Prolylcarboxypeptidase Deficiency Is a Risk Factor for Arterial Thrombosis and Hypertension

Prolylcarboxypeptidase (PRCP), an S28 serine protease, degrades bradykinin, angiotensin II, and alpha melanocyte stimulating hormone and activates prekallikrein to plasma kallikrein (Blood 103:4554, 2004). In GWAS, it has been recognized as a risk factor for metabolic syndrome, hypertension, and pre-eclampsia. We postulated that PRCP murine hypomorphs (PRCPgt/gt) have a cardiovascular phenotype.

PRCP is mostly found in kidney in proximal tubules. In arteries, it is found both on endothelium and in media. A gene-trap murine hypomorph was created with 7% mRNA and 23% PRCP antigen in renal tissue. Using the Rose Bengal carotid artery thrombosis models, PRCPgt/gt mice had shorter carotid artery occlusion times (24±3 min [mean±SD]) compared to wild type (52±8 min). On a 4% ferric chloride carotid artery thrombosis assay PRCPgt/gt occluded in 21±8 min whereas wild type do not occlude at 60 min. Pharmacologic inhibition of PRCP with Z-pro-prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-Chloromethylketone or PKSI-572 in 3 mouse strains also shortened the time to carotid artery occlusion. PRCPgt/gt were constitutively hypertensive during the late night cycle (122±5 mm Hg mean arterial pressure vs 114±6 mm Hg for wildtype) as measured by carotid artery telemetry. Treatment of these animals with the mitochondria specific antioxidant mitoTEMPO significantly reduced (113±7 mm Hg) the elevated BP. Plasma angiotensin II and bradykinin levels were unaltered in PRCPgt/gt. PRCPgt/gt plasma had a significant increase in contact activation-induced thrombin generation. Aortic and renal reactive oxygen species (ROS) were increased (3.2-fold and 2.8-fold, respectively) in PRCPgt/gt mice as determined by dihydroethidium (DHE) fluorescence. PRCPgt/gt aortic and renal superoxide measured by lucigenin luminescence also was increased (1.6-fold and 1.7-fold, respectively). In PRCPgt/gt kidneys Amplex Red fluorescence, a measure of hydrogen peroxide, was increased 2.4-fold. Renal tissue had 1.6-fold increased uncoupled eNOS on SDS-PAGE. Arterial occlusion times in PRCPgt/gt were corrected by treatment with antioxidant apocynin or tempol. PRCP siRNA knockdowns in HUVEC or mesenchymal embryonic fibroblasts prepared from PRCPgt/gt embryos had increased constitutive DHE fluorescent ROS (2.1-fold and 1.4-fold, respectively). PRCPgt/gt aorta had decreased expression of Kruppel-like factors 2 and 4, thrombomodulin and eNOS. Moreover, PRCP knockdowns in HUVEC had 36% reduced eNOS mRNA expression.

These investigations indicate that PRCP is a specific gene/protein target for arterial thrombosis risk and hypertension. Its presence modulates constitutive cell and tissue ROS. Arterial thrombosis risk is related to effect of ROS on endothelial cell anticoagulant mechanisms.

No relevant conflicts of interest to declare.