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.
Protein C is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts potent anticoagulant activity. In addition to its antithrombotic role as a plasma protease, ...pharmacologic APC is a pleiotropic protease that activates diverse homeostatic cell signaling pathways via multiple receptors on many cells. Engineering of APC by site-directed mutagenesis provided a signaling selective APC mutant with 3 Lys residues replaced by 3 Ala residues, 3K3A-APC, that lacks >90% anticoagulant activity but retains normal cell signaling activities. This 3K3A-APC mutant exerts multiple potent neuroprotective activities, which require the G-protein–coupled receptor, protease activated receptor 1. Potent neuroprotection in murine ischemic stroke models is linked to 3K3A-APC–induced signaling that arises due to APC's cleavage in protease activated receptor 1 at a noncanonical Arg46 site. This cleavage causes biased signaling that provides a major explanation for APC's in vivo mechanism of action for neuroprotective activities. 3K3A-APC appeared to be safe in ischemic stroke patients and reduced bleeding in the brain after tissue plasminogen activator therapy in a recent phase 2 clinical trial. Hence, it merits further clinical testing for its efficacy in ischemic stroke patients. Recent studies using human fetal neural stem and progenitor cells show that 3K3A-APC promotes neurogenesis in vitro as well as in vivo in the murine middle cerebral artery occlusion stroke model. These recent advances should encourage translational research centered on signaling selective APC's for both single-agent therapies and multiagent combination therapies for ischemic stroke and other neuropathologies.
Cytoprotection by activated protein C (aPC) after ischemia-reperfusion injury (IRI) is associated with apoptosis inhibition. However, IRI is hallmarked by inflammation, and hence, cell-death forms ...disjunct from immunologically silent apoptosis are, in theory, more likely to be relevant. Because pyroptosis (ie, cell death resulting from inflammasome activation) is typically observed in IRI, we speculated that aPC ameliorates IRI by inhibiting inflammasome activation. Here we analyzed the impact of aPC on inflammasome activity in myocardial and renal IRIs. aPC treatment before or after myocardial IRI reduced infarct size and Nlrp3 inflammasome activation in mice. Kinetic in vivo analyses revealed that Nlrp3 inflammasome activation preceded myocardial injury and apoptosis, corroborating a pathogenic role of the Nlrp3 inflammasome. The constitutively active Nlrp3A350V mutation abolished the protective effect of aPC, demonstrating that Nlrp3 suppression is required for aPC-mediated protection from IRI. In vitro aPC inhibited inflammasome activation in macrophages, cardiomyocytes, and cardiac fibroblasts via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Accordingly, inhibiting PAR-1 signaling, but not the anticoagulant properties of aPC, abolished the ability of aPC to restrict Nlrp3 inflammasome activity and tissue damage in myocardial IRI. Targeting biased PAR-1 signaling via parmodulin-2 restricted mTORC1 and Nlrp3 inflammasome activation and limited myocardial IRI as efficiently as aPC. The relevance of aPC-mediated Nlrp3 inflammasome suppression after IRI was corroborated in renal IRI, where the tissue protective effect of aPC was likewise dependent on Nlrp3 inflammasome suppression. These studies reveal that aPC protects from IRI by restricting mTORC1-dependent inflammasome activation and that mimicking biased aPC PAR-1 signaling using parmodulins may be a feasible therapeutic approach to combat IRI.
•aPC protects from myocardial and renal IRIs by restricting mTORC1-mediated activation of the Nlrp3 inflammasome.•Nlrp3 inflammasome suppression by aPC is independent of its anticoagulant effect, depends on PAR-1, and can be mimicked by parmodulin-2.
The cytoprotective protein C pathway Mosnier, Laurent O.; Zlokovic, Berislav V.; Griffin, John H.
Blood,
04/2007, Letnik:
109, Številka:
8
Journal Article
Recenzirano
Odprti dostop
Protein C is best known for its mild deficiency associated with venous thrombosis risk and severe deficiency associated with neonatal purpura fulminans. Activated protein C (APC) anticoagulant ...activity involves proteolytic inactivation of factors Va and VIIIa, and APC resistance is often caused by factor V Leiden. Less known is the clinical success of APC in reducing mortality in severe sepsis patients (PROWESS trial) that gave impetus to new directions for basic and preclinical research on APC. This review summarizes insights gleaned from recent in vitro and in vivo studies of the direct cytoprotective effects of APC that include beneficial alterations in gene expression profiles, anti-inflammatory actions, antiapoptotic activities, and stabilization of endothelial barriers. APC's cytoprotection requires its receptor, endothelial cell protein C receptor, and protease-activated receptor-1. Because of its pleiotropic activities, APC has potential roles in the treatment of complex disorders, including sepsis, thrombosis, and ischemic stroke. Although much about molecular mechanisms for APC's effects on cells remains unclear, it is clear that APC's structural features mediating anticoagulant actions and related bleeding risks are distinct from those mediating cytoprotective actions, suggesting the possibility of developing APC variants with an improved profile for the ratio of cytoprotective to anticoagulant actions.
Activated protein C (APC) exerts endothelial cytoprotective actions that require protease-activated receptor 1 (PAR1), whereas thrombin acting via PAR1 causes endothelial disruptive, proinflammatory ...actions. APC's activities, but not thrombin's, require PAR1 located in caveolae. PAR1 is a biased 7-transmembrane receptor because G proteins mediate thrombin's signaling, whereas β-arrestin 2 mediates APC's signaling. Here we elucidate novel mechanisms for APC's initiation of signaling. Biochemical studies of APC's protease specificity showed that APC cleaved PAR1 sequences at both Arg41 and Arg46. That PAR1 cleavage at Arg46 can occur on cells was supported by APC's cleavage of N-terminal-SEAP-tagged R41Q-PAR1 but not R41Q/R46Q-PAR1 mutants transfected into cells and by anti-PAR1 epitope mapping of APC-treated endothelial cells. A synthetic peptide composing PAR1 residues 47-66, TR47, stimulated protective signaling in endothelial cells as reflected in Akt and glycogen synthase kinase 3β phosphorylation, Ras-related C3 botulinum toxin substrate 1 activation, and barrier stabilization effects. In mice, the TR47 peptide reduced VEGF-induced vascular leakage. These in vitro and in vivo data imply that the novel PAR1 N-terminus beginning at residue Asn47, which is generated by APC cleavage at Arg46, mediates APC's cytoprotective signaling and that this unique APC-generated N-terminal peptide tail is a novel biased agonist for PAR1.
Essentials
Striated muscle myosins can promote prothrombin activation by FXa or FVa inactivation by APC.
Cardiac myosin and skeletal muscle myosin are pro‐hemostatic in murine tail cut bleeding ...models.
Infused cardiac myosin exacerbates myocardial injury caused by myocardial ischemia reperfusion.
Skeletal muscle myosin isoforms that circulate in human plasma can be grouped into 3 phenotypes.
Two striated muscle myosins, namely skeletal muscle myosin (SkM) and cardiac myosin (CM), may potentially contribute to physiologic mechanisms for regulation of thrombosis and hemostasis. Thrombin is generated from activation of prothrombin by the prothrombinase (IIase) complex comprising factor Xa, factor Va, and Ca++ ions located on surfaces where these factors are assembled. We discovered that SkM and CM, which are abundant motor proteins in skeletal and cardiac muscles, can provide a surface for thrombin generation by the prothrombinase complex without any apparent requirement for phosphatidylserine or lipids. These myosins can also provide a surface that supports the inactivation of factor Va by activated protein C/protein S, resulting in negative feedback downregulation of thrombin generation. Although the physiologic significance of these reactions remains to be established for humans, substantive insights may be gleaned from murine studies. In mice, exogenously infused SkM and CM can promote hemostasis as they are capable of reducing tail cut bleeding. In a murine myocardial ischemia‐reperfusion injury model, exogenously infused CM exacerbates myocardial infarction damage. Studies of human plasmas show that SkM antigen isoforms of different MWs circulate in human plasma, and they can be used to identify three plasma SkM phenotypes. A pilot clinical study showed that one SkM isoform pattern appeared to be linked to isolated pulmonary embolism. These discoveries enable multiple preclinical and clinical studies of SkM and CM, which should provide novel mechanistic insights with potential translational relevance for the roles of CM and SkM in the pathobiology of hemostasis and thrombosis.
Humans with ALS and transgenic rodents expressing ALS-associated superoxide dismutase (SOD1) mutations develop spontaneous blood-spinal cord barrier (BSCB) breakdown, causing microvascular ...spinal-cord lesions. The role of BSCB breakdown in ALS disease pathogenesis in humans and mice remains, however, unclear, although chronic blood-brain barrier opening has been shown to facilitate accumulation of toxic blood-derived products in the central nervous system, resulting in secondary neurodegenerative changes. By repairing the BSCB and/or removing the BSCB-derived injurious stimuli, we now identify that accumulation of blood-derived neurotoxic hemoglobin and iron in the spinal cord leads to early motor-neuron degeneration in SOD1(G93A) mice at least in part through iron-dependent oxidant stress. Using spontaneous or warfarin-accelerated microvascular lesions, motor-neuron dysfunction and injury were found to be proportional to the degree of BSCB disruption at early disease stages in SOD1(G93A) mice. Early treatment with an activated protein C analog restored BSCB integrity that developed from spontaneous or warfarin-accelerated microvascular lesions in SOD1(G93A) mice and eliminated neurotoxic hemoglobin and iron deposits. Restoration of BSCB integrity delayed onset of motor-neuron impairment and degeneration. Early chelation of blood-derived iron and antioxidant treatment mitigated early motor-neuronal injury. Our data suggest that BSCB breakdown contributes to early motor-neuron degeneration in ALS mice and that restoring BSCB integrity during an early disease phase retards the disease process.
Recent studies indicate that single-action–single-target agents are unlikely to cure CNS disorders sharing a pathogenic triad consisting of vascular damage, neuronal injury/neurodegeneration and ...neuroinflammation. Here we focus on a recent example of a multiple-action–multiple-target approach for CNS disorders based on newly discovered biological properties of activated protein C (APC), an endogenous plasma protease with antithrombotic, cytoprotective and anti-inflammatory activities in the CNS. We propose that APC-mediated signaling through the protease activated receptor-1 (PAR1) can favorably regulate multiple pathways within the neurovascular unit in non-neuronal cells and neurons during acute or chronic CNS insults, leading to stabilization of the blood–brain barrier (BBB), neuroprotection and control of neuroinflammation. Although much remains to be understood regarding the biology of APC, preclinical studies suggest that APC has promising applications as disease-modifying therapy for ischemic stroke and other neuropathologies whose underlying pathology involves deficits in the vasculo-neuronal-inflammatory triad.
COVID-19-associated coagulopathy (CAC) is a life-threatening complication of SARS-CoV-2 infection. However, the underlying cellular and molecular mechanisms driving this condition are unclear. ...Evidence supports the concept that CAC involves complex interactions between the innate immune response, the coagulation and fibrinolytic pathways, and the vascular endothelium, resulting in a procoagulant condition. Understanding of the pathogenesis of this condition at the genomic, molecular and cellular levels is needed in order to mitigate thrombosis formation in at-risk patients. In this Perspective, we categorize our current understanding of CAC into three main pathological mechanisms: first, vascular endothelial cell dysfunction; second, a hyper-inflammatory immune response; and last, hypercoagulability. Furthermore, we pose key questions and identify research gaps that need to be addressed to better understand CAC, facilitate improved diagnostics and aid in therapeutic development. Finally, we consider the suitability of different animal models to study CAC.
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