Summary The protein C pathway provides multiple important functions to maintain a regulated balance between haemostasis and host defence systems in response to vascular and inflammatory injury. The anticoagulant protein C pathway is designed to regulate coagulation, maintain the fluidity of blood within the vasculature, and prevent thrombosis, whereas the cytoprotective protein C pathway prevents vascular damage and stress. The cytoprotective activities of activated protein C (APC) include anti-apoptotic activity, anti-inflammatory activity, beneficial alterations of gene expression profiles, and endothelial barrier stabilisation. These cytoprotective activities of APC, which require the endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR1), have been a major research focus. Recent insights, such as non-canonical activation of PAR1 at Arg46 by APC and biased PAR1 signalling, provided better understanding of the molecular mechanisms by which APC elicits cytoprotective signalling through cleavage of PAR1. The discovery and development of anticoagulant-selective and cytoprotective-selective APC mutants provided unique opportunities for preclinical research that has been and may continue to be translated to clinical research. New mechanisms for the regulation of EPCR functionality, such as modulation of EPCR-bound lipids that affect APC’s cytoprotective activities, may provide new research directions to improve the efficacy of APC to convey its cytoprotective activities to cells. Moreover, emerging novel functions for EPCR expand the roles of EPCR beyond mediating protein C activation and APC-induced PAR1 cleavage. These discoveries increasingly develop our understanding of the protein C pathway, which will conceivably expand its physiological implications to many areas in the future.

Hemophilia A (HA) is a bleeding disorder resulting from deficient Factor VIII (FVIII), which normally functions as a cofactor to activated Factor IX (FIXa) that facilitates activation of Factor X (FX). To mimic this property in a bispecific antibody (biAb) format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting biAb (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant (KD) of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with KD-values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by four orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation with potencies 13 and 18 times higher than a sequence-identical analog of emicizumab, respectively. A similar potency difference was observed in a tail-vein transection model in hemophilia A mice, while reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetics of Mim8 were investigated and a half-life of 14 days demonstrated in cynomolgus monkey. In conclusion, Mim8 is a FVIIIa-mimetic with a potent and efficacious hemostatic effect based on preclinical data.

Summary. Background: Factor seven activating protease (FSAP) was initially reported as an activator of single-chain urokinase-type plasminogen activator (scuPA) and factor VII (FVII). Subsequently, numerous additional substrates have been identified, and multiple other biological effects have been reported. Due to the apparent lack of specificity, the physiological role of FSAP has become increasingly unclear. Rigorous studies have been limited by the difficulty of obtaining intact FSAP from blood or recombinant sources. Objectives: Our aim was to produce intact recombinant human FSAP, and to assess its role as a trigger of coagulation and fibrinolysis. Results: Expression of wild-type FSAP in various mammalian cells invariably resulted in the accumulation of degraded FSAP due to autoactivation and degradation. To overcome this problem, we constructed a variant in which Arg 313 at the natural activation site was replaced by Gln, creating a cleavage site for the bacterial protease thermolysin. HEK293 cells produced FSAP R313Q in its intact form. Thermolysin-activated FSAP displayed the same reactivity toward the substrate S-2288 as plasma-derived FSAP, and retained its ability to activate scuPA. Polyphosphate and heparin increased V max by 2-3-fold, without affecting K m (62 nM) of scuPA activation. Surprisingly, FVII activation by activated FSAP proved negligible, even in the presence of calcium ions, phospholipid vesicles and recombinant soluble tissue factor. On membranes of 100% cardiolipin FVII cleavage did occur, but this resulted in transient activation and rapid degradation. Conclusions: While FSAP indeed activates scuPA, FVII appears remarkably resistant to activation. Therefore, reappraisal of the putative role of FSAP in hemostasis seems appropriate.

Objective. Removal of dead cells is essential in the maintenance of tissue homeostasis, and efficient removal prevents exposure of intracellular content to the immune system, which could lead to autoimmunity. The plasma protease factor VII-activating protease (FSAP) can release nucleosomes from late apoptotic cells. FSAP circulates as an inactive single-chain protein, which is activated upon contact with either apoptotic cells or necrotic cells. The purpose of this study was to investigate the role of FSAP in the release of nucleosomes from necrotic cells.Methods. Necrotic Jurkat cells were incubated with serum, purified 2-chain FSAP, and/or DNase I. Nucleosome release was analyzed by flow cytometry, and agarose gel electrophoresis was performed to detect DNA breakdown.Results. Incubation with serum released nucleosomes from necrotic cells. Incubation with FSAPdeficient serum or serum in which FSAP was inhibited by a blocking antibody was unable to release nucleosomes from necrotic cells, confirming that FSAP is indeed the essential serum factor in this process. Together with serum DNase I, FSAP induced the release of DNA from the cells, the appearance of nucleosomes in the supernatant, and the fragmentation of chromatin into eventually mononucleosomes.Conclusion. FSAP and DNase I are the essential serum factors that cooperate in necrotic cell DNA degradation and nucleosome release. We propose that this mechanism may be important in the removal of potential autoantigens.

• Factor Xa activates PAR3 in the presence of EPCR by noncanonical cleavage at Arg41.• Noncanonical PAR3 activation induces Tie2 activation, upregulation and redistribution of ZO-1, and stabilization of tight junctions.Endothelial barrier protective effects of activated protein C (APC) require the endothelial protein C receptor (EPCR), protease-activated receptor (PAR) 1, and PAR3. In contrast, PAR1 and PAR3 activation by thrombin results in barrier disruption. Noncanonical PAR1 and PAR3 activation by APC vs canonical activation by thrombin provides an explanation for the functional selectivity of these proteases. Here we found that factor Xa (FXa) activated PAR1 at canonical Arg41 similar to thrombin but cleaved PAR3 at noncanonical Arg41 similar to APC. This unique PAR1-PAR3 activation profile permitted the identification of noncanonical PAR3 activation as a novel activation pathway for barrier protective tunica intima endothelial receptor tyrosine kinase 2 (Tie2). APC, FXa, and the noncanonical PAR3 tetheredligand peptide induced prolonged activation of Tie2, whereas thrombin and the canonical PAR3 tethered-ligand peptide did not. Tie2 activation by FXa required PAR3 and EPCR. FXa and the noncanonical PAR3 tethered-ligand peptide induced Tie2-and PAR3-dependent upregulation of tight-junction-associated protein zona occludens 1 (ZO-1), translocation of ZO-1 to cell-cell borders, and the formation of typical ZO-1 honeycomb patterns that are indicative of tight-junction stabilization. These data provide intriguing novel insights into the diversification of functional selectivity of protease signaling achievable by canonical and noncanonical PAR activation, such as the activation of vascular-protective Tie2 by noncanonical PAR3 activation. (Blood. 2014;124(23):3480-3489) Introduction Protease-activated receptors (PARs) are G protein-coupled receptors that comprise a subfamily of 4 receptors (PAR1, PAR2, PAR3, and PAR4). The PARs are unique in that they carry their own encrypted ligand encoded in the extracellular N-terminal tail. Proteolysis by coagulation or vascular proteases creates a new N-terminal tethered ligand that activates the PAR. Multiple proteases can activate PARs with each protease displaying a unique specificity for the different receptors.1 Efficient activation of PAR1 by thrombin is driven by binding of exosite I to the hirudin-like sequence of PAR1, thereby optimally positioning Arg41 in the active site of thrombin.2 Other proteases make use of coreceptors for efficient PAR activation. For instance, tissue factor permits PAR1 and PAR2 activation by the ternary complex, and the endothelial protein C receptor (EPCR) enhances activation of PAR1, PAR2, and PAR3 by activated protein C (APC). 3-5The requirement of PAR1 for APC's cytoprotective effects created a conundrum because PAR1 activation by thrombin generally results in opposite proinflammatory and endothelial barrier disruptive effects. 5,6The discordant effects of PAR1 activation by thrombin vs APC are perhaps most apparent for the regula...