immuno blot, using an antibody against VWF. Mature VWF and VWF cleavage fragments (*) are indicated by the arrows. (B) Purified plasma VWF (2 µg/well) was immobilized onto plastic and incubated with various purified recombinant granzymes (300 nM) in TBS for 1 hour at 37°C and analyzed by immuno blot, using an antibody against VWF. (C) Purified plasma VWF (5 µg/mL) was incubated with different concentrations of GrM (0–800 nM) or GrM-SA (800 nM) in TBS for 4 hours at 37°C and analyzed by immuno blot, using an antibody against VWF. (D) Purified plasma VWF (2 µg/well) was immobilized onto plastic and incubated with GrM (0–800 nM) or GrM-SA (800 nM) in TBS for 1 hour at 37°C and analyzed by immuno blot, using an antibody against VWF. (E) Purified plasma VWF (5 µg/mL) was incubated with GrM (100 nM) for 1 hour at 37°C in Tris (pH 7.4) in the presence or absence of increasing concentrations of NaCl (0–600 mM) and analyzed by immuno blot, using an antibody against VWF. (F) GrM, GrB, and ADAMTS13 cleavage sites in VWF are schematically indicated. Data are representative of at least three independent experiments.

Figure 2. GrM does not affect VWF multimerization.

Purified plasma VWF (2 µg/mL) was incubated with indicated concentrations of GrM, GrB, GrM-SA and GrB-SA in Tris (pH 7.4) for 1 hour at 37°C. (A) Samples analyzed at 6% SDS-PAGE and Western blot, using an antibody against VWF. Mature VWF and VWF cleavage fragments (*) are indicated by the arrows. (B) Samples were separated at a SDS agarose gel and multimers were visualized on blot. Data are representative of at least three independent experiments.

Figure 3. GrM does not affect VWF platelet binding affinity.

(A) Purified plasma VWF (2 µg/mL) was incubated with GrB (100 nM), GrM (100 nM), or buffer only in Tris (pH 7.4) at 37°C for 1 hour and active VWF was detected by AU/VWF-A11-based immunosorbent assay. Data represent the mean +/− SD of three independent experiments. (B) Purified plasma VWF (15 µg/mL) was incubated for 1 hour at 37°C with various concentrations of GrM (0–100 nM), GrM-SA (100 nM), or GrB (100 nM) in Tris (pH 7.4) and VWF ristocetin cofactor activity was determined. Data represent the mean +/− SD of three independent experiments. (C) Purified plasma VWF (6 µg/mL) was treated with GrM (0–600 nM) or buffer only in Tris (pH 7.4) for 1 hour at 37°C and subsequently incubated 1∶1 with washed platelets (5×10⁷ platelets/mL). Platelet aggregation was induced by the addition of ristocetin (1.5 mg/mL). (D) Purified plasma VWF (6 µg/mL) was treated with GrB (0–600 nM) or buffer only in Tris (pH 7.4) for 1 hour at 37°C and subsequently incubated 1∶1 with washed platelets (5×10⁷ platelets/mL). Platelet aggregation was induced by the addition of ristocetin (1.5 mg/mL). Data are representative of at least four independent experiments with four different donors.

Figure 4. GrM slightly affects VWF binding to collagen.

Purified plasma VWF (1 µg/mL) was incubated with indicated concentrations of GrB, GrB-SA, GrM, or GrM-SA in Tris (pH 7.4) for 1 hour at 37°C. Samples were assessed for collagen binding, using an immunosorbent assay. Data represent the mean +/− SD of three independent experiments. GrM (25 nM) versus GrM-SA (25 nM), p = 0.02; GrB (25 nM) versus GrB-SA (25 nM), p<0.001.

Figure 5. GrM disrupts FVIII binding to VWF.

(A) Purified plasma VWF (1 µg/mL) VWF was incubated with indicated concentration of GrM, GrB, GrM-SA or GrB-SA in Tris (pH 7.4) for 1 hour at 37°C and subsequent FVIII binding was assessed in an ELISA setup. Data represent the mean +/− SD of three independent experiments. GrM (25 nM) versus GrM-SA (25 nM), p = 0.009; GrB (25 nM) versus GrB-SA (25 nM), p = 0.009. (B) VWF (30 ng) was pre-incubated in the presence or absence of GrM (100 nM) or GrM-SA (100 nM) in 5 mM Tris (pH 7.4) for 1 hour at 37°C, followed by incubation with purified FVIII heterodimer in TBS, 10 mM CaCl₂ for 2 hours at 37°C. Protein mixtures were immunoprecipitated by ProtA/G sepharose pre-treated with anti-VWF antibody. Samples were analyzed by Western blotting, using antibodies against VWF and FVIII heterodimer. Full-length VWF, its GrM-cleaved form, and the FVIII heavy chain (HCh) and light chain (LCh) are indicated by the arrows. IP, immunoprecipitation; WB, Western blot. Data are representative of at least three independent experiments.

Figure 6. Plasma GrM and VWF/FVIII ratios positively correlate in patients with meningococcal disease.

(A) Representative example of GrM in plasma of patients with meningococcal disease without shock (1) and with shock (2–5), using immunoblotting with a monoclonal antibody against GrM. (B) VWF and FVIII levels in plasma were determined by ELISA and chromogenic activity, respectively. VWF/FVIII ratios were determined in meningococcal shock patients with or without detectable plasma GrM, patients with meningococcal disease without shock, and healthy controls. P values are indicated. (C) VWF/FVIII ratios are plotted against plasma GrM levels, as quantified by western blot. R_spearman = 0.92, p<0.001.