Thrombin Activation of Protein C Requires Prior Processing by a Liver Proprotein Convertase [Metabolism]

Protein C, a secretory vitamin K-dependent anticoagulant serine protease, inactivates factors Va/VIIIa. It is exclusively synthesized in liver hepatocytes as an inactive zymogen (proProtein C). In human, thrombin cleavage of the propeptide at PR221↓ results in activated Protein C (APC; residues 222-461). However, the propeptide is also cleaved by a furin-like proprotein convertase(s) at KKRSHLKR199↓, but the order of cleavage is unknown. Herein, we present evidence that at the cell surface of Cos-1 cells, mouse proProtein C is first cleaved by the convertases furin, PC5/6A and PACE4. In mouse, this cleavage occurs at the equivalent site KKRKILKR198↓, and requires the presence of Arg198 at P1, and a combination of two other basic residues at either P2 (Lys197), P6 (Arg193) or P8 (Lys191) positions. Notably, the thrombin-resistant R221A mutant is still cleaved by these convertases, revealing that convertase cleavage can precede thrombin activation. This conclusion was supported by the fact that the APC-specific activity in the medium of Cos-1 cells is exclusively dependent on prior cleavage by the convertases, as both R198A and R221A lack Protein C activity. Primary cultures of hepatocytes derived from wild type, or hepatocyte-specific furin, PC5/6 or complete PACE4 knockout mice suggested that the cleavage of overexpressed proProtein C is predominantly performed by furin intracellularly, and by all three PCs at the cell surface. Indeed, plasma analyses of single PC-knockout mice showed that loss of the convertases furin or PC5/6 in hepatocytes results in a ~30% decrease in APC levels, with no significant contribution from PACE4. We conclude that prior convertase-cleavage of Protein C in hepatocytes is critical for its thrombin-activation.

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2qtU5IO
via IFTTT