Large burns provoke profound pathophysiological changes. Survival rates of patients with large burns have improved significantly with the advancement of critical care and adaptation of early excision protocols. Nevertheless, care of large burn wounds remains challenging secondary to limited donor sites, prolonged time to wound closure, and immunosuppression. The development of skin substitutes and new grafting techniques decreased time to wound closure. Individually, these methods have limited success, but a combination of them may yield more successful outcomes. Early identification of patients with likely poor prognosis should prompt goals of care discussion and involvement of a palliative care team when possible.

Various preclinical and clinical studies have demonstrated the robust wound healing capacity of the natural anticoagulant activated protein C (APC). A bioengineered APC variant designated 3K3A-APC retains APC's cytoprotective cell signalling actions with <10% anticoagulant activity. This study was aimed to provide preclinical evidence that 3K3A-APC is efficacious and safe as a wound healing agent. 3K3A-APC, like wild-type APC, demonstrated positive effects on proliferation of human skin cells (keratinocytes, endothelial cells and fibroblasts). Similarly it also increased matrix metollaproteinase-2 activation in keratinocytes and fibroblasts. Topical 3K3A-APC treatment at 10 or 30 μg both accelerated mouse wound healing when culled on Day 11. And at 10 μg, it was superior to APC and had half the dermal wound gape compared to control. Further testing was conducted in excisional porcine wounds due to their congruence to human skin. Here, 3K3A-APC advanced macroscopic healing in a dose-dependent manner (100, 250 and 500 μg) when culled on Day 21. This was histologically corroborated by greater collagen maturity, suggesting more advanced remodelling. A non-interference arm of this study found no evidence that topical 3K3A-APC caused either any significant systemic side-effects or any significant leakage into the circulation. However the female pigs exhibited transient and mild local reactions after treatments in week three, which did not impact healing. Overall these preclinical studies support the hypothesis that 3K3A-APC merits future human wound studies.

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

Significance: Burns are debilitating, life threatening, and difficult to assess and manage. Recent advances in assessment and management have occurred since a comprehensive review of the care of patients with severe burns was last published, which may influence research and clinical practice. Recent Advances: Recent advances have occurred in the understanding of burn pathophysiology, which has led to the identification of potential biomarkers of burn severity, such as protein C. There is new evidence about the potential superiority of natural colloids over crystalloids during fluid resuscitation, and new evidence about components of initial and perioperative management, including an improved understanding of pain following burns. Critical Issues: The limitations of the clinical examination highlight the need for imaging and biomarkers to assist in estimations of burn severity. Fluid resuscitation reduces mortality, although there is conjecture over the ideal method. The subsequent perioperative period is associated with significant morbidity and the evidence for preventing and treating pain, infection, and fluid overload while maximizing wound healing potential is described. Future Directions: Promising developments are ongoing in imaging technology, histopathology, biomarkers, and wound healing adjuncts such as hyperbaric oxygen therapy, topical negative pressure therapy, stem cell treatments, and skin substitutes. The greatest benefit from further research on management of patients with burns would most likely be derived from the elucidation of optimal fluid resuscitation protocols, pain management protocols, and surgical techniques from randomized controlled trials.

Although suspected conceptually in the 60 s, Protein C and Protein S activities in hemostasis were investigated and reported from the mid-80 s, followed by the discovery of Thrombomodulin, an endothelial cell membrane associated protein, playing the most important heamostatic role. These 3 proteins act in regulating thrombogenesis and protecting against thrombo-embolic events. When blood is activated, any trace of circulating thrombin is captured by Thrombomodulin in the microcirculation, making thrombin become an anticoagulant through its capacity to activate Protein C to Activated Protein C, which operates as a sentinel in blood coagulation, in the form of a complex with free Protein S, to block any new blood activation site, and more especially circulating activated Factors V and VIII. Protein S not only acts as the Activated Protein C cofactor, but also as the cofactor of Tissue Factor Pathway Inhibitor. In addition, it has some functions in the complement pathway through its binding to C4b-BP. Another capability of activated protein C is to lower fibrinolytic activity, as the Activated Protein C Inhibitor is also known as Plasminogen Activator Inhibitor 3. The Protein C-Protein S system becomes less efficient in the presence of mutated Factor V (Factor V-Leiden or other variants), which is resistant to its inactivating effect. Other pathologies linked to this system concern the development of allo- or auto-antibodies to Protein S or to thrombin, which can generate severe thrombotic complications in affected patients. Some antithrombotic drugs have originated from this regulatory system. Protein C or Protein S concentrates are used for treating deficient patients. Activated Protein C (especially in patients with sepsis) or Thrombomodulin are proposed as antithrombotic medications. Most importantly, congenital or acquired Protein C or Protein S deficiencies are associated with severe recurrent thrombotic events. From the clinical standpoint most of the patients are heterozygous, as homozygosity is almost incompatible with life in the absence of a continuous and efficient treatment. Laboratory investigation of this highly complex system involves many different specialized assays for measuring these 3 proteins' activities, their antigenic content or their genetic sequence. The Protein S in-vitro anticoagulant activity is weak and contrasts with its high antithrombotic role in-vivo, showing that diagnostic assays have not yet succeeded in reproducing all the natural mechanisms for evidencing the anticoagulant role of Protein S. This paradoxal notion is discussed and illustrated in this manuscript as well is a revisit of the major characteristics and pathophysiological functions of the Protein C-Protein S-Thrombomodulin system; the associated pathologies; and the main laboratory tools available for clinical diagnosis. In respect to future perspectives, we also focused on developing more significant and relevant assays, especially for Protein S, thanks to the understanding of its biological roles.

Independent of its well-known anticoagulation effects, activated protein C (APC) exhibits pleiotropic cytoprotective properties. These include anti-inflammatory actions, anti-apoptosis, and endothelial and epithelial barrier stabilisation. Such beneficial effects have made APC an attractive target of research in a plethora of physiological and pathophysiological processes. Of note, the past decade or so has seen the emergence of its roles in cutaneous wound healing-a complex process involving inflammation, proliferation and remodelling. This review will highlight APC's functions and mechanisms, and detail its pre-clinical and clinical studies on cutaneous wound healing.

Diabetic foot ulcer (DFU) constitutes a burden to patients with diabetes deteriorating their quality of life. Health related quality of life (HRQoL) can now be quantified with the use of specific tools; some of them provide a holistic approach to patients' well-being, while others are disease specific or even region specific. Many of these tools are applicable to patients with DFU. This review will present current data about the impact different interventions in the management of DFU on quality of life related parameters.

We performed a search of literature using keywords 'diabetes mellitus', 'diabetic foot ulcer', 'diabetic foot', 'health related quality of life', 'quality of life' and 'SF-36' to identify studies that contained data about the relationship between different interventions and quality of life of patients with diabetic foot ulcers.

Available data are not sufficient to conclude on the impact of interventions aimed to heal DFU on HRQoL. There is need for more, better designed studies and meta-analysis to estimate the effect of treatments on HRQoL in patients with DFUs. The development of new, diabetic foot specific tools will help to improve our knowledge in this field.

While wound therapy remains a clinical challenge in current medical practice, much effort has focused on developing biological therapeutic approaches. This paper presents a comprehensive review of delivery systems for current biologicals for the treatment of chronic wounds and severe burns. The biologicals discussed here include proteins such as growth factors and gene modifying molecules, which may be delivered to wounds free, encapsulated, or released from living systems (cells, skin grafts or skin equivalents) or biomaterials. Advances in biomaterial science and technologies have enabled the synthesis of delivery systems such as scaffolds, hydrogels and nanoparticles, designed to not only allow spatially and temporally controlled release of biologicals, but to also emulate the natural extracellular matrix microenvironment. These technologies represent an attractive field for regenerative wound therapy, by offering more personalised and effective treatments.

In the management of acute ischemic stroke, vessel recanalization correlates with functional status, mortality, cost, and other outcome measures. Thrombolysis with intravenous tissue plasminogen activator has many limitations that restrict its applicability, but recent advances in the development of mechanical thrombectomy devices as well as improved systems of stroke care have resulted in greater likelihood of vessel revascularization. Nonetheless, there remains substantial discrepancy between rates of recanalization and rates of favorable outcome. The poor neurological recovery among some stroke patients despite successful recanalization confirms the need for adjuvant pharmacological therapy for neuroprotection and/or neurorestoration. Prior clinical trials of such drugs may have failed due to the inability of the agent to access the ischemic tissue beyond the occluded artery. A protocol that couples revascularization with concurrent delivery of a neuroprotectant drug offers the potential to enhance the benefit of thrombolysis. Analogs of activated protein C (APC) exert pleiotropic anti-inflammatory, anti-apoptotic, antithrombotic, cytoprotective, and neuroregenerative effects in ischemic stroke and thus appear to be promising candidates for this novel approach. A multicenter, prospective, double-blinded, dose-escalation Phase 2 randomized clinical trial has enrolled 110 patients to assess the safety, pharmacokinetics, and efficacy of human recombinant 3K3A-APC following endovascular thrombolysis. This article is part of the Special Issue entitled 'Cerebral Ischemia'.

Protease-activated receptors (PARs) are a ubiquitously expressed class of G-protein-coupled receptors (GPCRs) that enable cells to respond to proteases in the extracellular environment in a nuanced and dynamic manner. PAR1 is the archetypal family member and has been the object of large-scale drug development programs since the 1990s. Vorapaxar and drotrecogin-alfa are approved PAR1-targeted therapeutics, but safety concerns have limited the clinical use of vorapaxar and questions regarding the efficacy of drotrecogin-alfa led to its withdrawal from the market. New understanding of mechanisms of PAR1 function, discovery of improved strategies for modifying PAR1 function, and identification of novel indications for PAR1 modulators have provided new opportunities for therapies targeting PAR1. In this review, we critically evaluate prospects for the next generation of PAR1-targeted therapeutics.

A homeostatic function of the coagulation system in regard to hemostasis is well established. Homeostasis of blood coagulation depends partially on protease activated receptor (PAR)-signaling. Beyond coagulation proteases, numerous other soluble and cell-bound proteases convey cellular effects via PAR signaling. As we learn more about the mechanisms underlying cell-, tissue-, and context-specific PAR signaling, we concurrently gain new insights into physiological and pathophysiological functions of PARs. In this regard, regulation of cell and tissue homeostasis by PAR signaling is an evolving scheme. Akin to the control of blood clotting per se (the fibrin-platelet interaction) coagulation proteases coordinately regulate cell- and tissue-specific functions. This review summarizes recent insights into homeostatic regulation through PAR signaling, focusing on blood coagulation proteases. Considering the common use of drugs altering coagulation protease activity through either broad or targeted inhibitory activities, and the advent of PAR modulating drugs, an in-depth understanding of the mechanisms through which coagulation proteases and PAR signaling regulate not only hemostasis, but also cell and tissue homeostasis is required.

The homeostatic blood protease, activated protein C (APC), can function as (1) an antithrombotic on the basis of inactivation of clotting factors Va and VIIIa; (2) a cytoprotective on the basis of endothelial barrier stabilization and anti-inflammatory and antiapoptotic actions; and (3) a regenerative on the basis of stimulation of neurogenesis, angiogenesis, and wound healing. Pharmacologic therapies using recombinant human and murine APCs indicate that APC provides effective acute or chronic therapies for a strikingly diverse range of preclinical injury models. APC reduces the damage caused by the following: ischemia/reperfusion in brain, heart, and kidney; pulmonary, kidney, and gastrointestinal inflammation; sepsis; Ebola virus; diabetes; and total lethal body radiation. For these beneficial effects, APC alters cell signaling networks and gene expression profiles by activating protease-activated receptors 1 and 3. APC's activation of these G protein-coupled receptors differs completely from thrombin's activation mechanism due to biased signaling via either G proteins or β-arrestin-2. To reduce APC-associated bleeding risk, APC variants were engineered to lack >90% anticoagulant activity but retain normal cell signaling. Such a neuroprotective variant, 3K3A-APC (Lys191-193Ala), has advanced to clinical trials for ischemic stroke. A rich data set of preclinical knowledge provides a solid foundation for potential translation of APC variants to future novel therapies.

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p<0.01) compared to rhBMP-2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair.

Pressure ulcers present a major clinical challenge, are physically debilitating and place the patient at risk of serious comorbidities such as septic shock. Recombinant human activated protein C (APC) is an anticoagulant with anti-inflammatory, cytoprotective and angiogenic effects that promote rapid wound healing. Topical negative pressure wound therapy (TNP) has become widely used as a treatment modality in wounds although its efficacy has not been proven through randomised controlled trials. The aim of this study was to determine the preliminary efficacy and safety of treatment with APC for severe chronic pressure sores with and without TNP. This case presentation describes the history, management and outcome of two patients each with a severe chronic non-healing pressure ulcer that had failed to respond to conventional therapy. TNP was added to conservative management of both ulcers with no improvement seen. Then local application of small doses of APC was added to TNP and with conservative management, resulted in significant clinical improvement and rapid healing of both ulcers, displaying rapid growth of vascular granulation tissue with subsequent epithelialisation. Patients tolerated the treatment well and improvements suggested by long-term follow-up were provided. Randomised placebo-controlled double blind trials are needed to quantify the efficacy, safety, cost-effectiveness, optimal dose and quality of life changes seen from treatment with APC.

Pyoderma gangrenosum (PG) is a systemic disease that presents with cutaneous necrotizing ulceration, producing deep necrotic ulcers, usually with a raised, undermined, violaceous border. Treatment typically involves high dose immunosuppressive drugs, but more recently anti-tumour necrosis factor and monoclonal antibodies have been used. Activated protein C (APC) stimulates wound healing in patients with treatment-refractory skin ulcers, possibly by stimulating angiogenesis and re-epithelialization, and preventing inflammation.

To investigate whether APC may be beneficial as a treatment for ulcers related to cutaneous PG.

Two patients were recruited with a clinical history and physical and histopathological evidence of acute PG. A total of 400 μg (1.0 mL) of APC was injected subcutaneously into the dermal edge of necrotic PG ulcers weekly for a total treatment period of 6 weeks. Photographs were taken, and clinical progress, ulcer size and pain score were monitored during this period and after the cessation of treatment, at weeks 8 and 12.

Over the 12 weeks of the trial, APC led to a reduction in wound size from 3.8 cm(2) to 0.8 cm(2) in patient 1 (78.9% decrease) and from 41 cm(2) to 16 cm(2) in patient 2 (70.0% decrease, respectively), and a reduction in pain scores from 10 to 0 (100% decrease) in both patients.

Although this study has limited because of its small sample size and lack of a true placebo group, it does indicate that APC has potential as a therapeutic option for patients with chronic skin ulcers from PG.

The cellular mechanisms of AMP-Activated Protein Kinase (AMPK) activators in the treatment and prevention of diabetes, cancer, and cardiovascular disease.

Activated protein C (aPC) is a natural anticoagulant and a potent anti-inflammatory and cytoprotective agent. At the expense of increased bleeding risk aPC has been used - with some success - in sepsis. The design of cytoprotective-selective aPC variants circumvents this limitation of increased bleeding, reviving the interest in aPC as a therapeutic agent. Emerging studies suggest that aPC`s beneficial effects are not restricted to acute illness, but likewise relevant in chronic diseases, such as diabetic nephropathy, neurodegeneration or wound healing. Epigenetic regulation of gene expression, reduction of oxidative stress, and regulation of ROS-dependent transcription factors are potential mechanisms of sustained cytoprotective effects of aPC in chronic diseases. Given the available data it seems questionable whether a unifying mechanism of aPC dependent cytoprotection in acute and chronic diseases exists. In addition, the signalling pathways employed by aPC are tissue and cell specific. The mechanistic insights gained from studies exploring aPC`s effects in various diseases may hence lay ground for tissue and disease specific therapeutic approaches. This review outlines recent investigations into the mechanisms and consequences of long-term modulation of aPC-signalling in models of chronic diseases.