Publications by authors named "Laurent O Mosnier"

63 Publications

Activated protein C and PAR1-derived and PAR3-derived peptides are anti-inflammatory by suppressing macrophage NLRP3 inflammasomes.

J Thromb Haemost 2021 01 7;19(1):269-280. Epub 2020 Dec 7.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA.

Essentials Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective effects. We tested whether APC or non-canonical PAR-derived peptides suppress inflammasome activity. APC or PAR1- and PAR3-derived peptides restrict inflammasome-dependent caspase-1 activity. Combined PAR1-derived and PAR3-derived peptides synergistically suppress caspase-1 activity. ABSTRACT: Background Activated protein C (APC) has been shown to restrict murine inflammasome activity. However, whether APC can exert anti-inflammatory activity in part through suppression of inflammasome activation in human systems is unknown. Objectives Studies were made to determine whether either APC or protease activated receptor (PAR)-derived peptides can reduce NLRP3 inflammasome activity in differentiated human THP-1 macrophage-like cells or in primary human monocytes stimulated to activate the inflammasome. Methods Human THP-1 cells or primary human monocytes were differentiated, treated with APC or PAR-derived peptides, and then stimulated with lipopolysaccharide and ATP to induce caspase-1 activity, a product of inflammasome activation. Results Activated protein C or noncanonical PAR1-derived or PAR3-derived peptides significantly reduced caspase-1 activity, detection of fluorescent NLRP3, and IL-1β release from THP-1 cells. At low concentrations where no effect was observed for each individual peptide, combinations of the PAR1-derived peptide and the PAR3-derived peptide resulted in a significant synergistic decrease in caspase-1 and IL-1β release. Caspase-1 activity was also reduced in primary human monocytes. Studies using blocking antibodies and small molecule PAR1 inhibitors suggest that EPCR, PAR1, and PAR3 each play roles in the observed anti-inflammatory effects. Several shortened versions of the PAR1- and PAR3-derived peptide reduced caspase-1 activity and exhibited synergistic anti-inflammatory effects. Conclusions The results indicate that both APC and certain PAR1- and PAR3-derived peptides, which are biased agonists for PAR1 or PAR3, can reduce inflammasome activity in stimulated human monocytes as measured by caspase-1 activity and IL-1β release and that PAR-derived biased peptide agonist combinations are synergistically anti-inflammatory.
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http://dx.doi.org/10.1111/jth.15133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790994PMC
January 2021

An engineered factor Va prevents bleeding induced by direct-acting oral anticoagulants by different mechanisms.

Blood Adv 2020 08;4(15):3716-3727

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA.

Control of bleeding with direct-acting oral anticoagulants (DOACs) remains an unmet clinical need. Activated superFactor V (superFVa) is an engineered activated protein C (APC)-resistant FVa variant with enhanced procoagulant activity resulting from an A2/A3 domain disulfide bond and was studied here for control of DOAC-induced bleeding. SuperFVa reversed bleeding induced by FXa inhibitors (rivaroxaban, apixaban), and the FIIa inhibitor dabigatran in BalbC mice. The blocking anti-protein C and APC [(A)PC] antibody SPC-54 also reduced FXa inhibitor induced bleeding similar to superFVa, whereas dabigatran-induced bleeding was not affected. This indicated that sufficient APC was generated to contribute to bleeding in the presence of FXa inhibitors, but not in the presence of dabigatran, suggesting that mechanisms contributing to bleeding differed for FXa and FIIa inhibitors. Despite different mechanisms contributing to bleeding, superFVa effectively reduced bleeding for all DOACs, indicating the versatility of superFVa's properties that contribute to its universal prohemostatic effects for DOAC associated bleeding. Supported by thrombin generation assays on endothelial cells in normal plasma spiked with DOACs and patient plasma anticoagulated with DOACs, 3 complementary mechanisms were identified by which superFVa achieved DOAC class-independent prohemostatic efficiency. These mechanisms are resistance to inactivation by APC, overcoming the FV activation threshold, and maximizing the efficiency of the prothrombinase complex when the available FXa is increased by FVIIa-based prohemostatics. In summary, it is this versatility of superFVa that delineates it from other prohemostatic agents as a promising class-independent rescue agent in bleeding situations associated with DOACs.
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http://dx.doi.org/10.1182/bloodadvances.2020001699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422119PMC
August 2020

How much clotting is enough?

Blood 2020 08;136(6):651-652

The Scripps Research Institute.

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http://dx.doi.org/10.1182/blood.2020006443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414588PMC
August 2020

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Nat Commun 2020 06 12;11(1):2992. Epub 2020 Jun 12.

Pathology/Toxicology, Bayer AG, 42096, Wuppertal, Germany.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.
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http://dx.doi.org/10.1038/s41467-020-16720-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293249PMC
June 2020

Cardiac Myosin Promotes Thrombin Generation and Coagulation In Vitro and In Vivo.

Arterioscler Thromb Vasc Biol 2020 04 27;40(4):901-913. Epub 2020 Feb 27.

From the Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA (J.Z.-R., H.D., M.S., J.N.O., Z.G., T.W., L.O.M., Z.M.R., J.H.G.).

Objective: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM's ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM's in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM's procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor).

Conclusions: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM's procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM's pathophysiology and its mechanistic influences on hemostasis or thrombosis.
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http://dx.doi.org/10.1161/ATVBAHA.120.313990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135739PMC
April 2020

C-terminal residues of activated protein C light chain contribute to its anticoagulant and cytoprotective activities.

J Thromb Haemost 2020 05 5;18(5):1027-1038. Epub 2020 Mar 5.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA.

Background: Activated protein C (APC) is an important homeostatic blood coagulation protease that conveys anticoagulant and cytoprotective activities. Proteolytic inactivation of factors Va and VIIIa facilitated by cofactor protein S is responsible for APC's anticoagulant effects, whereas cytoprotective effects of APC involve primarily the endothelial protein C receptor (EPCR), protease activated receptor (PAR)1 and PAR3.

Objective: To date, several binding exosites in the protease domain of APC have been identified that contribute to APC's interaction with its substrates but potential contributions of the C-terminus of the light chain have not been studied in detail.

Methods: Site-directed Ala-scanning mutagenesis of six positively charged residues within G142-L155 was used to characterize their contributions to APC's anticoagulant and cytoprotective activities.

Results And Conclusions: K151 was involved in protein S dependent-anticoagulant activity of APC with some contribution of K150. 3D structural analysis supported that these two residues were exposed in an extended protein S binding site on one face of APC. Both K150 and K151 were important for PAR1 and PAR3 cleavage by APC, suggesting that this region may also mediate interactions with PARs. Accordingly, APC's cytoprotective activity as determined by endothelial barrier protection was impaired by Ala substitutions of these residues. Thus, both K150 and K151 are involved in APC's anticoagulant and cytoprotective activities. The differential contribution of K150 relative to K151 for protein S-dependent anticoagulant activity and PAR cleavage highlights that binding exosites for protein S binding and for PAR cleavage in the C-terminal region of APC's light chain overlap.
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http://dx.doi.org/10.1111/jth.14756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380734PMC
May 2020

Activated protein C anticoagulant activity is enhanced by skeletal muscle myosin.

Haematologica 2020 08 19;105(8):e424-e427. Epub 2019 Dec 19.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA

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http://dx.doi.org/10.3324/haematol.2019.242982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395292PMC
August 2020

Role of thrombomodulin expression on hematopoietic stem cells.

J Thromb Haemost 2020 01 6;18(1):123-135. Epub 2019 Nov 6.

Blood Research Institute, BloodCenter of Wisconsin: Part of Versiti, Milwaukee, WI, USA.

Background: Activation of protease-activated receptor 1 (PAR1) by either thrombin or activated protein C (aPC) differentially regulate the quiescence and bone marrow (BM) retention of hematopoietic stem cells (HSC). Murine HSC co-express THBD, PAR1, and endothelial protein C receptor (EPCR), suggesting that HSC sustain quiescence in a quasi-cell autonomous manner due to the binding of thrombin present in the microenvironment to THBD, activation of EPCR-bound protein C by the thrombin-THBD-complex, and subsequent activation of PAR1 by the aPC-EPCR complex.

Objective: To determine the role of THBD expression on HSC for sustaining stem cell quiescence and BM retention under homeostatic conditions.

Methods: Hematopoietic stem cell function was analyzed in mice with constitutive or temporally controlled complete THBD-deficiency by flow cytometry, functional assays, and single cell RNA profiling.

Results: THBD was expressed in mouse, but not human, HSC, progenitors, and immature B cells. Expression in vascular endothelium was conserved in humans' BM. Mice with constitutive THBD deficiency had a normal peripheral blood profile, altered BM morphology, reduced numbers of progenitors and immature B cells, pronounced extramedullary hematopoiesis, increased HSC frequency, and marginally altered transcriptionally defined HSC stemness. Transplantation experiments indicated near normal engraftment and repopulating ability of THBD-deficient HSC. Transgenic aPC supplementation normalized BM histopathology and HSC abundance, and partially restored transcriptional stemness, but had no effect on B cell progenitors and extramedullary hematopoiesis. Temporally controlled THBD gene ablation in adult mice did not cause the above abnormalities.

Conclusion: THBD expression on HSPC has minor effects on homeostatic hematopoiesis in mice, and is not conserved in humans.
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http://dx.doi.org/10.1111/jth.14663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940513PMC
January 2020

TAFI deficiency causes maladaptive vascular remodeling after hemophilic joint bleeding.

JCI Insight 2019 10 3;4(19). Epub 2019 Oct 3.

Deptartment of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA.

Excessive vascular remodeling is characteristic of hemophilic arthropathy (HA) and may contribute to joint bleeding and the progression of HA. Mechanisms for pathological vascular remodeling after hemophilic joint bleeding are unknown. In hemophilia, activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is impaired, which contributes to joint bleeding and may also underlie the aberrant vascular remodeling. Here, hemophilia A (factor VIII-deficient; FVIII-deficient) mice or TAFI-deficient mice with transient (antibody-induced) hemophilia A were used to determine the role of FVIII and TAFI in vascular remodeling after joint bleeding. Excessive vascular remodeling and vessel enlargement persisted in FVIII-deficient and TAFI-deficient mice, but not in transient hemophilia WT mice, after similar joint bleeding. TAFI-overexpression in FVIII-deficient mice prevented abnormal vessel enlargement and vascular leakage. Age-related vascular changes were observed with FVIII or TAFI deficiency and correlated positively with bleeding severity after injury, supporting increased vascularity as a major contributor to joint bleeding. Antibody-mediated inhibition of uPA also prevented abnormal vascular remodeling, suggesting that TAFI's protective effects include inhibition of uPA-mediated plasminogen activation. In conclusion, the functional TAFI deficiency in hemophilia drives maladaptive vascular remodeling in the joints after bleeding. These mechanistic insights allow targeted development of potentially new strategies to normalize vascularity and control rebleeding in HA.
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http://dx.doi.org/10.1172/jci.insight.128379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795396PMC
October 2019

Activated protein C in neuroprotection and malaria.

Curr Opin Hematol 2019 09;26(5):320-330

Department of Molecular Medicine (IMM-315), The Scripps Research Institute, La Jolla, California, USA.

Purpose Of Review: Activated protein C (APC) is a homeostatic coagulation protease with anticoagulant and cytoprotective activities. Focusing on APC's effects in the brain, this review discusses three different scenarios that illustrate how APC functions are intimately affecting the physiology and pathophysiology of the brain.

Recent Findings: Cytoprotective APC therapy holds promise for the treatment of ischemic stroke, and a recently completed trial suggested that cytoprotective-selective 3K3A-APC reduced bleeding in ischemic stroke patients. In contrast, APC's anticoagulant activity contributes to brain bleeding as shown by the disproportional upregulation of APC generation in cerebral cavernous malformations lesions in mice. However, too little APC generation also contributes to maladies of the brain, such as in case of cerebral malaria where the binding of infected erythrocytes to the endothelial protein C receptor (EPCR) may interfere with the EPCR-dependent functions of the protein C pathway. Furthermore, discoveries of new activities of APC such as the inhibition of the NLRP3-mediated inflammasome and of new applications of APC therapy such as in Alzheimer's disease and graft-versus-host disease continue to advance our knowledge of this important proteolytic regulatory system.

Summary: APC's many activities or lack thereof are intimately involved in multiple neuropathologies, providing abundant opportunities for translational research.
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http://dx.doi.org/10.1097/MOH.0000000000000528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840173PMC
September 2019

Mechanisms of vascular permeability and remodeling associated with hemarthrosis in factor VIII-deficient mice.

J Thromb Haemost 2019 11 9;17(11):1815-1826. Epub 2019 Aug 9.

Division of Hematology/Oncology, Department of Medicine, University of California San Diego, La Jolla, CA.

Background: Vascular remodeling associated with hemophilic arthropathy (HA) may contribute to bleed propagation, but the mechanisms remain poorly understood.

Objectives: To explore molecular mechanisms of HA and the effects of hemostasis correction on synovial vascular remodeling after joint injury in hypocoagulable mice.

Methods: Factor VIII (FVIII)-deficient mice +/- FVIII treatment and hypocoagulable wild-type mice ( BALB/c) were subjected to subpatellar puncture. BALB/c mice were treated with warfarin and anti-FVIII before injury, after which warfarin was continued for 2 weeks or reversed +/- continuous anti-FVIII until harvest. Synovial vascularity was analyzed at baseline and 2 to 4 weeks post injury by histology, musculoskeletal ultrasound with power Doppler (microvascular flow), and Evans blue extravasation (vascular permeability). Synovial gene expression and systemic markers of vascular collagen turnover were studied in FVIII-deficient mice by RNA sequencing and enzyme-linked immunosorbent assay.

Results: Vascular changes occurred in FVIII-deficient and BALB/c mice after injury with minimal effect of hemostasis correction. Increased vascular permeability was only significant in FVIII-deficient mice, who exhibited more pronounced vascular remodeling than BALB/c mice despite similar bleed volumes. FVIII-deficient mice exhibited a strong transcriptional response in synovium that was only partially affected by FVIII treatment and involved genes relating to angiogenesis and extracellular matrix remodeling, with vascular collagen turnover markers detected systemically.

Conclusions: Intact hemostasis at the time of hemarthrosis and during healing are both critical to prevent vascular remodeling, which appears worse with severe and prolonged FVIII deficiency. Unbiased RNA sequencing revealed potential targets for intervention and biomarker development to improve management of HA.
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http://dx.doi.org/10.1111/jth.14567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824926PMC
November 2019

Cerebral cavernous malformations form an anticoagulant vascular domain in humans and mice.

Blood 2019 01 15;133(3):193-204. Epub 2018 Nov 15.

Department of Medicine, University of California, San Diego, La Jolla, CA.

Cerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of ( ) or ( ), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of or Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the gene decreases brain hemorrhage in mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.
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http://dx.doi.org/10.1182/blood-2018-06-856062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337879PMC
January 2019

Erratum to: Vascular Permeability and Remodelling Coincide with Inflammatory and Reparative Processes after Joint Bleeding in Factor VIII-Deficient Mice.

Thromb Haemost 2019 09 2;119(9):1546. Epub 2018 Aug 2.

Division of Hematology/Oncology, Department of Medicine, University of California San Diego, La Jolla, California, United States.

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http://dx.doi.org/10.1055/s-0038-1668499DOI Listing
September 2019

Defective TAFI activation in hemophilia A mice is a major contributor to joint bleeding.

Blood 2018 10 19;132(15):1593-1603. Epub 2018 Jul 19.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA.

Joint bleeds are common in congenital hemophilia but rare in acquired hemophilia A (aHA) for reasons unknown. To identify key mechanisms responsible for joint-specific bleeding in congenital hemophilia, bleeding phenotypes after joint injury and tail transection were compared in aHA wild-type (WT) mice (receiving an anti-factor VIII [FVIII] antibody) and congenital HA (FVIII) mice. Both aHA and FVIII mice bled severely after tail transection, but consistent with clinical findings, joint bleeding was notably milder in aHA compared with FVIII mice. Focus was directed to thrombin-activatable fibrinolysis inhibitor (TAFI) to determine its potentially protective effect on joint bleeding in aHA. Joint bleeding in TAFI mice with anti-FVIII antibody was increased, compared with WT aHA mice, and became indistinguishable from joint bleeding in FVIII mice. Measurements of circulating TAFI zymogen consumption after joint injury indicated severely defective TAFI activation in FVIII mice in vivo, consistent with previous in vitro analyses in FVIII-deficient plasma. In contrast, notable TAFI activation was observed in aHA mice, suggesting that TAFI protected aHA joints against bleeding. Pharmacological inhibitors of fibrinolysis revealed that urokinase-type plasminogen activator (uPA)-induced fibrinolysis drove joint bleeding, whereas tissue-type plasminogen activator-mediated fibrinolysis contributed to tail bleeding. These data identify TAFI as an important modifier of hemophilic joint bleeding in aHA by inhibiting uPA-mediated fibrinolysis. Moreover, our data suggest that bleed protection by TAFI was absent in congenital FVIII mice because of severely defective TAFI activation, underscoring the importance of clot protection in addition to clot formation when considering prohemostatic strategies for hemophilic joint bleeding.
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http://dx.doi.org/10.1182/blood-2018-01-828434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182268PMC
October 2018

Warfarin, a juggler's demise.

Blood 2018 06;131(25):2742-2743

The Scripps Research Institute.

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http://dx.doi.org/10.1182/blood-2018-05-843151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014355PMC
June 2018

Activated protein C, protease activated receptor 1, and neuroprotection.

Blood 2018 07 4;132(2):159-169. Epub 2018 Jun 4.

The Scripps Research Institute, La Jolla, CA.

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.
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http://dx.doi.org/10.1182/blood-2018-02-769026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6043978PMC
July 2018

Vascular Permeability and Remodelling Coincide with Inflammatory and Reparative Processes after Joint Bleeding in Factor VIII-Deficient Mice.

Thromb Haemost 2018 06 30;118(6):1036-1047. Epub 2018 May 30.

Division of Hematology/Oncology, Department of Medicine, University of California San Diego, La Jolla, California, United States.

Vascular remodelling is a prominent feature of haemophilic arthropathy (HA) that may underlie re-bleeding, yet the nature of vascular changes and underlying mechanisms remain largely unknown. Here, we aimed to characterize synovial vascular remodelling and vessel integrity after haemarthrosis, as well as temporal changes in inflammatory and tissue-reparative pathways. Thirty acutely painful joints in patients with haemophilia (PWH) were imaged by musculoskeletal ultrasound with Power Doppler (MSKUS/PD) to detect vascular abnormalities and bloody effusions. Nineteen out of 30 painful joint episodes in PWH were associated with haemarthrosis, and abnormal vascular perfusion was unique to bleeding joints. A model of induced haemarthrosis in factor VIII (FVIII)-deficient mice was used for histological assessment of vascular remodelling (α-smooth muscle actin [αSMA] expression), and monitoring of in vivo vascular perfusion and permeability by MSKUS/PD and albumin extravasation, respectively. Inflammatory (M1) and reparative (M2) macrophage markers were quantified in murine synovium over a 10-week time course by real-time polymerase chain reaction. The abnormal vascular perfusion observed in PWH was recapitulated in FVIII-deficient mice after induced haemarthrosis. Neovascularization and increased vessel permeability were apparent 2 weeks post-bleed in FVIII-deficient mice, after a transient elevation of inflammatory macrophage M1 markers. These vascular changes subsided by week 4, while vascular remodelling, evidenced by architectural changes and pronounced αSMA expression, persisted alongside a reparative macrophage M2 response. In conclusion, haemarthrosis leads to transient inflammation coupled with neovascularization and associated vascular permeability, while subsequent tissue repair mechanisms coincide with vascular remodelling. Together, these vascular changes may promote re-bleeding and HA progression.
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http://dx.doi.org/10.1055/s-0038-1641755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191040PMC
June 2018

PAR1 biased signaling is required for activated protein C in vivo benefits in sepsis and stroke.

Blood 2018 03 17;131(11):1163-1171. Epub 2018 Jan 17.

The Scripps Research Institute, La Jolla, CA.

Activated protein C (APC) cleaves protease-activated receptor 1 (PAR1) in vitro at R46 to initiate beneficial cell signaling; however, thrombin and APC can cleave at R41. To elucidate PAR1-dependent aspects of the pharmacologic in vivo mechanisms of APC, we generated C57BL/6 mouse strains carrying QQ41 or QQ46 point mutations in PAR1 ( gene). Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against ischemic stroke when mice carried PAR1-homozygous mutations that prevent cleavage at either R41 or R46. Intercrossing PAR1/R46Q mice generated expected numbers of PAR1, PAR1/R46Q, and R46Q/R46Q offspring whereas intercrossing PAR1/R41Q mice gave decreased R41Q/R41Q homozygotes (resembling intercrossing PAR1/PAR1-knockout mice). QQ41-PAR1 and QQ46-PAR1 brain endothelial cells showed the predicted retention or loss of cellular responses to thrombin receptor-activating peptide, thrombin, or APC for each PAR1 mutation. In sepsis studies, exogenous APC reduced mortality from 50% to 10% in -induced pneumonia for wild-type (Wt) PAR1 and QQ41-PAR1 mice ( < .01) but had no benefit for QQ46-PAR1 mice. In transient distal middle cerebral artery occlusion stroke studies, exogenous APC significantly reduced infarct size, edema, and neuronal apoptosis for Wt mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb-asymmetry and foot-fault tests at 24 hours after stroke induction, signaling-selective APC was beneficial for Wt and QQ41-PAR1 mice but not QQ46-PAR1 mice. These results support the concept that APC-induced, PAR1-dependent biased signaling following R46 cleavage is central to the in vivo benefits of APC.
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http://dx.doi.org/10.1182/blood-2017-10-810895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855020PMC
March 2018

Activated protein C light chain provides an extended binding surface for its anticoagulant cofactor, protein S.

Blood Adv 2017 Aug 7;1(18):1423-1426. Epub 2017 Aug 7.

Department of Molecular Medicine and.

Protein S anticoagulant cofactor sensitivity and PAR1 cleavage activity were assayed for 9 recombinant APC mutants.Residues L38, K43, I73, F95, and W115 on one face of the APC light chain define an extended surface containing the protein S binding site.
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http://dx.doi.org/10.1182/bloodadvances.2017007005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727852PMC
August 2017

2016 Scientific Sessions Sol Sherry Distinguished Lecturer in Thrombosis: Thrombotic Stroke: Neuroprotective Therapy by Recombinant-Activated Protein C.

Arterioscler Thromb Vasc Biol 2016 11 6;36(11):2143-2151. Epub 2016 Oct 6.

From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA (J.H.G., L.O.M., J.A.F.); Division of Hematology/Oncology, Department of Medicine, University of California, San Diego (J.H.G.); and Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles (B.V.Z.).

APC (activated protein C), derived from the plasma protease zymogen, is antithrombotic and anti-inflammatory. In preclinical injury models, recombinant APC provides neuroprotection for multiple injuries, including ischemic stroke. APC acts directly on brain endothelial cells and neurons by initiating cell signaling that requires multiple receptors. Two or more major APC receptors mediate APC's neuroprotective cell signaling. When bound to endothelial cell protein C receptor, APC can cleave protease-activated receptor 1, causing biased cytoprotective signaling that reduces ischemia-induced injury. Pharmacological APC alleviates bleeding induced by tissue-type plasminogen activator in murine ischemic stroke studies. Remarkably, APC's signaling promotes neurogenesis. The signaling-selective recombinant variant of APC, 3K3A-APC, was engineered to lack most of the APC's anticoagulant activity but retain APC's cell signaling actions. Recombinant 3K3A-APC is in ongoing National Institutes of Health (NIH)-funded clinical trials for ischemic stroke.
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http://dx.doi.org/10.1161/ATVBAHA.116.308038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5119536PMC
November 2016

The role of EPCR in the pathogenesis of severe malaria.

Thromb Res 2016 May;141 Suppl 2:S46-9

Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, University of Copenhagen and Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark.

Of the five Plasmodium species that infect humans, infection with P. falciparum is the most lethal, causing severe malaria syndromes, that result in over half a million annual deaths. With parasites becoming increasingly resistant to artemisinin there is an urgent need for new preventative and therapeutic options, for which understanding of the mechanisms that cause death and disability in malaria is essential. The recent discoveries that certain variants of P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on infected erythrocytes are intimately linked to the precipitation of severe malaria syndromes and that these PfEMP1 variants contain EPCR binding domains provides new opportunities to improve our understanding of the molecular mechanisms responsible for the pathogenesis of severe malaria. EPCR is known for its essential role in the protein C (PC) system and for its ability to support the cytoprotective effects of activated protein C (APC) that result in vascular and tissue protective effects in many organ systems of the body, including the brain, lung, kidney, and liver. Observations that binding of PfEMP1 to EPCR results in an acquired functional PC system deficiency support the new paradigm that EPCR plays a central role in the pathogenesis of severe malaria. Thus, targeting of the PfEMP1-EPCR interaction and restoring the functionality of the PC system may provide new strategies for the development of novel adjuvant therapies for severe malaria.
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http://dx.doi.org/10.1016/S0049-3848(16)30364-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481197PMC
May 2016

Safety, Stability and Pharmacokinetic Properties of (super)Factor Va, a Novel Engineered Coagulation Factor V for Treatment of Severe Bleeding.

Pharm Res 2016 06 9;33(6):1517-26. Epub 2016 Mar 9.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, California, USA.

Purpose: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here.

Methods: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline.

Results: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 μg/mL), (super)FVa did not (6.7 μg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 μg/mL) or saline (5.6 μg/mL).

Conclusion: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.
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http://dx.doi.org/10.1007/s11095-016-1895-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854778PMC
June 2016

Heterozygous congenital Factor VII deficiency with the 9729del4 mutation, associated with severe spontaneous intracranial bleeding in an adolescent male.

Blood Cells Mol Dis 2016 Mar 10;57:8-12. Epub 2015 Nov 10.

University of California San Diego, Department of Medicine, 9500 Gillman Drive, San Diego, CA 92093, USA; The Scripps Research Institute, Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Background: In congenital Factor (F) VII deficiency bleeding phenotype and intrinsic FVII activity levels don't always correlate. Patients with FVII activity levels <30% appear to have a higher bleeding propensity, but bleeding can also occur at higher FVII activity levels. Reasons for bleeding at higher FVII activity levels are unknown, and it remains challenging to manage such patients clinically.

Case: A 19year old male with spontaneous intracranial hemorrhage and FVII activity levels of 44%, requiring emergent surgical intervention and a strategy for FVII replacement. Genotyping showed the rare heterozygous FVII 9729del4 mutation. Bleed evacuation was complicated by epidural abscess requiring craniectomy, bone graft procedures, and prolonged administration of recombinant human (rh) activated FVII (FVIIa). The patient recovered without neurological deficits, and remains on prophylactic low dose treatment with rhFVIIa in relation to risky athletic activities.

Conclusion: For clinicians, it is important to recognize that effects of rhFVIIa within these pathways are independent of its contribution to blood clot formation and cannot be assessed by clotting assays. Reduced FVII levels should therefore not be dismissed, as even a mild reduction may result in spontaneous bleeding. Treatment of mild FVII deficiency requires a careful case-by-case approach, based on the clinical scenario.
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http://dx.doi.org/10.1016/j.bcmd.2015.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874642PMC
March 2016

Advances and challenges in hemophilic arthropathy.

Semin Hematol 2016 Jan 26;53(1):10-9. Epub 2015 Oct 26.

The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, CA, USA; University of California at San Diego, Department of Medicine, San Diego, CA, USA. Electronic address:

Hemophilic arthropathy is a form of joint disease that develops secondary to joint bleeding and presents with synovial hypertrophy, cartilage and bony destruction. The arthropathy can develop despite clotting factor replacement and is especially disabling in the aging population. Pathobiological tissue changes are triggered by release of hemoglobin and iron deposition in the joint, but the sequence of events and the molecular mechanisms resulting in joint deterioration are incompletely understood. Treatment options other than clotting factor replacement are limited. Improvements in the treatment of hemophilia necessitate a better understanding of the processes that lead to this disabling condition and better diagnostic tools. Towards that end, studies of the molecular mechanisms leading to the arthropathy, as well as the development of sensitive imaging techniques and biomarkers are needed. These will pave the way to identify the cause of acute pain such as joint bleeding or synovitis, detect early, potentially reversible structural changes, and predict progression of disease. This review describes current imaging techniques and the development of high resolution musculoskeletal ultrasound with power Doppler to afford point-of-care diagnosis and management, the potential utility of diagnostic biomarkers, and summarizes our current knowledge of the pathobiology of hemophilic arthropathy.
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http://dx.doi.org/10.1053/j.seminhematol.2015.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034876PMC
January 2016

Apolipoprotein E Receptor 2 Mediates Activated Protein C-Induced Endothelial Akt Activation and Endothelial Barrier Stabilization.

Arterioscler Thromb Vasc Biol 2016 Mar 21;36(3):518-24. Epub 2016 Jan 21.

From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA.

Objective: Activated protein C (APC), a plasma serine protease, initiates cell signaling that protects endothelial cells from apoptosis and endothelial barrier disruption. Apolipoprotein E receptor 2 (ApoER2; LRP8) is a receptor known for mediating signaling initiated by reelin in neurons. ApoER2 contributes to APC-initiated signaling in monocytic U937 cells. The objective was to determine whether ApoER2 is required for APC's beneficial signaling in the endothelial cell surrogate EA.hy926 line.

Approach And Results: We used small interfering RNA and inhibitors to probe requirements for specific receptors for APC's antiapoptotic activity and for phosphorylation of disabled-1 by Src family kinases and of Akt. When small interfering RNA for ApoER2 or endothelial cell protein C receptor or protease activated receptor 1 was used, APC's antiapoptotic activity was ablated, indicating that each of these receptors was required. In EA.hy926 cells, APC induced a 2- to 3-fold increased phosphorylation of Ser473-Akt and Tyr232-disabled-1, a phosphorylation known to trigger disabled-1-mediated signaling in other cell types. Ser473-Akt phosphorylation was inhibited by ApoER2 small interfering RNA or by inhibitors of Src (PP2), phosphatidylinositol-3 kinase (LY303511), and protease activated receptor 1 (SCH79797). ApoER2 small interfering RNA blocked the ability of APC to prevent thrombin-induced endothelial barrier disruption in TransEndothelial Resistance assays. Binding studies using purified APC and purified immobilized wild-type and mutated ApoER2 ectodomains suggested that APC binding involves Lys49, Asp50, and Trp64 on the surface of the N-terminal LA1 domain of ApoER2.

Conclusions: ApoER2 contributes cooperatively with endothelial cell protein C receptor and protease activated receptor 1 to APC-initiated endothelial antiapoptotic and barrier protective signaling.
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http://dx.doi.org/10.1161/ATVBAHA.115.306795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816599PMC
March 2016

Improved coagulation and haemostasis in haemophilia with inhibitors by combinations of superFactor Va and Factor VIIa.

Thromb Haemost 2016 Mar 15;115(3):551-61. Epub 2015 Oct 15.

Annette von Drygalski, MD, Pharm D, The Scripps Research Institute, Department of Molecular and Experimental Medicine, MEM 180, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA, Tel.: +1 858 784 8220, Fax: +1 858 784 2243, E-mail:

Bypassing inhibitors in haemophilia patients is limited to activated (a) Factor(F)VII products. We introduced "FVa activity augmentation" as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilising properties of superFVa and recombinant human (rh)FVIIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in haemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. SuperFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e. g. 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalise clot lysis times could be reduced by 100-fold (e. g. from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In haemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of haemophilia with inhibitors and it merits further evaluation for new bypassing strategies.
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http://dx.doi.org/10.1160/TH15-07-0525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775351PMC
March 2016

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

Thromb Haemost 2015 Nov 13;114(6):1144-55. Epub 2015 Aug 13.

Laurent O. Mosnier, Department of Molecular and Experimental Medicine (MEM-180), The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA, Tel.: +1 858 784 8220, Fax: +1 858 784 2243, E-mail:

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053618PMC
http://dx.doi.org/10.1160/TH15-01-0079DOI Listing
November 2015

Vascular remodeling underlies rebleeding in hemophilic arthropathy.

Am J Hematol 2015 Nov 8;90(11):1027-35. Epub 2015 Oct 8.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California.

Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal, and cartilage changes in response to a single induced hemarthrosis in the FVIII-deficient mouse. We found soft-tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft-tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII-deficient mice involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105), and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α-SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis and osteoarthritis and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees, and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease.
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http://dx.doi.org/10.1002/ajh.24133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618067PMC
November 2015

Protein C system defects inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant.

Thromb Haemost 2015 Nov 9;114(5):1038-48. Epub 2015 Jul 9.

Jens E. V. Petersen, Centre for Medical Parasitology, Dept. of International Health, Immunology & Microbiology, University of Copenhagen and Dept. of Infectious Diseases, Rigshospitalet, 1014 Copenhagen, Denmark, Tel.: +45 35327549, Fax: +45 35327851, E-mail:

The Endothelial Protein C receptor (EPCR) is essential for the anticoagulant and cytoprotective functions of the Protein C (PC) system. Selected variants of the malaria parasite protein, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) associated with severe malaria, including cerebral malaria, specifically target EPCR on vascular endothelial cells. Here, we examine the cellular response to PfEMP1 engagement to elucidate its role in malaria pathogenesis. Binding of the CIDRα1.1 domain of PfEMP1 to EPCR obstructed activated PC (APC) binding to EPCR and induced a loss of cellular EPCR functions. CIDRα1.1 severely impaired endothelial PC activation and effectively blocked APC-mediated activation of protease-activated receptor-1 (PAR1) and associated barrier protective effects of APC on endothelial cells. A soluble EPCR variant (E86A-sEPCR) bound CIDRα1.1 with high affinity and did not interfere with (A)PC binding to cellular EPCR. E86A-sEPCR used as a decoy to capture PfEMP1, permitted normal PC activation on endothelial cells, normal barrier protective effects of APC, and greatly reduced cytoadhesion of infected erythrocytes to brain endothelial cells. These data imply important contributions of PfEMP1-induced protein C pathway defects in the pathogenesis of severe malaria. Furthermore, the E86A-sEPCR decoy provides a proof-of-principle strategy for the development of novel adjunct therapies for severe malaria.
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http://dx.doi.org/10.1160/TH15-01-0018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142631PMC
November 2015

Activated protein C: biased for translation.

Blood 2015 May 30;125(19):2898-907. Epub 2015 Mar 30.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA;

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.
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http://dx.doi.org/10.1182/blood-2015-02-355974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424414PMC
May 2015
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