Debates of the European Parliament. Report of Proceedings from 15 to 19 September 1980. No. 1-260. 1980-1981 Session

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K(D) of 0.11 ± 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg(506) and not Arg(306) and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg(506) cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg(506) interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation

Identification of novel small molecule inhibitors of activated protein C

Introduction: Activated protein C (APC) is the central enzyme of the anticoagulant protein C pathway. Low concentrations of APC circulate in plasma and are believed to contribute to the maintenance of a normal haemostatic balance. Materials and Methods: We have used a structure-based virtual screening approach to discover small drug-like molecules that inhibit the interaction between APC and its substrate FVa through inhibition of a predominant APC exosite, known to be involved in FVa substrate binding. We have combined in silico selection with functional screening and direct binding analysis to identify novel molecules and to ascertain and characterize the inhibition of the interaction between APC and FVa. Results: We have identified a number of novel molecules that bind to APC and protein C with Kd values in the range of 10(-3)-10(-5) M. Inhibition by these molecules is incomplete, which most likely reflects the extended surface that is involved in the interaction between APC and its substrates. Direct binding of hit molecules to variant APC molecules that were mutated in the targeted binding site revealed that several of the molecules presented a 100-500 fold lower affinity for the variant molecule, suggesting that these molecules indeed bind the exosite of APC. Conclusions: The protein-protein interaction inhibitors discovered here, could function as starting molecules for further development of small molecules with anti-APC properties. Such molecules may be of clinical interest, in particular in individuals where thrombin formation is compromised and the haemostatic balance is tipped towards bleeding tendencies, such as in haemophilia A

The structure-function relationship of activated protein C Lessons from natural and engineered mutations

Protein C is the central enzyme of the natural anticoagulant pathway and its activated form APC (activated protein C) is able to proteolyse non-active as well as active coagulation factors V and VIII. Proteolysis renders these cofactors inactive, resulting in an attenuation of thrombin formation and overall down-regulation of coagulation. Presences of the APC cofactor, protein S, thrombomodulin, endothelial protein C receptor and a phospholipid surface are important for the expression of anticoagulant APC activity. Notably, APC also has direct cytoprotective effects on cells: APC is able to protect the endothelial barrier function and expresses anti-inflammatory and anti-apoptotic activities. Exact molecular mechanisms have thus far not been completely described but it has been shown that both the protease activated receptor 1 and EPCR are essential for the cytoprotective activity of APC. Recently it was shown that also other receptors like sphingosine 1 phosphate receptor 1, Cd11b/CD18 and tyrosine kinase with immunoglobulin-like and EGF-like domains 2 are likewise important for APC signalling. Mutagenesis studies are being performed to map the various APC functions and interactions onto its 3D structure and to dissect anticoagulant and cytoprotective properties. The results of these studies have provided a wealth of structure-function information. With this review we describe the state-of-the-art of the intricate structure-function relationships of APC, a protein that harbours several important functions for the maintenance of both humoral and tissue homeostasi

Design and characterization of novel activated protein C variants for the proteolysis of cytotoxic extracellular histone H3

BACKGROUND: Extracellular histone H3 is implicated in several pathologies including inflammation, cell death, and organ failure. Neutralization of histone H3 is a strategy that was shown beneficial in various diseases, such as rheumatoid arthritis, myocardial infarction, and sepsis. It was shown that activated protein C (APC) can cleave histone H3, which reduces histone cytotoxicity. However, due to the anticoagulant properties of APC, the use of APC is not optimal for the treatment of histone-mediated cytotoxicity, in view of its associated bleeding side effects. OBJECTIVES: This study aimed to investigate the detailed molecular interactions between human APC and human histone H3, and subsequently use molecular docking and molecular dynamics simulation methods to identify key interacting residues that mediate the interaction between APC and histone H3 and to generate novel optimized APC variants. METHODS: After molecular simulations, the designed APC variants 3D2D-APC (Lys37-39Asp and Lys62-63Asp) and 3D2D2A-APC (Lys37-39Asp, Lys62-63Asp, and Arg74-75Ala) were recombinantly expressed and their abilities to function as anticoagulant, to bind histones, and to cleave histones were tested and correlated with their cytoprotective properties. RESULTS: Compared with wild type-APC, both the 3D2D-APC and 3D2D2A-APC variants showed a significantly decreased anticoagulant activity, increased binding to histone H3, and similar ability to proteolyze histone H3. CONCLUSIONS: Our data show that it is possible to rationally design APC variants that may be further developed into therapeutic biologicals to treat histone-mediated disease, by proteolytic reduction of histone-associated cytotoxic properties that do not induce an increased bleeding risk

Extracellular histone H3 levels are inversely correlated with antithrombin levels and platelet counts and are associated with mortality in sepsis patients

Sepsis is a leading cause of death worldwide. Extracellular histones are cytotoxic compounds mediating death in murine sepsis and circulating nucleosome levels predict mortality in human inflammation and sepsis. Whether or not circulating extracellular histone H3 correlates with other plasma parameters and/or ICU scoring systems has not been completely established, nor if levels of circulating extracellular histones can be used as predictive markers for clinical outcome in sepsis. We measured plasma histone H3 (H3) levels in the plasma of 43 sepsis patients who were admitted to the Intensive Care Unit and determined their correlation with disease severity, organ failure, mortality and coagulation- and tissue homeostasis parameters including LDH levels, thrombin potential (ETP), prothrombin levels, antithrombin levels and platelet counts. Median H3 levels of sepsis patients at the ICU were significantly increased in non-survivors as compared to survivors with levels found being 3.15μg/ml versus 0.57μg/ml respectively, P=0.04. H3 levels are positively correlated with lactate dehydrogenase (LDH) activity (Spearman's rho=0.49, P <0.001), and negatively correlated with antithrombin levels (rho=-0.34, P=0.027) and platelet counts (rho=-0.33, P=0.031). We conclude that circulating H3 levels correlate with mortality in sepsis patients and inversely correlate with antithrombin levels and platelet count

Effects of Exogenous Recombinant APC in Mouse Models of Ischemia Reperfusion Injury and of Atherosclerosis

<div><p>Activated protein C (APC) is a serine protease that has both anticoagulant and cytoprotective properties. The cytoprotective effects are protease activated receptor 1 (PAR-1) and endothelial protein C receptor (EPCR) dependent and likely underlie protective effects of APC in animal models of sepsis, myocardial infarction and ischemic stroke. S360A-(A)PC, a variant (A)PC that has no catalytic activity, binds EPCR and shifts pro-inflammatory signaling of the thrombin-PAR-1 complex to anti-inflammatory signaling. In this study we investigated effects of human (h)wt-PC, hS360A-PC, hwt-APC and hS360A-APC in acute (mouse model of acute myocardial ischemia/reperfusion (I/R) injury) and chronic inflammation (apoE^−/− mouse model of atherosclerosis). All h(A)PC variants significantly reduced myocardial infarct area (p<0.05) following I/R injury. IL-6 levels in heart homogenates did not differ significantly between sham, placebo and treatment groups in I/R injury. None of the h(A)PC variants decreased number and size of atherosclerotic plaques in apoE^−/− mice. Only hS360A-APC slightly affected phenotype of plaques. IL-6 levels in plasma were significantly (p<0.001) decreased in hwt-APC and hS360A-PC treated mice. In the last group levels of monocyte chemotactic protein 1 (MCP-1) were significantly increased (p<0.05). In this study we show that both hwt and hS360A-(A)PC protect against acute myocardial I/R injury, which implies that protection from I/R injury is independent of the proteolytic activity of APC. However, in the chronic atherosclerosis model hwt and hS360-(A)PC had only minor effects. When the dose, species and mode of (A)PC administration will be adjusted, we believe that (A)PC will have potential to influence development of chronic inflammation as occurring during atherosclerosis as well.</p></div

Only S360A-APC treatment influences plaque phenotype.

<p>Images of plaques stained for collagen (<b>A</b>), macrophages (<b>B</b>), leukocytes (<b>C</b>), and T cells (<b>D</b>). <b>E)</b> After sirius red staining, the percentage of collagen was determined by dividing the area of collagen by the total plaque area. <b>F)</b> After Mac-3 staining, the percentage of macrophages as a percentage of the total number of cells per plaque was calculated. <b>G)</b> After CD45 staining, the percentage of leukocytes as a percentage of the total number of cells per plaque was calculated. <b>H)</b> After CD3 staining, the percentage of T cells of the total number of cells per plaque was calculated. All graphs show mean +/− SEM, statistical significance was tested using one-way analysis of variance with Dunnett post hoc test, *P<0.05, **P<0.01.</p

Treatment with wt- or S360A-(A)PC does not alter plaque area or # of plaques.

<p>ApoE^−/− mice were injected with saline or 0.2 mg/kg of wt-PC, S360A-PC, wt-APC or S360A-APC. Images of an initial (<b>A</b>) and an advanced (<b>B</b>) plaque after hematoxylin and eosin (H&E) staining. <b>C)</b> Atherosclerotic lesions were classified as initial (black bars) or advanced (white bars) and numbers of both type of plaques per mouse were determined. <b>D)</b> Total plaque area in the aortic arch as determined by H&E staining. <b>E)</b> Surface area of individual initial plaques. <b>F)</b> Surface area of individual advanced plaques. All graphs show mean +/− SEM, statistical significance was tested using one-way analysis of variance with Dunnett post hoc test, *P<0.05.</p

Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis

Extracellular histones are considered to be major mediators of death in sepsis. Although sepsis is a condition that may benefit from low-dose heparin administration, medical doctors need to take into consideration the potential bleeding risk in sepsis patients who are already at increased risk of bleeding due to a consumption coagulopathy. Here, we show that mechanisms that are independent of the anticoagulant properties of heparin may contribute to the observed beneficial effects of heparin in the treatment of sepsis patients. We show that nonanticoagulant heparin, purified from clinical grade heparin, binds histones and prevents histone-mediated cytotoxicity in vitro and reduces mortality from sterile inflammation and sepsis in mouse models without increasing the risk of bleeding. Our results demonstrate that administration of nonanticoagulant heparin is a novel and promising approach that may be further developed to treat patients suffering from sepsi

Treatment with wt- or S360A-(A)PC decreases infarct area.

<p>Mice were subjected to 1(0.4 mg/kg) or placebo (saline) were administered i.v. 15 min after induction of ischemia and a second time 5 minutes after induction of reperfusion. <b>A)</b> Representative picture of the Evans Blue/TTC staining after 2 h reperfusion. The normal tissue (non infarcted area) is stained dark blue, whereas the ischemic area (area at risk (AAR)) is stained brick red and the non-viable infarct area (area of infarction (AOI)) did not stain and remained pale. <b>B)</b> Infarct area (percentage AOI of AAR) as determined by Evans Blue/TTC staining. <b>C)</b> IL-6 concentrations were determined in heart tissue homogenates (5 mg/ml protein content) by ELISA and IL-6 per mg heart homogenate was calculated. All graphs show mean +/− SEM, statistical significance was tested using one-way analysis of variance with Dunnett post hoc test, ***P<0.001.</p