A factor VIIIa-mimetic bispecific antibody, Mim8, ameliorates bleeding upon severe vascular challenge in hemophilia A mice.

Hemophilia A 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 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 bispecific antibody (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with equilibrium dissociation constant 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 4 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 analogue of emicizumab. A similar potency difference was observed in a tail vein transection model in hemophilia A mice, whereas reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetic parameters of Mim8 were investigated and a half-life of 14 days shown in cynomolgus monkeys. In conclusion, Mim8 is an activated FVIII mimetic with a potent and efficacious hemostatic effect based on preclinical data.

Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2.

HABP2 (hyaluronan-binding protein 2) is a Ca2+-dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg I polymorphism, reportedly affects HABP2 function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to activate the single-chain urokinase-type plasminogen activator, and the G221A and G221S variants displayed moderately reduced single-chain urokinase-type plasminogen activator activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A being the least defective. In the absence of Ca2+, S-2288 cleavage by wild-type HABP2 was Na+-dependent, with Km decreasing from 3.0 to 0.6 mm upon titration from 0 to 0.3 m Na+ In the presence of 5 mm Ca2+, Km was further reduced to 0.05 mm, but without an appreciable contribution of Na+ At physiological concentrations of Na+ and Ca2+, the three HABP2 variants, and particularly G221E, displayed a major Km increase for S-2288. Chemical footprinting revealed that Ile-16 is significantly less protected from chemical modification in G221E than in wild-type HABP2, suggesting impaired insertion of the N terminus into the G221E protease domain, with a concomitant impact on catalytic activity. Homology modeling suggested that the Glu-221 side chain could sterically hinder insertion of the N terminus into the HABP2 protease domain, helping to explain the detrimental effects of Glu-221 substitution on HABP2 activity.

Noncanonical PAR3 activation by factor Xa identifies a novel pathway for Tie2 activation and stabilization of vascular integrity.

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 tethered-ligand 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.

Phosphorylation of protein S by platelet kinases enhances its activated protein C cofactor activity.

Protein S (PS) is a multifunctional plasma protein of the hemostatic and inflammatory pathways, although mechanisms for its regulation are poorly understood. Since certain plasma proteins are regulated through extracellular phosphorylation, we investigated whether the anticoagulant activity of PS is regulated through phosphorylation by platelet-secreted kinases. PS was phosphorylated on exposure to activated platelets or their releasates, as judged by immunoblotting for phospho-amino acids and PS. PS phosphorylation was reduced by specific inhibitors of casein kinase 1 (CK1) and casein kinase 2 (CK2) (10 µM D4476, 100 µM CK2-inhibitory peptide YNLKSKSSEDIDESS). Involvement of CKs in PS phosphorylation was confirmed using purified CK1/CK2. Phosphorylation of PS by purified CK1 did not affect its activated protein C (APC) cofactor activity in activated partial thromboplastin time assays in PS-depleted plasma. However, phosphorylation of PS by CK2 or by CK1/CK2 increased PS cofactor activity ∼1.5-fold (158.7±4.8%, P<0.01) or ∼2-fold (191.5±6.4%, P<0.0001), respectively. The APC cofactor activity of PS in PS-depleted plasma exposed to platelet-secreted kinases was enhanced, while CK2 but not CK1 inhibitors reduced APC cofactor activity. Mass spectrometry revealed a phosphorylated CK2 site at Thr37 within the N-terminal Gla-domain. Thus, platelet-mediated extracellular phosphorylation of PS is a potential mechanism by which its activity is regulated.

Cell painting with an engineered EPCR to augment the protein C system.

The protein C (PC) system conveys beneficial anticoagulant and cytoprotective effects in numerous in vivo disease models. The endothelial protein C receptor (EPCR) plays a central role in these pathways as cofactor for PC activation and by enhancing activated protein C (APC)-mediated protease-activated receptor (PAR) activation. During inflammatory disease, expression of EPCR on cell membranes is often diminished thereby limiting PC activation and APC's effects on cells. Here a caveolae-targeting glycosylphosphatidylinositol (GPI)-anchored EPCR (EPCR-GPI) was engineered to restore EPCR's bioavailability via "cell painting." The painting efficiency of EPCR-GPI on EPCR-depleted endothelial cells was time- and dose-dependent. The EPCR-GPI bioavailability after painting was long lasting since EPCR surface levels reached 400 % of wild-type cells after 2 hours and remained > 200 % for 24 hours. EPCR-GPI painting conveyed APC binding to EPCR-depleted endothelial cells where EPCR was lost due to shedding or shRNA. EPCR painting normalised PC activation on EPCR-depleted cells indicating that EPCR-GPI is functional active on painted cells. Caveolin-1 lipid rafts were enriched in EPCR after painting due to the GPI-anchor targeting caveolae. Accordingly, EPCR painting supported PAR1 and PAR3 cleavage by APC and augmented PAR1-dependent Akt phosphorylation by APC. Thus, EPCR-GPI painting achieved physiological relevant surface levels on endothelial cells, restored APC binding to EPCR-depleted cells, supported PC activation, and enhanced APC-mediated PAR cleavage and cytoprotective signalling. Therefore, EPCR-GPI provides a novel tool to restore the bioavailability and functionality of EPCR on EPCR- depleted and -deficient cells.

Cooperation of factor VII-activating protease and serum DNase I in the release of nucleosomes from necrotic cells.

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 FSAP-deficient 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.

Down-regulation of the clotting cascade by the protein C pathway.

The protein C pathway provides important biological activities to maintain the fluidity of the circulation, prevent thrombosis, and protect the integrity of the vasculature in response to injury. Activated protein C (APC), in concert with its cofactors and cell receptors, assembles in specific macromolecular complexes to provide efficient proteolysis of multiple substrates that result in anticoagulant and cytoprotective activities. Numerous studies on APC's structure-function relation with its cofactors, cell receptors, and substrates provide valuable insights into the molecular mechanisms and presumed assembly of the macromolecular complexes that are responsible for APC's activities. These insights allow for molecular engineering approaches specifically targeting the interaction of APC with one of its substrates or cofactors. Thus far, these approaches resulted in several anticoagulant-selective and cytoprotective-selective APC mutants, which provide unique insights into the relative contributions of APC's anticoagulant or cytoprotective activities to the beneficial effects of APC in various murine injury and disease models. Because of its multiple physiological and pharmacological activities, the anticoagulant and cytoprotective protein C pathway have important implications for the (patho)physiology of vascular disease and for translational research exploring novel therapeutic strategies to combat complex medical disorders such as thrombosis, inflammation, ischemic stroke and neurodegenerative disease.

Platelet protein S directly inhibits procoagulant activity on platelets and microparticles.

Anticoagulant plasma protein S (PS) is essential for maintaining haemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the "thrombin-sensitive region", abolishing activated protein C (APC) cofactor activity. However, we showed that cleaved PS retains APC-independent anticoagulant activities ("PS-direct"). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p<0.0004, n=20) more thrombin generation upon addition of FXa and prothrombin. PltPS exerted PS-direct that was similar to or greater than that of Zn(2+)-containing pdPS and much greater than that of Zn(2+)-deficient pdPS. Findings were confirmed using purified pltPS. Platelet-bound pltPS and microparticle-bound pltPS had similar PS-direct. Finally, platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 1.5 ± 0.2-fold (p<0.0001, n=11) more FXa generation upon addition of TF/FVIIa and FX. Thus, pltPS inhibits both prothrombinase and extrinsic FXase activities. Neutralising antibodies against APC and TFPI had no effect on the PS-direct of pltPS or pdPS on platelets. This study indicates that pltPS may be an essential pool of PS that counterbalances procoagulant activities on platelets.