In the resting state the presence of neutrophils in

In the resting state the presence of neutrophils in glomeruli would be minimal and thus VWF-cleaving activity by neutrophil proteases negligible. During infection and inflammation neutrophil influx will occur and proteases released into the glomerular capillary lumen will migrate towards the subendothelium, as demonstrated here. Thus, neutrophil proteases may prevent thrombosis from occurring on a thrombogenic vascular surface. In the presence of functional ADAMTS13, released locally from the activated glomerular endothelium (Tati et al., 2011) or circulating in the plasma, a combined anti-thrombotic effect would be achieved. In addition to proteases, neutrophils may also secrete peptides that regulate the anti-thrombotic activity of ADAMTS13 (Pillai et al., 2016). Elevated neutrophil elastase occurring during infectious episodes (Mikes et al., 2014) could explain why certain TTP patients are partially protected and remain asymptomatic for long periods in spite of ADAMTS13 deficiency. TTP recurrences are often triggered by infections during which neutrophils infiltrate tissue. Although lack of ADAMTS13 predisposes the endothelium to a prothrombotic state the presence of proteases released by neutrophils could offer partial protection. VWF cleavage mediated by neutrophil proteases could prevent the fulminant formation of microthrombi. In spite of development of thrombotic microangiopathy many TTP patients do not develop renal failure, which could be due to the phenotype of their mutation (Rurali et al., 2015), as well as neutrophil protease activity in glomeruli. The main inhibitor of elastase and PR3 in plasma is α1-antitrypsin (Ohlsson, 1971). The VWF-cleaving activity of neutrophil elastase, demonstrated under flow conditions, was maintained after PGECs were in contact with TTP plasma (lacking ADAMTS13 activity). The presence of an elastase inhibitor would most probably be minimal under the conditions used, as the plasma had been washed away in the perfusion process. In the glomerular subendothelium and the GBM the concentration of plasma would be very low and thus elastase-induced VWF cleavage could proceed in an uninhibited manner and prevent formation of large VWF-platelet thrombi thereby protecting the kidney from fulminant damage during mild inflammation. The effects of neutrophil proteases are most probably related to their local concentrations. During massive neutrophil-mediated inflammation excessive release of neutrophil proteases will occur and incur injury due to the destructive properties of these proteases. Cultured endothelial phospholipase a2 inhibitor from umbilical veins and the glomerular microvasculature release ULVWF (Dong et al., 2002). The release of VWF strings from histamine-stimulated PGECs to which platelets bound, confirmed this finding in this study. Elastase alone, and within the GBM, was shown to cleave the VWF-platelet strings, as was also demonstrated for rADAMTS13. ADAMTS13 cleaves VWF at the Y1605-M1606 bond resulting in 176 and 140kDa fragments (Dent et al., 1990; Furlan et al., 1996). Similarly, elastase and PR3 were shown to cleave VWF at the V1607-T1608 bond, cathepsin G at Y1605-M1606 and MMP9 at the M1606-V1607 cleavage site (Raife et al., 2009). GBM samples incubated with exogenous VWF generated similar cleavage fragments in the current study. The findings suggest that neutrophil proteases cleave large VWF multimers and could do so within the glomerulus. The GBM possesses a unique position between the fenestrated glomerular endothelium and podocytes allowing it to function as part of the filtration barrier and as a recipient of factors secreted from both adjacent cells. Interestingly, ADAMTS13 is present within the GBM, as we have demonstrated previously (Manea et al., 2007) and in the current study, but apparently does not contribute to VWF cleavage. ADAMTS13 is a 190kDa protein synthesized in both glomerular endothelial cells (Tati et al., 2011) and podocytes (Manea et al., 2007) both of which contribute to the constitution of the GBM. ADAMTS13 in the GBM would most probably originate from podocytes, as secretion from glomerular endothelial cells is apical (Shang et al., 2006). Podocyte-derived growth factors such as vascular endothelial-derived factor and angiopoietin-1 have been shown to affect glomerular endothelial cells (Eremina et al., 2006; Fogo and Kon, 2010; Haraldsson and Nystrom, 2012; Satchell et al., 2004). Interactions between podocyte-derived factors and the glomerular endothelium have been suggested to occur by transfer of substances counter to the direction of filtration via sub-podocyte spaces (Fogo and Kon, 2010; Salmon et al., 2009). Thus the ADAMTS13 present in the GBM could originate from podocytes although it is unclear if such a large protein could transfer through the GBM and cleave VWF at the GBM-endothelial interface. The lack of ADAMTS13 VWF-cleaving activity in our GBM preparations may be due to inherently low levels in the GBM, alternatively to the process of GBM isolation or the relative concentration of proteins remaining in the GBM after storage. Nevertheless, the results indicate that there are additional VWF-cleaving proteases within the GBM and that these proteases can replace the function of ADAMTS13 at this site.