Coagulation protease signaling via protease-activated receptors (PARs) is essential for maintenance of cellular homeostasis. Perturbed or aberrant activation of protease-dependent signaling via PARs propagates inflammation and pathological responses in disease models such as sepsis, neurological diseases and metabolic diseases including atherosclerosis, obesity and diabetic nephropathy (dNP). Disruption of protease-activated protein C (aPC) signaling in renal epithelial cells, i.e. podocytes, compromises adaptive endoplasmic reticulum (ER) signaling, promoting maladaptive ER-stress and ultimately dysfunction of the glomerular filtration barrier and dNP. While these results demonstrate that aPC-dependent ER-reprogramming is an essential component of its cytoprotective effect, currently the co-receptors that are involved in mediating aPC's cytoprotective effects in podocytes remain unclear. In endothelial cells, endothelial protein C receptor (EPCR) facilitates the binding and alternative proteolytic cleavage of PAR1 propagating biased anti-inflammatory and cytoprotective effects. Intriguingly, unlike in endothelial cells, podocytes do not express EPCR and the cytoprotective effects of aPC requires PAR3/PAR2 heterodimer (human podocytes) and PAR3/PAR1 heterodimer (mouse podocytes). Podocytes express integrins β1 and β3, which could potentially mediate biased protease signaling.


To characterize the receptor-dependent mechanism through which aPC-mediates its cytoprotective effects in podocytes, we generated transgenic mice that expresses constitutively higher levels of wild type-human aPC (APChigh mice) and integrin binding deficient mutant (RGE-APChigh mice). Additionally, the receptor-dependent mechanisms in dNP were evaluated in mouse models with podocyte specific genetic deletion of the integrin β3. After 26 weeks of persistent hyperglycaemia (streptozotocin-induced diabetes), markers of dNP were determined. In wild type diabetic mice, integrin β3 antagonist Cyclo-RGDfv and PAR1 antagonist (SCH79797) was administered to pharmacologically inhibit integrin and PAR1 signaling, respectively.


In vitro data show that integrin αvβ3 in podocytes controls protease-dependent spatial and temporal regulation of PAR signaling by aPC. This protease binds to integrin αvβ3 and temporally regulates PAR signaling. Abolishing aPC-αvβ3 interaction or knockdown of αvβ3 integrin perturbs endosomal trafficking of PARs resulting in aberrant cell-disruptive signaling. Likewise, in vivo overexpression of integrin binding deficient aPC-mutant or podocyte-specific genetic deletion of integrin β3 abrogates aPC-dependent cytoprotective effects in a mouse model of dNP. Akin to wild type aPC, pharmacological inhibition of aberrant PAR1 signaling protects against dNP. Corroborating our results in murine dNP, analysis of human dNP samples revealed increased expression and activation of αvβ3 associated with altered PAR receptor localization specifically within the podocytes.


Our results show that aPC-αvβ3 interaction on podocytes modulates endosomal trafficking of PAR receptors and that loss of this interaction drives aberrant protease-dependent signaling and pathological podocyte migration. Complementary in vivo studies in newly developed integrin binding deficient aPC mutant mice demonstrate that integrin αvβ3 ligation is essential for nephroprotective aPC signaling. These results identify integrin αvβ3 as a rheostat that provides temporal and spatial regulation of PAR signaling in diabetic disease.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.