Leukocyte proteases cleave von Willebrand factor at or near the ADAMTS13 cleavage site

Abstract

The function of von Willebrand factor (VWF) is regulated by proteolysis, which limits its multimeric size and ability to tether platelets. The importance of ADAMTS13 metalloprotease in VWF regulation is demonstrated by the association between severe deficiency of ADAMTS13 and thrombotic thrombocytopenic purpura (TTP). However, ADAMTS13 activity levels do not always correlate with the clinical course of TTP, suggesting that other proteases could be important in regulating VWF. We identified 4 leukocyte proteases that cleave the synthetic VWF substrate FRETS-VWF73 and multimeric VWF. Elastase and proteinase 3 (PR3) cleave multimeric VWF and FRETS-VWF73 at the V(1607)-T(1608) peptide bond; cathepsin G and matrix metalloprotease 9 cleave VWF substrates at the Y(1605)-M(1606) and M(1606)-V(1607) bonds, respectively. Isolated intact human neutrophils cleave FRETS-VWF73 at the V(1607)-T(1608) peptide bond, suggesting that elastase or PR3 expressed on leukocyte surfaces might cleave VWF. In the presence of normal or ADAMTS13-deficient plasma, cleavage of FRETS-VWF73 by resting neutrophils is abolished. However, activated neutrophils retain proteolytic activity toward FRETS-VWF73 in the presence of plasma. Although the in vivo relevance remains to be established, these studies suggest the existence of a "hot spot" of VWF proteolysis in the VWF A2 domain, and support the possibility that activated leukocytes may participate in the proteolytic regulation of VWF.

Figure 1

Kinetic cleavage of rFRETS-VWF73 by leukocyte proteases. Leukocyte elastase, 10 nM (A), cathepsin G, 15 nM (B), MMP9, 10 nM (C), PR3, 10 nM (D), and recombinant ADAMTS13, 10 nM (E) were incubated with increasing concentrations (0-16 μM) of rFRETS-VWF73 peptide in 5 mM Bis-Tris, pH 6.0, 25 mM CaCl₂, 0.005% Tween-20. The proteolytic cleavage was monitored by the initial rate of fluorescent generation (v_max) at the excitation 485 nm and the emission 530 nm on a Vector 3 fluorescent microtiter reader. The data (slope vs substrate concentrations) were fitted into a Michaelis-Menten equation to determine the catalytic constant (k_cat) and Michaelis constant (k_m) using SigmaPlot software. The curves represent mean values of 2 independent experiments at the same concentration of for each enzyme. Slope refers to the change in fluorescence units per second.

Figure 2

Mass spectrometry analysis of FRETS-VWF73 cleaved by leukocyte proteases. Commercial FRETS-VWF73 peptide was incubated without (A), and with recombinant ADAMTS13 (rADAMTS13; B), neutrophil elastase (C), PR3 (D), cathepsin G (E), and MMP9 (F) at 30°C for 60 minutes. The digested materials were analyzed by MALDI-TOF mass spectrometry. The masses of the major peaks were compared with the calculated molecular weights of carboxyl terminus fragments of FRETS-VWF73. The mass of the major peak arising from rADAMTS13 cleavage and cathepsin G cleavage corresponds to cleavage at the M¹⁶⁰⁵-Y¹⁶⁰⁶ peptide bond. The major peaks resulting from neutrophil elastase cleavage and PR3 cleavage correspond to cleavage at the V¹⁶⁰⁷-T¹⁶⁰⁸ peptide bond. The major peak resulting from cleavage by MMP9 corresponds with cleavage at M¹⁶⁰⁶-V¹⁶⁰⁷. The mass spectrometry margin of error is 0.2%.

Figure 3

Cleavage of denatured VWF substrate by leukocyte proteases. Purified VWF (Humate P) was incubated with elastase (138 nM; A-B), cathepsin G (8.6 nM; C), and PR3 (172 nM; D) for the times indicated in each panel at 37°C in 0.005 M Tris buffer, pH 8.0, with (A,C-D) or without (B) 1.5 M urea. Proteolytic cleavage products were analyzed by 5% SDS–polyacrylamide gel electrophoresis under denaturing and reducing conditions, and stained with Coomassie blue. The positions of molecular mass standards are marked. ━ indicates the intact VWF polypeptide (250 kDa), whereas the indicates the cleavage products of various sizes under denaturing and reducing conditions.

Figure 4

Cleavage of multimeric VWF by leukocyte proteases under fluid shear stress. Purified plasma VWF (pVWF; 37.5 μg/mL) was incubated for 15 minutes and subjected to no vortexing (shear −) or constant vortexing at 2500 rpm for 5 minutes (shear +) in the presence of various concentrations of elastase (A), cathepsin G (B), MMP9 (C), or PR3 (D) as indicated in each panel. The reaction was performed in a polymerase chain reaction tube with a total volume of 20 μL in buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCl₂, and 1 mg/mL BSA. The proteolytic cleavage products were determined by 5% SDS–polyacrylamide gel under denaturing and nonreducing conditions. Western blotting was performed with anti-VWF IgG and infrared fluorescent dye labeled anti–rabbit IgG as described in “Cleavage of multimeric VWF under fluid shear stress.” → (left borders) indicate positions of a 250-kDa molecular weight marker. (right borders) indicates positions of cleavage products. The asterisk in panel C denotes 20 mM EDTA added to the reaction mixture.

Figure 5

Cleavage of FRETS-VWF73 by membrane-bound proteases on neutrophils. (A) The (commercial) FRETS-VWF-73–cleaving activity of 2.5% normal pooled plasma (NPP) or 2.5% ADAMTS13-deficient TTP plasma in the presence of resting or PMA-activated neutrophils (10⁵ cells/mL). Resting or activated cells were resuspended in 2.5% plasma mixed in reaction buffer. Activities are represented relative to 2.5% NPP alone, which is designated as 100% activity. (B) The cleaving activity of 2.5% wild-type (WT) or 2.5% ADAMTS13-deficient (ADAMTS13^−/−) mouse plasma in the presence of resting or PMA-activated neutrophils (10⁵ cells/mL). Activities are represented relative to WT mouse plasma, which is designated as 100% activity. Results shown in panels A and B are mean values (± SD) from 2 combined representative experiments (N = 4). ▨ represents activated neutrophils. The P values were determined by Student t tests.

Figure 6

Mass spectrometry analysis of cleavage site on FRETS-VWF73 by membrane-bound proteases on neutrophils. A commercial FRETS-VWF73 peptide cleaved by (A) normal pooled plasma (NPP), (B) PMA-activated neutrophils in assay buffer, (C) activated neutrophils combined with NPP, (D) or activated neutrophils combined with ADAMTS13-deficient TTP plasma were analyzed by MALDI-TOF mass spectrometry. The doublet peaks (shown by asterisks above brackets) observed when NPP or TTP plasmas were present differed by 157 Da, and are consistent with the removal of a carboxyl terminal arginine of the FRETS-VWF73 substrate by plasma carboxypeptidase B. NPP alone generated a doublet peak consistent with cleavage at the Y¹⁶⁰⁵-M¹⁶⁰⁶ peptide bond (A). Activated neutrophils combined with NPP or TTP plasma generated a major peak of 6649 Da, consistent with cleavage at the V¹⁶⁰⁷-T¹⁶⁰⁸ bond and the removal of the carboxyl terminal arginine (C-D).

Figure 7

Potential cleavage sites in the VWF-A2 domain of leukocyte proteases and ADAMTS13. A schematic diagram of the VWF A domains is shown with the peptide bond cleavage sites in the A2 domain shown by arrows for neutrophil elastase, PR3, cathepsin G, ADAMTS13, MMP9, and neutrophils. The cleavage sites were determined by mass spectrometry analysis of the carboxyl terminal fragments of cleaved FRETS-VWF73, and confirmed by amino terminal sequencing of approximately 175 kDa electrophoretic bands of cleaved multimeric VWF under reducing conditions for neutrophil elastase, PR3, and cathepsin G.