Alireza R. Rezaie

Protein C Inhibitor Is a Potent Inhibitor of the Thrombin-Thrombomodulin Complex

Protein C inhibitor (PCI), a plasma serine protease inhibitor, inhibits several proteases including the anticoagulant enzyme, activated protein C (APC), and the coagulation enzymes, thrombin and factor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin accelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates inhibition of thrombin by PCI ≈140-fold (k = 2.4 × 10 in the presence of TM compared to 1.7 × 10M s in the absence of TM). Most of this effect is mediated by protein-protein interactions since the active fragment of TM composed of epidermal growth factor-like domains 4-6 (TM 4-6) accelerates inhibition by PCI ≈59-fold (k = 1.0 × 10M s). The mechanism by which TM alters reactivity with PCI appears to reside in part in an alteration of the S2 specificity pocket. Replacing Phe with Pro at the P2 position in the reactive loop of PCI yields a mutant that inhibits thrombin better in the absence of TM (k = 6.3 × 10M s), but TM 4-6 enhances inhibition by this mutant ≈9-fold (k = 5.8 × 10M s) indicating that TM alleviates the inhibitory effect of the less favored Phe residue. These results indicate that PCI is a potent inhibitor of the protein C anticoagulant pathway at the levels of both zymogen activation and enzyme inhibition.

Identification and Characterization of the Sodium-binding Site of Activated Protein C

Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221–225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221–225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (K d (app)) = 44.1 ± 8.6 mm. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5–6-fold withK d (app) = 2.3 ± 0.3 mm. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221–225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.

Activated Protein C Attenuates Severe Inflammation by Targeting VLA-3high Neutrophil Subpopulation in Mice

The host injury involved in multiorgan system failure during severe inflammation is mediated, in part, by massive infiltration and sequestration of hyperactive neutrophils in the visceral organ. A recombinant form of human activated protein C (rhAPC) has shown cytoprotective and anti-inflammatory functions in some clinical and animal studies, but the direct mechanism is not fully understood. Recently, we reported that, during endotoxemia and severe polymicrobial peritonitis, integrin VLA-3 (CD49c/CD29) is specifically upregulated on hyperinflammatory neutrophils and that targeting the VLA-3high neutrophil subpopulation improved survival in mice. In this article, we report that rhAPC binds to human neutrophils via integrin VLA-3 (CD49c/CD29) with a higher affinity compared with other Arg-Gly-Asp binding integrins. Similarly, there is preferential binding of activated protein C (PC) to Gr1highCD11bhighVLA-3high cells isolated from the bone marrow of septic mice. Furthermore, specific binding of rhAPC to human neutrophils via VLA-3 was inhibited by an antagonistic peptide (LXY2). In addition, genetically modified mutant activated PC, with a high affinity for VLA-3, shows significantly improved binding to neutrophils compared with wild-type activated PC and significantly reduced neutrophil infiltration into the lungs of septic mice. These data indicate that variants of activated PC have a stronger affinity for integrin VLA-3, which reveals novel therapeutic possibilities.

Whole-exome sequencing in evaluation of patients with venous thromboembolism

Genetics play a significant role in venous thromboembolism (VTE), yet current clinical laboratory-based testing identifies a known heritable thrombophilia (factor V Leiden, prothrombin gene mutation G20210A, or a deficiency of protein C, protein S, or antithrombin) in only a minority of VTE patients. We hypothesized that a substantial number of VTE patients could have lesser-known thrombophilia mutations. To test this hypothesis, we performed whole-exome sequencing (WES) in 64 patients with VTE, focusing our analysis on a novel 55-gene extended thrombophilia panel that we compiled. Our extended thrombophilia panel identified a probable disease-causing genetic variant or variant of unknown significance in 39 of 64 study patients (60.9%), compared with 6 of 237 control patients without VTE (2.5%) (P < .0001). Clinical laboratory-based thrombophilia testing identified a heritable thrombophilia in only 14 of 54 study patients (25.9%). The majority of WES variants were either associated with thrombosis based on prior reports in the literature or predicted to affect protein structure based on protein modeling performed as part of this study. Variants were found in major thrombophilia genes, various SERPIN genes, and highly conserved areas of other genes with established or potential roles in coagulation or fibrinolysis. Ten patients (15.6%) had >1 variant. Sanger sequencing performed in family members of 4 study patients with and without VTE showed generally concordant results with thrombotic history. WES and extended thrombophilia testing are promising tools for improving our understanding of VTE pathogenesis and identifying inherited thrombophilias.

TR47, a PAR1-based peptide, inhibits melanoma cell migration in vitro and metastasis in vivo

Activated Protein C (APC) is a serine-protease that displays antithrombotic and anti-inflammatory properties. In addition, cleavage of protease-activated receptor 1 (PAR1) by APC exerts endothelial cytoprotective actions. The effects of APC on endothelial cells may be reproduced by TR47, a PAR1-based peptide that mimics the novel N-terminus of PAR1 generated upon cleavage at Arg-46 by APC. In this study we demonstrate that wild-type APC and its signaling-proficient mutant, APC-2Cys (which has dramatically reduced anticoagulant activity), display similar inhibitory effects towards the transendothelial migration of A375 human melanoma cells. Consistent with this observation, APC and APC-2Cys significantly reduced the inxa0vivo metastatic potential of the B16F10 murine melanoma cells. TR47 recapitulated the inxa0vitro and inxa0vivo protective profiles of APC and APC-2Cys. Treatment of EA.hy926 endothelial cells with TR47 (20xa0μM) significantly decreased the A375xa0cell migration. In addition, treatment of C57/BL6 mice with a single TR47 dose (125 μg/animal) strongly reduced the metastatic burden of B16F10xa0cells. Together, our results suggest that protection of the endothelial barrier by APC/TR47-mediated signaling pathways might be a valuable therapeutic approach to prevent metastasis.

Activated protein C protects against pressure overload-induced hypertrophy through AMPK signaling

We found that the anticoagulant plasma protease, activated protein C (APC), stimulates the energy sensor kinase, AMPK, in the stressed heart by activating protease-activated receptor 1 (PAR1) on cardiomyocytes. Wild-type (WT) and AMPK-kinase dead (KD) transgenic mice were subjected to transverse aortic constriction (TAC) surgery. The results demonstrated that while no phenotypic differences can be observed between WT and AMPK-KD mice under normal physiological conditions, AMPK-KD mice exhibit significantly larger hearts after 4 weeks of TAC surgery. Analysis by echocardiography suggested that the impairment in the cardiac function of AMPK-KD hearts is significantly greater than that of WT hearts. Immunohistochemical staining revealed increased macrophage infiltration and ROS generation in AMPK-KD hearts after 4 weeks of TAC surgery. Immunoblotting results demonstrated that the redox markers, pShc66, 4-hydroxynonenal and ERK, were all up-regulated at a higher extent in AMPK-KD hearts after 4 weeks of TAC surgery. Administration of APC-WT and the signaling selective APC-2Cys mutant, but not the anticoagulant selective APC-E170A mutant, significantly attenuated pressure overload-induced hypertrophy and fibrosis. Macrophage infiltration and pShc66 activation caused by pressure overload were also inhibited by APC and APC-2Cys but not by APC-E170A. Therefore, the cardiac AMPK protects against pressure overload-induced hypertrophy and the signaling selective APC-2Cys may have therapeutic potential for treating hypertension-related hypertrophy without increasing the risk of bleeding.