Stroke Treatment With PAR-1 Agents to Decrease Hemorrhagic Transformation

Abstract

Ischemic stroke is the most widespread cause of disability and a leading cause of death in developed countries. To date, the most potent approved treatment for acute stroke is recanalization therapy with thrombolytic drugs such as tissue plasminogen activator (rt-PA or tPA) or endovascular mechanical thrombectomy. Although tPA and thrombectomy are widely available in the United States, it is currently estimated that only 10-20% of stroke patients get tPA treatment, in part due to restrictive selection criteria. Recently, however, tPA and thrombectomy selection criteria have loosened, potentially allowing more patients to qualify. The relatively low rate of treatment may also reflect the perceived risk of brain hemorrhage following treatment with tPA. In translational research and a single patient study, protease activated receptor 1 (PAR-1) targeted therapies given along with thrombolysis and thrombectomy appear to reduce hemorrhagic transformation after recanalization. Such adjuncts may likely enhance the availability of recanalization and encourage more physicians to use the recently expanded selection criteria for applying recanalization therapies. This narrative review discusses stroke therapies, the role of hemorrhagic transformation in producing poor outcomes, and presents the data suggesting that PAR-1 acting agents show promise for decreasing hemorrhagic transformation and improving outcomes.

Keywords: activated protein C; bleeding; hemorrhagic transformation; intracranial hemorrhage; ischemic stroke; stroke therapy; thrombectomy; tissue plasminogen activator.

Figure 1

Anticoagulant and cell-signaling pathways of APC and the structure of signaling-selective 3K3A-APC. APC, activated protein C; BBB, blood–brain barrier; EGF, endothelial growth factor; EPCR, endothelial protein C receptor; GLA, gamma-carboxyglutamic acid; PAR, protease-activated receptor. Reprinted from blood, vol. 132(2), Griffin et al. (67) activated protein C, protease activated receptor 1 and neuroprotection; 159–169, 2018, with permission from the American Society of Hematology. (A) Anticoagulant activity of APC involves the proteolytic inactivation of factors Va and VIIIa on membrane surfaces containing phospholipids that are derived from cells, platelets, lipoproteins, or cellular microparticles. The irreversible inactivation of factors Va and VIIIa to yield inactive factors Vi and VIIIi by APC is accelerated by a variety of lipid and protein cofactors (e.g., glucosyl ceramide, protein S, etc). (B) Beneficial direct effects of APC on cells require the EPCR and PAR-1. One distinction between pro-inflammatory thrombin signaling and cytoprotective APC signaling is the localization of APC signaling in the caveolin-1–rich microdomains (caveolae). (C) Neuroprotective mechanisms for APC effects on cells may also involve other receptors including PAR-3. APC-initiated signaling effects on cells can include anti-apoptotic activities, anti-inflammatory activities, inhibition of the inflammasome, stabilization of endothelial barrier functions, including the BBB, and neurogenesis. (D) The polypeptide structure of APC comprises an N-terminal GLA domain (green) that binds to negatively charged lipids and EPCR, 2 EGF-like domains (light blue and dark blue), and the protease domain containing the active site triad of serine, histidine, and aspartic acid residues (red). Four glycosylation sites are indicated by gray-shaded moieties. Substrate selectivity of this protease is determined by interactions between the targeted substrates and the active site and also by multiple unique binding exosites on APC that vary for different substrates. The protease domain space–filled model (see insert in D) highlights in the yellow box 3 positively charged lysine (K) residues within the so-called 37 loop (KKK 191–193), which is an exosite for APC's recognition of factors Va and VIIIa. Mutation of these 3 lysine residues to alanine (3K3A-APC) reduces APC's anticoagulant activity by >90% but does not affect its interactions with the cytoprotective substrates, PAR-1, PAR-3, or its other known cell-signaling receptors. Thus, 3K3A-APC is very “signaling-selective”.

Figure 2

Cell-Specific APC protective signaling pathways. Akt, protein kinase B; APC, activated protein C; BBB, blood–brain barrier; EPCR, endothelial protein C receptor; MMP, matrix metallopeptidase; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; PAR, protease-activated receptor; Rac1, Ras-related C3 botulinum toxin substrate 1; S1PR1, sphingosine 1-phosphate receptor 1. Reprinted from neuropharmacology, vol. 134, Amar et al. (69) can adjunctive therapies augment the efficacy of endovascular thrombolysis? A potential role for activated protein C, 293–301, 2018, with permission from Elsevier. 3D structure reprinted from blood, vol. 132(2), Griffin et al. (67) activated protein C, protease activated receptor 1 and neuroprotection; 159–169, 2018, with permission from the American Society of Hematology. (A) In endothelial cells, APC helps to seal the BBB and is vasculoprotective. APC/EPCR activates PAR-1 and inhibits caspase-8 activation of caspase-3, thereby limiting the extrinsic apoptotic pathway in endothelium. APC/EPCR-dependent PAR-1 activation suppresses the pro-apoptotic p53 transcription factor inhibiting caspase-3 activation blocking the intrinsic apoptotic pathway. Also, APC suppresses the NFkB-dependent transcriptional activation of MMP-9, thereby blocking degradation of the BBB basement membrane. Furthermore, APC blocks the expression of pro-inflammatory cytokines, limiting inflammation by controlling NFkB nuclear translocation. APC's cytoprotective effects on endothelial cells require EPCR and PAR-1 to cross-activate S1PR1. Cross-activation of S1PR1 activates Rac1, leading to stabilization of the BBB cytoskeleton, thereby supporting the integrity of the BBB. (B) In neurons, APC/EPCR is cytoprotective via PAR-1 and PAR-3, which inhibits caspase-8 upstream of caspase-3 and thereby limits the extrinsic apoptotic pathway. Also, an APC-PAR-1-PAR-3 pathway blocks p53 activation in injured neurons, thereby blocking the caspase-9-dependent intrinsic apoptotic pathway. Furthermore, APC promotes neurogenesis via a PAR-1-PAR-3-S1PR1-Akt pathway. (C) APC's inhibition of NFkB-dependent transcriptional expression of different pro-inflammatory cytokines suppresses microglial activation. Suppression of NLRP3 inflammasome development by APC is another activity but is not shown in this figure.

Figure 3

Biased Agonism of PAR-1 by APC. Akt, protein kinase B; APC, activated protein C; BBB, blood–brain barrier; PAR, protease-activated receptor; P13K, phosphoinositide 3-kinase; Rac, Ras-related C3 botulinum toxin substrate; RhoA, ras homolog gene family member A; TRAP, thrombin-receptor activated peptide. Reprinted from Blood, vol. 120(26), Mosnier et al. (68) biased agonism of protease-activated receptor 1 by activated protein C caused by noncanonical cleavage at Arg46; 5237–5246, 2012, with permission from the American Society of Hematology. Activation of PAR-1 by APC and its cytoprotective analogs involves cleavage of PAR-1 N-terminal domain at Arg46, which reveals a tethered ligand peptide that begins at Asn47 causing APC's biased, β-arrestin-2-dependent cytoprotective signaling. Activation of PAR-1 by thrombin involves cleavage at Arg41, which generates a tethered ligand that begins at Thr42, initiating cytotoxic effects via G-protein-dependent signaling causing human platelet activation, inflammatory changes, vascular leakage, and CNS toxicity.

Figure 4

Effects of 3K3A-APC and 3K3A-APC combined with tPA on hemorrhage (upper panel) and neuropathological (hematoxylin and eosin staining; infarct volume) and neurological (neurological score) outcomes (lower panel) in young male spontaneously hypertensive rats within 7 days after embolic stroke. APC, activated protein C; SD, standard deviation; tPA, tissue plasminogen activator. Reprinted from Stroke, vol. 44(12), Wang et al. (56) activated protein C analog protects from ischemic stroke and extends the therapeutic window of tissue-type plasminogen activator in aged female mice and hypertensive rats, 3529–3536, 2013, with permission from Wolters Kluwer Health, Inc. 3K3A-APC and tPA were administered 4 h after embolic stroke. 3K3A-APC was administered for 3 consecutive days afterward. Mean + SD, N = 8–9 rats per group.

Figure 5

Unmet medical needs concerning physician satisfaction with tPA therapy in the United States and Europe. IV, intravenous; tPA, tissue plasminogen activator. The terms in the figure were provided to the physician interviewees without further definitions, and interviewees were asked to rank each term on a 10-point scale. For importance: 0 = unimportant and 10 = essential. For satisfaction: 0 = fully unsatisfactory and 10 = entirely satisfactory. The numbers are mean responses of the 32 interviewees.