Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli – An Anti-thrombotic Mechanism in the Kidney

Abstract

Adequate cleavage of von Willebrand factor (VWF) prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM) was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3), cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

Contribution of ADAMTS13‐independent VWF regulation in sickle cell disease

“…25 Leukocyte-derived enzymes within the subendothelial glomerular space may help to maintain the balance of pro-thrombotic and anti-thrombotic forces within the kidney. 31 However, to our knowledge, data generated from this SCD cohort are the first demonstrations of ADAMTS13-independent cleavage contributing presented here should therefore be interpreted within the context of this population and we are currently looking to replicate these findings within independent SCD cohorts. Nonetheless, given that leukocyte and endothelial activation appear to be fundamental components of SCD pathophysiology, we anticipate that these findings could be more applicable to the general SCD population.…”

“…Neutrophil proteases have shown capacity to cleave VWF in vitro 25 . Leukocyte‐derived enzymes within the subendothelial glomerular space may help to maintain the balance of pro‐thrombotic and anti‐thrombotic forces within the kidney 31 . However, to our knowledge, data generated from this SCD cohort are the first demonstrations of ADAMTS13‐independent cleavage contributing to VWF regulation in a specific patient population.…”

“…Given the assay‐discrepant findings of ADAMTS13 activity, we hypothesized that additional proteases may be present in SCD plasma with capacity for VWF cleavage. Prior studies have shown that neutrophil enzymes are able to cleave VWF in vitro 25,31 . Knowing that neutrophil activation is pervasive in SCD, 32,33 we measured levels of three neutrophil proteases within SCD plasma: polymorphonuclear (PMN) elastase, proteinase 3 (PR3), and matrix metalloproteinase‐9 (MMP‐9).…”

“…Prior studies have shown that neutrophil enzymes are able to cleave VWF in vitro. 25,31 Knowing that neutrophil activation is pervasive in SCD, 32,33 we measured levels of three neutrophil proteases within SCD plasma: polymorphonuclear (PMN) elastase, proteinase 3 (PR3), and matrix metalloproteinase-9 (MMP-9). Levels of MMP-9 and PR3 measured by ELISA were elevated in SCD plasma relative to controls.…”

Activity-based protein profiling guided identification of urine proteinase 3 activity in subclinical rejection after renal transplantation

“…PR3/PRTN3 can also proteolytically activate TNF-α and extracellular IL-1β [43,44]. It has been suggested that PR3/PRTN3 activity may play a protective role in the glomerular basement membrane by cleavage of von Willebrand factor helping to prevent thrombus formation [45]. There is also some evidence to suggest that PR3/PRTN3 activity may contribute to the pathology of other disease states [41,46].…”