The Protein C Pathway

doi:10.1378/chest.124.3_suppl.26S

Chest

Volume 124, Issue 3, Supplement, September 2003, Pages 26S-32S

https://doi.org/10.1378/chest.124.3_suppl.26S Get rights and content

The protein C anticoagulant pathway serves as a major system for controlling thrombosis, limiting inflammatory responses, and potentially decreasing endothelial cell apoptosis in response to inflammatory cytokines and ischemia. The essential components of the pathway involve thrombin, thrombomodulin, the endothelial cell protein C receptor (EPCR), protein C, and protein S. Thrombomodulin binds thrombin, directly inhibiting its clotting and cell activation potential while at the same time augmenting protein C (and thrombin activatable fibrinolysis inhibitor [TAFI]) activation. Furthermore, thrombin bound to thrombomodulin is inactivated by plasma protease inhibitors > 20 times faster than free thrombin, resulting in increased clearance of thrombin from the circulation. The inhibited thrombin rapidly dissociates from thrombomodulin, regenerating the anticoagulant surface. Thrombomodulin also has direct anti-inflammatory activity, minimizing cytokine formation in the endothelium and decreasing leukocyte-endothelial cell adhesion. EPCR augments protein C activation approximately 20-fold in vivo by binding protein C and presenting it to the thrombin-thrombomodulin activation complex. Activated protein C (APC) retains its ability to bind EPCR, and this complex appears to be involved in some of the cellular signaling mechanisms that down-regulate inflammatory cytokine formation (tumor necrosis factor, interleukin-6). Once APC dissociates from EPCR, it binds to protein S on appropriate cell surfaces where it inactivates factors Va and VIIIa, thereby inhibiting further thrombin generation. Clinical studies reveal that deficiencies of protein C lead to microvascular thrombosis (purpura fulminans). During severe sepsis, a combination of protein C consumption, protein S inactivation, and reduction in activity of the activation complex by oxidation, cytokine-mediated down-regulation, and proteolytic release of the activation components sets in motion conditions that would favor an acquired defect in the protein C pathway, which in turn favors microvascular thrombosis, increased leukocyte adhesion, and increased cytokine formation. APC has been shown clinically to protect patients with severe sepsis. Protein C and thrombomodulin are in early stage clinical trials for this disease, and each has distinct potential advantages and disadvantages relative to APC.

Section snippets

The Activation Complex

The key feature of the protein C pathway resides in the ability of the pathway to respond to the presence of thrombin. As the thrombin concentration rises, much of the thrombin binds to thrombomodulin, primarily on the endothelial cell surface (Fig 1), leading to the activation of protein C.

Protein C activation is enhanced approximately 20-fold in vivo when protein C is bound to the endothelial cell protein C receptor (EPCR).² Relative to free thrombin, thrombin bound to thrombomodulin is

Other Functions of the Activation Complex

Thrombomodulin is a complex molecule with multiple distinct domains (Fig 2). Different functions have been ascribed to these domains. The N-terminal domain has sequence similarity to the lectins, which are carbohydrate-binding proteins, and is not involved in protein C activation. The lectin domain has been shown to have anti-inflammatory activity.¹⁰ Whether infused, added as the soluble isolated domain, or present on cellular thrombomodulin (ie, native), the presence of the domain

Functions of APC

In addition to inactivating factors Va and VIIIa (Fig 1),²⁴²⁵ APC has been shown to have anti-inflammatory, antiapoptotic, and anticoagulant activity at the cellular level. The antiapoptotic activity appears to be mediated, at least in part, by the APC-EPCR complex cleaving PAR-1. Since EPCR is expressed primarily on endothelium,²⁶ but PAR is located on many cells, the EPCR dependence probably allows for preferential activation of this receptor on endothelium,⁵ thereby avoiding platelet and

Physiology of the Protein C Pathway

Thrombomodulin is located on the endothelium; in general, the number of copies per endothelial cell appear by immunohistochemistry to vary < 10-fold among vascular beds (the brain microcirculation being an exception, where thrombomodulin expression is very low).³¹ From simple geometry, the endothelial cell to blood volume ratio increases hundreds of fold as blood moves from the large vessels to the capillaries. Assuming 100,000 copies of thrombomodulin per endothelial cell, a reasonable

Down-regulation

Both thrombomodulin and EPCR can be down-regulated at the transcriptional level by inflammatory cytokines like IL-1β and TNF-α.¹⁶³⁸ In addition, thrombomodulin activity can be dramatically reduced by oxidants released from leukocytes.³⁹ Finally, leukocyte elastase rapidly releases soluble forms of thrombomodulin⁴⁰ that have considerably less activity than the cellular form because they no longer have the EPCR acceleration effect and they do not contain the chondroitin for high-affinity thrombin

The Protein C Pathway in Sepsis

The combined anticoagulant and anti-inflammatory activities of APC have been recognized for a long time as an attractive combination for treating severe sepsis. In initial studies in baboons, APC was shown to prevent death from infusion of normally lethal concentrations of E coli. APC infusion was associated with a reduced drop in BP, decreased markers of inflammation, and a decreased coagulant response.⁴⁶ When endogenous protein C activation was prevented, the animals responded to low-level E

Summary

The protein C pathway is important for the negative control of both coagulation and inflammation. The recently described inhibition of apoptosis by APC may be particularly important in the treatment of ischemic diseases. Modulation of the pathway can be therapeutically beneficial. Furthermore, the pathway may be down-regulated in a variety of disease states to such an extent that the down-regulation contributes to the severity of the disease. Future studies will likely provide new methods to

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The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates.
To establish if thrombin can cleave at Trp residues.
The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage.
Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond.
The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.
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Patients with hematological malignancies (HM) frequently present thrombocytopenia and higher risk of bleeding. Although transfusion is associated with higher risk of adverse events and poor outcomes, prophylactic transfusion of platelets is a common practice to prevent hemorrhagic complications. Thromboelastometry has been considered a better predictor for bleeding than isolated platelet counts in different settings. In early stages of sepsis, hypercoagulability may occur due to higher fibrinogen levels.
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We performed a unicentric analytical cross-sectional study with 60 adult patients with HM and severe thrombocytopenia, of whom 30 had sepsis (sepsis group) and 30 had no infections (control group). Coagulation conventional tests and specific coagulation tests, including thromboelastometry, were performed. The main outcome evaluated was MCF.
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Patients with sepsis and HM presented higher concentrations of fibrinogen than uninfected patients, resulting in greater MCF amplitudes even in the presence of thrombocytopenia.
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Sepsis is a systemic syndrome involving physiological, pathological, and biochemical abnormalities precipitated by infection and is a major global public health problem. Endothelial cells (ECs) dysfunction is a major contributor to sepsis-induced multiple organ failure. This bibliometric analysis aimed to identify and characterize the status, evolution of the field, and new research trends of ECs and sepsis over the past 20 years.
For this analysis, the Web of Science Core Collection database was searched to identify relevant publications on ECs in sepsis published between January 1, 2002, and December 31, 2022. Microsoft Excel 2021, VOSviewer software, CiteSpace software, and the online analysis platform of literature metrology (http://bibliometric.com) were used to visualize the trends of publications’ countries/regions, institutions, authors, journals, and keywords.
In total, 4200 articles were identified and screened, primarily originating from 86 countries/regions and 3489 institutions. The USA was the leading contributor to this research field, providing 1501 articles (35.74 %). Harvard University's scientists were the most prolific, with 129 articles. Overall, 21,944 authors were identified, among whom Bae Jong Sup was the most prolific, contributing 129 publications. Additionally, Levi Marcel was the most frequently co-cited author, appearing 538 times. The journals that published the most articles were SHOCK, CRITICAL CARE MEDICINE, and PLOS ONE, accounting for 10.79 % of the total. The current emerging hotspots are concentrated on "endothelial glycocalyx," "NLRP3 inflammasome," "extracellular vesicle," "biomarkers," and "COVID-19," among others.
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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.
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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.
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.
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.
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Rebalance of coagulation and anticoagulation to achieve a hemostatic effect has recently gained attention as an alternative therapeutic strategy for hemophilia. We engineered a humanized chimeric antibody, SR604, based on a previously published murine antibody, HAPC1573, which selectively blocks the anticoagulant activity of human activated protein C (APC). SR604 effectively blocked the anticoagulation activities of APC in human plasma deficient in various coagulation factors in vitro with affinities ∼60 times greater than that of HAPC1573. SR604 exhibited prophylactic and therapeutic efficacy in the tail-bleeding and knee-injury models of hemophilia A and B mice expressing human APC (humanized hemophilic mice). SR604 did not interfere with the cytoprotection and endothelial barrier function of APC, nor were there obvious toxicity effects in humanized hemophilic mice. Pharmacokinetic study showed a high bioavailability (106%) of subcutaneously injected SR604 in cynomolgus monkeys. These results demonstrate that SR604 is expected to be a safe and effective therapeutic and/or prophylactic agent with a prolonged half-life for patients with congenital factor deficiencies including hemophilia A and B.
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Current assays that monitor thrombin generation in plasma rely on fluorogenic substrates to follow the kinetics of zymogen activation, which may be complicated by substrate cleavage from other proteases. In addition, these assays depend on activation following cleavage at the prothrombin R320 site and fail to report the cleavage at the alternative R271 site, leading to the shedding of the auxiliary Gla and kringle domains of prothrombin.
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: Dr. Esmon holds patents and licenses related to the use of protein C and activated protein C in the treatment of sepsis and thrombosis, as well as patents related to diagnostic aspects of defects in the protein C pathway.

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