Thrombin activation of protein C requires prior processing by a liver proprotein convertase

Abstract

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 humans, thrombin cleavage of the propeptide at PR↓ results in activated protein C (APC; residues 222-461). However, the propeptide is also cleaved by a furin-like proprotein convertase(s) (PCs) at KSHL↓ (underlined basic residues critical for the recognition by PCs), but the order of cleavage is unknown. Herein, we present evidence that at the surface of COS-1 cells, mouse proprotein C is first cleaved by the convertases furin, PC5/6A, and PACE4. In mice, this cleavage occurs at the equivalent site, KKIL↓, and requires the presence of Arg at P1 and a combination of two other basic residues at either P2 (Lys), P6 (Arg), or P8 (Lys) positions. Notably, the thrombin-resistant R221A mutant is still cleaved by these PCs, 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, because 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 knock-out mice suggested that the cleavage of overexpressed proprotein C is predominantly performed by furin intracellularly and by all three proprotein convertases at the cell surface. Indeed, plasma analyses of single-proprotein convertase-knock-out mice showed that loss of the convertase 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.

Protease-Activated Receptor 1 Contributes to Microcirculation Failure and Tubular Damage in Renal Ischemia-Reperfusion Injury in Mice

“…In our study, a possible explanation for its attributed role was that PAR-1 was activated by filtered thrombin. The size of protein C is over 60 kDa, which is similar to that of albumin [ 22 , 23 ] and much bigger than that of thrombin (36 kDa). Furthermore, its plasma concentration (<100 nM) is less than 10% of thrombin (>1 μ M), indicating that protein C undergoes less filtration and produced a weaker signal compared to thrombin.…”

Comprehensive Cell Type Specific Transcriptomics of the Human Kidney

“…Interestingly, we found that PT cells expressed plasma protein encoding genes (ALB, RBP4, GC, and PROC), which are reported to be synthesized and secreted by the liver [15][16][17] . Urinary ALB and RBP4 are biomarkers of albuminuria and CKD (nephritis) and renal Fanconi syndrome, respectively 10,17 .…”

“…ALB, RBP4, GC and PROC), normally thought to be liver-derived, were expressed in PT ( Supplementary Fig. 3) [15][16][17] .…”

Can adjunctive therapies augment the efficacy of endovascular thrombolysis? A potential role for activated protein C

“…PC circulates at 70 nM in the blood as an inactive zymogen of the natural anticoagulant serine protease APC (Gruber and Griffin, 1992; Mosnier et al, 2007). PC binds to the endothelial cell protein C receptor (EPCR) at the endothelial cell surface (Fukudome and Esmon, 1994) and is activated by thrombomodulin (TM) receptor-bound thrombin (IIa) by cleavage at Arg169 and removal of a peptide fragment at the amino-terminal of PC heavy chain (Esmon, 2003; Essalmani et al, 2017) (Fig. 1).…”