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Keywords:

  • activated protein C;
  • disseminated intravascular coagulation;
  • high-mobility group box 1 protein;
  • sepsis;
  • thrombomodulin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References

Summary.  Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated activation of protein C. Recently, we conducted a multicentre, double-blind, randomized trial to evaluate the efficacy and safety of recombinant human soluble thrombomodulin (rhsTM, also known as ART-123) for the treatment of disseminated intravascular coagulation (DIC), and found that rhsTM therapy is more effective and safer than low-dose heparin therapy. Thus, in 2008, rhsTM (Recomodulin) was approved for the treatment of DIC in Japan. Here we re-evaluate the therapeutic basis of this drug from the view of its anticoagulant, anti-inflammatory, and cytoprotective properties. Structurally, the extracellular portion of TM is composed of three domains: an N-terminal lectin-like domain (TM-D1), followed by an epidermal growth factor (EGF)-like domain (TM-D2), and an O-glycosylation–rich domain (TM-D3). TM-D2 and TM-D3 are important for the protein’s anticoagulant cofactor activities, i.e. inhibition of thrombin and activation of protein C. TM-D1 plays an important role in attenuation of inflammatory responses, through inhibition of leukocyte adhesion to endothelial cells, inhibition of complement pathways, neutralization of lipopolysaccharide (LPS), and sequestration and degradation of pro-inflammatory high-mobility group box 1 protein (HMGB1). Thus, TM on the surface of endothelial cells prevents dissemination of pro-coagulant and pro-inflammatory molecules, and by doing so, allows these molecules to act locally at the site of injury. In patients with sepsis and DIC, TM expression is down-regulated, which may result in dissemination of pro-coagulant and pro-inflammatory molecules throughout the systemic circulation. Replacement with rhsTM may offer therapeutic value in such conditions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References

Arthropods, such as insects, have an open circulatory system in which blood is pumped by a heart and is delivered to an open cavity where the tissue is directly bathed by the blood/haemolymph. Vertebrates, such as humans, have a closed circulatory system in which blood remains in a network of vessels and does not leave it. The closed circulatory system carries blood at a high pressure and allows for rapid and efficient transport of oxygen, nutrients, and waste, which is suitable for animals with a fast metabolism. It also allows blood to be directed precisely to where it is needed. However, the closed circulatory system could also contain risks of massive loss of blood (haemorrhage) and obstruction in the vessels (thrombosis), which can cause perfusion deficiency and tissue necrosis. The defensive mechanisms against them include the haemostatic system and the anticoagulant system. The former prevents haemorrhage by sealing defects rapidly with platelets and fibrin clots, whereas the latter prevents thrombosis by maintaining blood in a fluid state. Under physiological conditions, the anticoagulant system is dominant within blood vessels since anticoagulant molecules, such as thrombomodulin (TM), are rich on the luminal surface of endothelial cells [1], and the haemostatic system is dominant outside blood vessels since haemostatic molecules, such as tissue factor, are rich in the subendothelial space [2]. However, under pathological conditions, the thrombotic system can override the anticoagulant system even within blood vessels, resulting in intravascular thrombus growth. In some cases, microvessel thrombus formation can be a host-protective mechanism against infection because it suppresses dissemination of pathogens [3,4]. However, if it proceeds out of control, serious adverse events, including disseminated intravascular coagulation (DIC), can occur.

DIC is characterised by the widespread activation of coagulation within blood vessels, which results in thrombotic occlusion of microvessels [5]. DIC occurs in association with a variety of clinical conditions, including sepsis, malignancy, and trauma. The essence of the management of DIC is the treatment of the underlying disorder. However, it is often not easy to remove the trigger for DIC promptly, and supportive therapy directed at DIC itself, such as anticoagulant therapy, is useful to ameliorate the hypercoagulable state in DIC. Indeed, anticoagulant therapy has been shown to have beneficial effects in animal models of DIC [6–8]. Among anticoagulant drugs, rhsTM is a promising drug for the management of DIC because it has anti-inflammatory as well as anticoagulant properties with less bleeding complications [6,9–12]. In this review, we summarise the pleiotropic effects of TM in the management of DIC and sepsis.

Anticoagulant properties of thrombomodulin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References

TM is a thrombin-binding anticoagulant cofactor, which is expressed on the surface of endothelial cells [13–15]. Structurally, TM is composed of five domains: N-terminal lectin-like domain (TM-D1), EGF-like domain (TM-D2), O-glycosylation–rich domain (TM-D3), transmembrane domain, and short cytoplasmic domain (Fig. 1). TM binds to thrombin via TM-D2, and therefore this domain is critical for anticoagulant activity.

image

Figure 1.  Domain structure of TM. D1: N-terminal lectin-like domain, D2: EGF-like domain, D3: O-glycosylation–rich domain, D4: transmembrane domain, D5: cytoplasmic domain. TM-D2 and TM-D3 are important for the protein’s anticoagulant cofactor activities, i.e. inhibition of thrombin and activation of protein C. TM-D1 is important for the protein’s anti-inflammatory activities.

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Thrombin is a critical pro-thrombotic factor. In the absence of TM, thrombin activates fibrinogen to generate fibrin. Thrombin activates other coagulation factors, such as factors V, VIII, XI, and XIII, and also activates platelets. However, in the presence of TM, a high-affinity complex with thrombin is formed, inhibiting thrombin interaction with these pro-coagulant substrates. Instead, thrombin-TM complexes activate protein C, and activated protein C (APC) inactivates coagulation factors Va and VIIIa, thereby suppressing further thrombin generation (Fig. 2). Thus, TM not only inhibits procoagulant thrombin, but also converts it to an anticoagulant agent. Considering that TM is expressed on the surface of endothelial cells, TM may play a role in preventing intravascular thrombus formation. This idea is supported by the observation that endothelium-specific loss of TM in mice causes spontaneous and fatal thrombosis in the arterial as well as venous circulation [16]. As a result, 40% of mutant mice die before birth, and the remaining 60% die within a month after birth because of massive thrombosis, indicating that TM is indispensable to prevent intravascular thrombus growth. In humans, the endothelial TM expression can be down-regulated in certain pathologic conditions, such as meningococcal sepsis and graft rejection, resulting in thrombotic complications [17–19]. It is suggested that infusion of TM into these patients may be a therapeutic option to prevent thrombotic complications.

image

Figure 2.  Anticoagulant activities of TM. TM forms a high-affinity complex with thrombin, inhibiting fibrin generation. Thrombin-TM complexes activate protein C, and activated protein C (APC) inactivates coagulation factors Va and VIIIa, thereby suppressing further thrombin generation. The dotted line indicates a negative feedback mechanism.

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A new drug for the treatment of DIC: recombinant human soluble thrombomodulin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References

The recombinant human soluble TM (rhsTM, Asahi Kasei Pharma, Tokyo, Japan), derived from Chinese hamster ovary (CHO) cells, is composed of the extracellular domain of TM [20]. Similar to membrane-bound TM, rhsTM binds to thrombin to activate protein C. Theoretically, rhsTM has less risk of bleeding complications than other anticoagulant drugs, such as argatroban, because rhsTM does not inhibit the initial phase of thrombin generation [9]. Furthermore, rhsTM may have less risk of bleeding complications than activated protein C (APC) because rhsTM exerts its anti-coagulant effect in a thrombin-dependent manner. In other words, rhsTM acts as an anticoagulant preferentially where and when much thrombin exists (Fig. 2, negative feedback). In a tissue factor-induced DIC model in rats, rhsTM therapy reverses the prolongation of bleeding time. Notably, rhsTM therapy does not complicate bleeding symptoms even at higher doses, whereas the heparin therapy complicates bleeding symptoms at higher doses (Fig. 3A). These data suggest that the rhsTM therapy may be effective and safe in DIC patients with frequent bleeding complications.

image

Figure 3.  Protective effects of rhsTM on DIC. (A) Efficacy and safety of rhsTM in a rat DIC model. Intravenous injection of tissue factor (50 mg kg−1) causes DIC-like changes, including the prolongation of bleeding time. Infusion of rhsTM (190–1900 U kg−1) reverses the prolongation of bleeding time. Notably, rhsTM therapy does not complicate bleeding symptoms even at higher doses, whereas the heparin therapy complicates bleeding symptoms at higher doses. (B) and (C) Efficacy of rhsTM in the phase III clinical trial. DIC resolution rates at day 7 (B) and disappearance rates of bleeding symptoms at day 7 (C) are shown. rhsTM is superior to heparin.

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To evaluate the efficacy and safety of rhsTM for the treatment of DIC, we conducted a multicentre, randomized, double-blind clinical trial [21]. Subjects were DIC patients with haematologic malignancy or infection. DIC patients were assigned to receive rhsTM or heparin for 6 days. The primary endpoint was DIC resolution rate at day 7, and the secondary endpoints included disappearance rate of bleeding symptoms at day 7 and mortality rate at day 28. In this trial, 116 patients received rhsTM, and the same number of patients received heparin. Of these, 112 patients each have been assessed for the primary endpoint. The DIC resolution rates were 66.1% for the rhsTM group and 49.9% for the heparin group, and rhsTM was significantly superior to heparin (Fig. 3B). The disappearance rates of bleeding symptoms were 35.2% for the rhsTM group, and 20.9% for the heparin group (Fig. 3C). The mortality rates of DIC patients associated with infection were 28.0% for the rhsTM group and 34.6% for the heparin group. In the case of haematologic malignancy, the mortality rates were 17.2% for the rhsTM group and 18.0% for the heparin group. Decreases in plasma thrombin-antithrombin complex (TAT) levels and D-dimer levels were significantly greater in the rhsTM group, suggesting that rhsTM is superior to heparin in the attenuation of hypercoagulable states. As for safety, the incidence of bleeding-related adverse events was lower in the rhsTM group. Thus, the rhsTM therapy is more effective and safer than the heparin therapy [21], and in 2008, rhsTM was approved in Japan for the treatment of DIC. This study is the first double-blind clinical study, which shows the superiority of a new anticoagulant therapy over the heparin therapy for the treatment of DIC.

Anti-inflammatory properties of thrombomodulin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References

Recent studies have suggested that TM plays an important role in attenuation of inflammation through APC-dependent and -independent mechanisms [22–26]. The anti-inflammatory properties of TM-D1 are considered as APC-independent since this domain is dispensable for APC generation [22,27,28]. Mice lacking the D1 domain of TM display reduced survival after lipopolysaccharide (LPS) exposure and accumulate more neutrophils in their lungs while they can activate protein C normally [22]. TM-D1 attenuates inflammation through multiple mechanisms (Fig. 4). First, TM-D1 interferes with complement activation. Mice lacking the D1 domain of TM display enhanced deposition of complement factor C3 on their joint surfaces and develop more severe inflammatory arthritis than wild-type counterparts [24]. TM provides additional protection from complement activation, by enhancing complement factor I-mediated inactivation of C3b, and by promoting activation of thrombin-activatable fibrinolysis inhibitor (TAFI), which inactivates complement-derived anaphylatoxins C5a and C3a [29–31]. In this context, a recent study has shown that about 5% of patients with atypical haemolytic-uremic syndrome carry heterozygous mutations, which impair the function of TM [31]. Cells expressing these variants are less efficient in inactivating C3b and less efficient in activating TAFI [31,32]. Second, TM-D1 attenuates inflammation by neutralizing LPS. TM-D1 binds to Lewis Y antigen in LPS, and this may contribute to the protective effect of TM-D1 against inflammatory responses induced by LPS and Gram-negative bacteria [33]. Third, TM attenuates inflammation by binding to high-mobility group box 1 protein (HMGB1) and subsequently promoting degradation of HMGB1 [23,34].

image

Figure 4.  Anti-inflammatory activities of TM. TM plays an important role in attenuation of inflammatory responses, through inhibition of complement pathways, neutralization of endotoxin, and sequestration and degradation of HMGB1. TM provides additional protection from complement activation, by promoting activation of TAFI, which inactivates complement-derived anaphylatoxins C5a and C3a.

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Sequestration and degradation of HMGB1 by thrombin-thrombomodulin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References

HMGB1 is an abundant protein in the nucleus, and is highly conserved through evolution. This protein is expressed in almost all eukaryotic cells, and serves a dual function as an intracellular DNA-binding protein and as an extracellular signal of tissue damage [35]. Inside the cells, HMGB1 binds to DNA without sequence specificity, which promotes interaction of DNA with transcription factors such as glucocorticoid receptors. HMGB1 deficient mice die shortly after birth because of, at least in part, hypoglycaemia caused by deficient glucocorticoid receptor function [36]. Once released into the extracellular milieu, HMGB1 acts as a signal of tissue damage. HMGB1 is passively released from necrotic cells and actively secreted by inflammatory cells [37,38]. Extracellular HMGB1 stimulates target cells by acting on pattern recognition receptors, such as receptor for advanced glycation end-products (RAGE, also known as AGER), Toll-like receptor 2 (TLR2), and TLR4 [35], and promotes inflammation [39], immune responses [40], and tissue regeneration [41]. Thus, HMGB1 is important for controlling infection and for promoting tissue repair especially at sites of tissue injury. But when HMGB1 is disseminated throughout the systemic circulation, HMGB1 promotes the development of systemic inflammatory response syndrome (SIRS) and DIC, and acts as a lethal mediator [42,43]. Serum or plasma HMGB1 levels are higher in patients with sepsis or DIC than those in healthy individuals, and are correlated with the disease severity [42,44]. In animal experiments, HMGB1 promotes fibrin deposition in kidneys and increases mortality rate [42,43]. Treatment with anti-HMGB1 neutralizing antibody rescues mice from lethal endotoxemia, suggesting that HMGB1 is responsible for endotoxin lethality [23,42].

TM on the surface of endothelial cells may prevent dissemination of HMGB1. TM, via its D1 domain, binds to HMGB1, thereby preventing HMGB1 from engaging its receptor RAGE [23]. TM also binds to thrombin via the D2 domain, and promotes the degradation of HMGB1 by thrombin, which can decrease the proinflammatory activity of HMGB1 [34]. In experimental endotoxemia, treatment with rhsTM decreases plasma HMGB1 levels, increases HMGB1 degradation product levels, and rescues endotoxemic animals [12,23,34]. Thus, TM binds to HMGB1 as well as thrombin, thereby promoting thrombin-mediated cleavage of HMGB1, and preventing dissemination of these pro-inflammatory and procoagulant molecules throughout the systemic circulation.

Cytoprotective properties of activated protein C

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References

TM exerts cytoprotective effects, in part through APC-dependent mechanisms. APC’s cytoprotective effects include anti-apoptotic activity [45–47], endothelial barrier stabilisation [48,49], and inhibition of inflammation [25,50], and are mainly mediated by protease activated receptor-1 (PAR-1) and endothelial protein C receptor (EPCR) expressed on the surface of endothelial cells and leukocytes [51,52]. APC also exerts cytoprotective effects through proteolytic cleavage of extracellular histones, which are, like HMGB1, mediators of endothelial dysfunction, organ failure, and death during sepsis [53]. Although the source of extracellular histones is not defined yet, necrotic cells or extracellular traps (NETs)-forming neutrophils may be responsible [54]. NETs promote platelet aggregation as well as fibrin deposition, and serves as a scaffold for intravascular thrombus formation [3,55]. Since histones are important constituents of NETs, APC-mediated degradation of histones may ameliorate tissue damage evoked by NETs.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References

Inflammation and coagulation are self-protective responses, and they are beneficial at sites of tissue injury. TM, expressed on the surface of endothelial cells, prevents dissemination of procoagulant and proinflammatory molecules, including thrombin, complement factors, LPS, HMGB1, and possibly histones, and by doing so, allows these molecules to act locally at the site of injury. However, TM expression is down-regulated in patients with sepsis and DIC, which may result in dissemination of procoagulant and proinflammatory molecules throughout the body. In such conditions, replacement with rhsTM may offer therapeutic value.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Anticoagulant properties of thrombomodulin
  5. A new drug for the treatment of DIC: recombinant human soluble thrombomodulin
  6. Anti-inflammatory properties of thrombomodulin
  7. Sequestration and degradation of HMGB1 by thrombin-thrombomodulin
  8. Cytoprotective properties of activated protein C
  9. Conclusion
  10. Disclosure of Conflict of Interests
  11. References
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