von Willebrand factor cleaved from endothelial cells by ADAMTS13 remains ultralarge in size.

Von Willebrand factor (vWF) is synthesized in all vascular endothelial cells and megakaryocytes 1. The mature vWF is stored in the Weibel-Palade bodies of endothelial cells and the α-granules of platelets. A small fraction of vWF is constitutively secreted from the cells. Upon stimulation by thrombin, hormones, and inflammatory cytokines, vWF is released from the Weibel-Palade bodies of the endothelial cells as ultra large (UL) vWF, which forms polymers and remains attached to the endothelial cell surface via poorly characterized mechanisms, although recent reports have suggested that UL-vWF might be anchored on endothelial cells via interactions with P-selectin 2 and αvβ3 integrin 3. Proteolytic cleavage of the newly synthesized UL-vWF on endothelial cells by ADAMTS13 may be critical to maintain normal hemostasis and inflammatory response. For instance, certain mutations in the vwf gene may result in increased proteolysis of vWF by ADAMTS13 and significant reduction of vWF multimer sizes, leading to compromised hemostatic function as seen in patients with von Willebrand disease 4. Conversely, an inability to cleave the newly released UL-vWF strings 5;6 due to hereditary or acquired deficiency of ADAMTS13 may result in an accumulation of UL-vWF that results in formation of disseminated thrombi in the microvasculature as in patients with thrombotic thrombocytopenic purpura (TTP) 7. Moreover, ADAMTS13 deficiency appears to result in increased leukocyte rolling on unstimulated veins, leukocyte adhesion and extravasation of neutrophils in the inflamed veins 8. All these processes seem to depend on the presence of UL-vWF multimers 8. Therefore, the removal of cell bound UL-vWF by ADAMTS13 may help attenuate systemic inflammatory responses in addition to arterial thrombosis. In contrast to proteolytic cleavage of vWF in solution, which requires denaturant (urea 9 or guanidine 10) in a low ionic and alkaline buffer or high shear stress 11;12, the removal of UL-vWF polymers on cultured endothelial cells by ADAMTS13 occurs very efficiently under various shear stresses 5;6. The rapid removal of the UL-vWF strings by infused recombinant ADAMTS13 also occurs in both arteries (high shear) and venules (low shear) in Adamts13-/- mice 13. A recent study has shown that UL-vWF strings on histamine stimulated endothelial cells can be removed by ADAMTS13 in the absence of flow shear stress 14. It remains poorly understood what ADAMTS13 domains are required for cleavage of cell bound UL-vWF, and whether proteolytic cleavage of UL-vWF by ADAMTS13 on endothelial cell membranes is sufficient to reduce the multimer sizes of UL-vWF in the presence or absence of flow shear stress. In this study, we demonstrated by immunofluorescent microscopy that UL-vWF polymers were readily formed on the membrane of human umbilical vein endothelial cells (HUVECs) after being stimulated for 2 min with histamine (100 μM) in phosphate buffered saline (Fig. 1A-i). Recombinant ADAMTS13 (10 nM) (Fig. 1A-ii) or normal human plasma (NHP) (1:2 dilution) (not shown) in an assay buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 5 mM CaCl2) within 5 min could efficiently remove all surface bound UL-vWF polymers in the absence of flow shear stress. Plasma (1:2 dilution) from a patient with acquired idiopathic TTP with known high titer of anti-ADAMTS13 IgG autoantibodies, however, did not result in the removal of the cell bound UL-vWF polymers under the same conditions (not shown). These results are consistent with what has been reported by Turner et al 14, and are the premises for our further study of the structural and functional relationship of ADAMTS13 in cleavage of UL-vWF at the cellular levels. Figure 1 Cleavage of cell bound UL-vWF by recombinant ADAMTS13 and variants To better quantify the amount of UL-vWF released from endothelial cells, we employed an enzyme linked immunoassay (ELISA) using two polyclonal anti-vWF antibodies as described previously 15. In these experiments, HUVECs cultured on 6-well plate were stimulated with histamine (100 μM) for 2 min, washed with PBS and then incubated for 0~60 minutes without or with a fixed concentration (10 nM) of purified recombinant ADAMTS13 in 20 mM HEPES, 150 mM NaCl and 5 mM CaCl2, pH 7.4 (Fig. 1B-i) or for 5 min with various concentrations of recombinant ADAMTS13 (0~ 40 nM) (Fig. 1B-ii). The amount of vWF antigen in the conditioned media was increased in an incubation time and ADAMTS13 concentration dependent manner (Fig. 1B). After 60 min of incubation with purified recombinant ADAMTS13 (10 nM), the vWF antigen in the conditioned media (~24 ng/ml) was approximately 4-fold higher than in the buffer-treated control (~6 ng/ml) (Fig. 1B-i). The concentration of ADAMTS13 that achieved the half maximal levels of vWF released from endothelial cells was approximately 3.0 nM (Fig. 1B-ii), which is within the physiological range of ADAMTS13 in plasma. To determine whether or not the C-terminal domains of ADAMTS13 were required for cleavage of cell bound UL-vWF, histamine-stimulated and washed HUVECs were incubated with 10 nM of purified ADAMTS13 and various C-terminal truncated variants that were well characterized previously 12. The remaining UL-vWF on endothelial cells was determined by immunofluorescent microscopy using polyclonal anti-vWF IgG (Dako). We showed that the variants lacking the CUB domains (delCUB) (Fig. 1D-i) and truncated after the spacer domain (MDTCS) (Fig. 1D-ii) also efficiently removed UL-vWF polymers from histamine-stimulated endothelial cells, similar to full-length ADAMTS13 (Fig. 1A-ii). However, the variant truncated after the first thrombospondin type 1 (TSP1) repeat (MDT) (Fig. 1D-iii) or the metalloprotease domain (M) (Fig. 1D-iv) exhibited markedly impaired activity in removing the cell bound UL-vWF under the same conditions. The removal of cell bound UL-vWF polymers was consistent with the about 3~4 fold of increase in vWF antigen in the conditioned medium after incubation of the histamine-stimulated endothelial cells with full-length ADAMTS13, delCUB and MDTCS, but not with MDT and M or buffer alone (Fig. 1C-i). The proteolytic cleavage products (176kDa and 140 kDa) were also detectable in the conditioned media of HUVECs treated with FL-A13, delCUB and MDTCS, but not with MDT and M or buffer alone (Fig. 1C-ii). The amount of vWF antigen detected in the MDTCS-treated medium appeared to be slightly low compared with the FL-A13 and delCUB-treated media (Fig. 1C-ii), but such a difference was not statistically significant (p>0.05). The reduced cleavage products in the MDTCS-treated medium compared with full-length ADAMTS13 and delCUB may be a result of imperfect protein loading, rather than the lower proteolytic activity. Thus, our results suggest that the Cys-rich and spacer domains are required for recognition of the cell bound UL-vWF, but the TSP1 2-8 repeats and the CUB domains are dispensable. These results, however, do not fully agree with those reported 14. The reason for the discrepancy is not completely understood, but may be related to the assay methodologies being used. The domain requirement for proteolytic cleavage of cell bound UL-vWF is reminiscent of that seen in cleavage of soluble vWF by ADAMTS13 under denaturing conditions 16;17, suggesting that membrane association of UL-vWF results in a conformational change that permits ADAMTS13 binding in the absence of fluid shear stress. The mechanism underlying such a conformational change remains to be determined. To determine whether proteolytic cleavage of cell bound UL-vWF by ADAMTS13 was sufficient to reduce UL-vWF multimer size, the conditioned media were assessed by SDS-agarose (1%) gel electrophoresis and Western blot as previously described 15. Unexpectedly, whether in the absence of flow (Fig. 1E-i) or in the presence of flow (2.5 dynes/cm2) (Fig. 1E-ii), vWF multimers released into the conditioned media after being incubated with ADAMTS13 (10 nM) were indistinguishable from those in the conditioned media after being incubated with buffer alone or immediately after histamine stimulation (Fig. 1E-i & ii). When compared with normal human plasma, the conditioned media were highly enriched with UL-vWF as indicated by UL (Fig. 1E-i & ii). Incubation of the histamine-stimulated endothelial cells with recombinant ADAMTS13 (10 nM) for up to 60 min in the absence of flow (Fig. 1E-i, lanes 2-8) or for 5 min under various flow shear stresses up to ~25 dynes/cm2 (Fig. 1E-ii, lanes 3-7) did not appear to alter the vWF multimer distribution in the conditioned media, as compared with the UL-vWF released from the Weibel-Palade bodies after histamine stimulation (Fig. 1E-i, lane 9 & Fig. 1E-ii, lane 8), although some of the low molecular weight vWF (L) may be resulted from proteolytic degradation of the UL-vWF in the conditioned medium during storage. Addition of 0.1% protease inhibitor cocktail (Sigma) reduced the intensity of the smaller vWF bands. Nevertheless, the vWF cleaved off from endothelial cells by ADAMTS13 remains ultra large in size. This suggests further proteolytic processing downstream by ADAMTS13 or other leukocyte proteases 18, likely to occur in the small arteries and capillaries, where high fluid shear stress and perhaps physiological cofactors such as factor VIII 19 and platelets 20 are present, may be necessary to eliminate the ultra large vWF polymers.