Publications by authors named "John H Griffin"

169 Publications

Sex-dependent effects of genetic upregulation of activated protein C on delayed effects of acute radiation exposure in the mouse heart, small intestine, and skin.

PLoS One 2021 24;16(5):e0252142. Epub 2021 May 24.

Department of Pharmaceutical Sciences, Division of Radiation Health, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America.

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart, small intestine and dorsal skin were collected for tissue analysis. Female APCHi mice showed the most severe radiation-induced deposition of cardiac collagens but were protected against a radiation-induced loss of microvascular density. Both male and female APCHi mice were protected against a radiation induced upregulation of toll-like receptor 4 in the heart, but this did not translate into a clear protection against immune cell infiltration. In the small intestine, the APCHi genotype had no effect on an increase in the number of myeloperoxidase positive cells (seen mostly in females) or an increase in the expression of T-cell marker CD2 (males). Lastly, both male and female APCHi mice were protected against radiation-induced epidermal thickening and increase in 3-nitrotyrosine positive keratinocytes. In conclusion, prolonged high levels of APC in a transgenic mouse model had little effects on indicators of DEARE in the heart, small intestine and skin, with some differential effects in male compared to female mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252142PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143413PMC
May 2021

Stroke Treatment With PAR-1 Agents to Decrease Hemorrhagic Transformation.

Front Neurol 2021 15;12:593582. Epub 2021 Mar 15.

Department of Physiology and Neuroscience, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, United States.

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.
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http://dx.doi.org/10.3389/fneur.2021.593582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005555PMC
March 2021

Addendum: American College of Medical Genetics consensus statement on factor V Leiden mutation testing.

Genet Med 2021 Mar 5. Epub 2021 Mar 5.

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

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http://dx.doi.org/10.1038/s41436-021-01108-xDOI Listing
March 2021

Activated Protein C (APC) and 3K3A-APC-Induced Regression of Choroidal Neovascularization (CNV) Is Accompanied by Vascular Endothelial Growth Factor (VEGF) Reduction.

Biomolecules 2021 02 26;11(3). Epub 2021 Feb 26.

Rabin Medical Center, Ophthalmology Department and Laboratory of Eye Research Felsenstein Medical Research Center, Petah-Tikva 49100, Israel.

The activated protein C (APC) ability to inhibit choroidal neovascularization (CNV) growth and leakage was recently shown in a murine model. A modified APC, 3K3A-APC, was designed to reduce anticoagulant activity while maintaining full cytoprotective properties, thus diminishing bleeding risk. We aimed to study the ability of 3K3A-APC to induce regression of CNV and evaluate vascular endothelial growth factor (VEGF) role in APC's activities in the retina. CNV was induced by laser photocoagulation on C57BL/6J mice. APC and 3K3A-APC were injected intravitreally after verification of CNV presence. CNV volume and vascular penetration were evaluated on retinal pigmented epithelium (RPE)-choroid flatmount by fluorescein isothiocyanate (FITC)-dextran imaging. VEGF levels were measured using immunofluorescence anti-VEGF staining. We found that 3K3A-APC induced regression of pre-existing CNV. VEGF levels, measured in the CNV lesion sites, significantly decreased upon APC and 3K3A-APC treatment. Reduction in VEGF was sustained 14 days post a single APC injection. As 3K3A-APC retained APCs' activities, we conclude that the anticoagulant properties of APC are not mandatory for APC activities in the retina and that VEGF reduction may contribute to the protective effects of APC and 3K3A-APC. Our results highlight the potential use of 3K3A-APC as a novel treatment for CNV and other ocular pathologies.
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http://dx.doi.org/10.3390/biom11030358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996919PMC
February 2021

Factor VIIa induces extracellular vesicles from the endothelium: a potential mechanism for its hemostatic effect.

Blood 2021 Jun;137(24):3428-3442

Department of Cellular and Molecular Biology, The University of Texas Health Science Center at Tyler, Tyler, TX.

Recombinant factor FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type (WT), EPCR-overexpressing, and PAR1-R46Q-mutant mice, but not EPCR-deficient or PAR1-R41Q-mutant mice. In vivo studies revealed that administration of FVIIa to WT, EPCR-overexpressing, and PAR1-R46Q-mutant mice, but not EPCR-deficient or PAR1-R41Q-mutant mice, increased the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.
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http://dx.doi.org/10.1182/blood.2020008417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8212509PMC
June 2021

Different DOACs Control Inflammation in Cardiac Ischemia-Reperfusion Differently.

Circ Res 2021 Feb 23;128(4):513-529. Epub 2020 Dec 23.

Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital, Leipzig, Germany (I.G., S.F., A.E., S.N., M.M.A.-D., R.R., J.M., D.G., R.B., S.K., B.I., K.S.).

Rationale: While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa (factor Xa) or aPC (activated protein C), independently modulate intracellular signaling via partially distinct receptors.

Objectives: To study the differential effects of fXa or fIIa (factor IIa) inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury.

Methods And Results: Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail-bleeding assay and FeCl-induced thrombosis). Myocardial ischemia-reperfusion injury was induced via left anterior descending ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNA sequencing, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL [interleukin]-1β, IL-6, and TNFα [tumor necrosis factor alpha]), as well as NF-κB (nuclear factor kappa B) and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post-myocardial ischemia-reperfusion injury. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect.

Conclusions: We showed that specific inhibition of coagulation via direct oral anticoagulants had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8293866PMC
February 2021

PAR1 regulation of CXCL1 expression and neutrophil recruitment to the lung in mice infected with influenza A virus.

J Thromb Haemost 2021 04 19;19(4):1103-1111. Epub 2021 Feb 19.

Department of Medicine, Division of Hematology and Oncology, UNC Blood Research Institute, University of North Carolina, Chapel Hill, NC, USA.

Background: Protease-activated receptor 1 (PAR1) is expressed in various immune cells and in the lung. We showed that PAR1 plays a role in Coxsackievirus B3 infection by enhancing toll-like receptor 3-dependent interferon- expression in cardiac fibroblasts.

Objectives: We investigated the role of PAR1 in a mouse model of influenza A virus (IAV) infection.

Methods: We used mice with either a global deficiency of PAR1, cell type-specific deficiencies of PAR1, or mutation of PAR1 at the R41 or R46 cleavage sites.

Results: PAR1-deficient mice had increased CXCL1 expression in the lung, increased neutrophil recruitment, increased protein levels in the bronchoalveolar lavage fluid, and increased mortality after IAV infection compared with control mice infected with IAV. Results from mice with cell type-specific deletion of PAR1 indicated that PAR1 expression by hematopoietic cells suppressed CXCL1 expression, whereas PAR1 expression by endothelial cells enhanced CXCL1 expression in response to IAV infection. PAR1 activation also enhanced polyinosinic:polycytodylic acid induction of interleukin-8 in a human endothelial cell line. Mutation of the R46 cleavage site of PAR1 was associated with increased CXCL1 expression in the lung in response to IAV infection, which suggested that R46 signaling suppresses CXCL1 expression.

Conclusions: These results indicate that PAR1 expression by different cell types and activation by different proteases modulates the immune response during IAV infection.
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http://dx.doi.org/10.1111/jth.15221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8048419PMC
April 2021

Skeletal muscle myosin and cardiac myosin attenuate heparin's antithrombin-dependent anticoagulant activity.

J Thromb Haemost 2021 02 10;19(2):470-477. Epub 2020 Dec 10.

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

Background: Heparin enhances the ability of the plasma protease inhibitor, antithrombin, to neutralize coagulation factor Xa and thrombin. Skeletal muscle myosin binds unfractionated heparin.

Objectives: The aim of this study was to investigate the influence of myosin binding to heparin on antithrombin's anticoagulant activity.

Methods: Inhibition of factor Xa and thrombin by antithrombin in the presence of different heparins and skeletal muscle myosin or cardiac myosin was studied by measuring inhibition of each enzyme's chromogenic substrate hydrolysis.

Results And Conclusions: Skeletal muscle myosin and cardiac myosin neutralized unfractionated heparin's enhancement of antithrombin's inhibition of purified factor Xa and thrombin. Skeletal muscle myosin also reduced the inhibition of factor Xa and thrombin by antithrombin in the presence of heparan sulfate. These two myosins did not protect factor Xa from antithrombin inhibition when tested in the presence of smaller heparins (eg, low molecular weight heparin, heparin pentasaccharide). This chain length dependence for skeletal muscle myosin's ability to reduce heparin's anticoagulant activity might have potential implications for therapy for patients who experience increases in plasma myosin levels (eg, acute trauma patients). In addition to the chain length, the type and extent of sulfation of glycosaminoglycans influenced the ability of skeletal muscle myosin to neutralize the polysaccharide's ability to enhance antithrombin's activity. In summary, these studies show that skeletal muscle myosin and cardiac myosin can influence antithrombin's anticoagulant activity against factor Xa and thrombin, implying that they may significantly influence the hemostatic balance involving bleeding vs clotting.
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http://dx.doi.org/10.1111/jth.15169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902397PMC
February 2021

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

Novel blood coagulation molecules: Skeletal muscle myosin and cardiac myosin.

J Thromb Haemost 2021 01 25;19(1):7-19. Epub 2020 Oct 25.

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

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. ABSTRACT: 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.
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http://dx.doi.org/10.1111/jth.15097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819347PMC
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

3K3A-Activated Protein C Variant Does Not Interfere With the Plasma Clot Lysis Activity of Tenecteplase.

Stroke 2020 07 17;51(7):2236-2239. Epub 2020 Jun 17.

Department of Molecular Medicine, Scripps Research Institute, La Jolla, CA (P.M., J.A.F., J.H.G.).

Background And Purpose: A recombinant engineered variant of APC (activated protein C), 3K3A-APC, lacks anticoagulant properties (<10%) while preserving APCs anti-inflammatory, anti-apoptotic, and neuroprotective functions and is very promising in clinical trials for ischemic stroke. Therapeutic intervention with single bolus administration of the third-generation tPA (tissue-type plasminogen activator), tenecteplase, is anticipated to be widely adopted for treatment of acute ischemic stroke. 3K3A-APC is well-tolerated in stroke patients dosed with alteplase, and in vitro studies show 3K3A-APC does not interfere with alteplase-induced clot lysis. The purpose of this in vitro study was to assess the influence of 3K3A-APC on tenecteplase-induced clot lysis.

Methods: Tenecteplase-mediated lysis of thrombin generated plasma clots of human normal pooled plasma was monitored in the presence of varying doses of 3K3A-APC. The effects on fibrinolysis by tenecteplase and alteplase were compared.

Results: The presence of 3K3A-APC shortened the time for clot lysis induced by tenecteplase at very low levels but not at higher therapeutic concentrations of tenecteplase. Comparisons of alteplase-mediated clot lysis to tenecteplase clot lysis showed that both thrombolytic agents behaved similarly in the presence of 3K3A-APC.

Conclusions: These results indicate that 3K3A-APC does not interfere with tenecteplase's clot lysis function.
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http://dx.doi.org/10.1161/STROKEAHA.120.028793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380733PMC
July 2020

COVID-19 hypothesis: Activated protein C for therapy of virus-induced pathologic thromboinflammation.

Res Pract Thromb Haemost 2020 May 12;4(4):506-509. Epub 2020 Jun 12.

Department of Neurology Cedars-Sinai Medical Center Los Angeles California USA.

Seriously ill patients with coronavirus disease 2019 (COVID-19) at risk for death exhibit elevated cytokine and chemokine levels and D-dimer, and they often have comorbidities related to vascular dysfunctions. In preclinical studies, activated protein C (APC) provides negative feedback downregulation of excessive inflammation and thrombin generation, attenuates damage caused by ischemia-reperfusion in many organs including lungs, and reduces death caused by bacterial pneumonia. APC exerts both anticoagulant activities and direct cell-signaling activities. Preclinical studies show that its direct cell-signaling actions mediate anti-inflammatory and anti-apoptotic actions, mortality reduction for pneumonia, and beneficial actions for ischemia-reperfusion injury. The APC mutant 3K3A-APC, which was engineered to have diminished anticoagulant activity while retaining cell-signaling actions, was safe in phase 1 and phase 2 human trials. Because of its broad spectrum of homeostatic effects in preclinical studies, we speculate that 3K3A-APC merits consideration for clinical trial studies in appropriately chosen, seriously ill patients with COVID-19.
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http://dx.doi.org/10.1002/rth2.12362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292662PMC
May 2020

Activated Protein C Attenuates Experimental Autoimmune Encephalomyelitis Progression by Enhancing Vascular Integrity and Suppressing Microglial Activation.

Front Neurosci 2020 15;14:333. Epub 2020 Apr 15.

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

Background: Activated protein C (APC), a serine protease with antithrombotic effects, protects in animal models of ischemic stroke by suppressing inflammation and enhancing vascular integrity, angiogenesis, neurogenesis and neuroprotection. A small number of animal studies suggest it might also have therapeutic potential in multiple sclerosis (MS), though results have been mixed. Based on these conflicting data, the goals of this study were to clarify the therapeutic potential of APC in the experimental autoimmune encephalomyelitis (EAE) model of MS and to determine mechanistically how APC mediates this protective effect.

Methods: The protective potential of APC was examined in a chronic progressive model of EAE. Vascular breakdown, tight junction protein expression and vascular expression of fibronectin and α5β1 integrin as well as vascularity and glial activation were analyzed using immunofluorescence (IF) of spinal cord sections taken from mice with established EAE. The direct influence of APC on microglial activation was evaluated by a combination of morphology and MMP-9 expression.

Results: APC attenuated the progression of EAE, and this was strongly associated at the histopathological level with reduced levels of leukocyte infiltration and concomitant demyelination. Further analysis revealed that APC reduced vascular breakdown which was associated with maintained endothelial expression of the tight junction protein zonula occludens-1 (ZO-1). In addition, APC suppressed microglial activation in this EAE model and studies revealed that APC strongly inhibited microglial activation at both the morphological level and by the expression of the pro-inflammatory protease MMP-9.

Conclusion: These findings build on the work of others in demonstrating strong therapeutic potential for APC in the treatment of inflammatory demyelinating disease and suggest that enhancement of vascular integrity and suppression of microglial activation may be important mediators of this protection.
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http://dx.doi.org/10.3389/fnins.2020.00333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174764PMC
April 2020

Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus.

Blood 2020 05;135(22):1969-1982

Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, and.

Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre- mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.
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http://dx.doi.org/10.1182/blood.2019003630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256357PMC
May 2020

Novel exomic rare variants associated with venous thrombosis.

Br J Haematol 2020 09 30;190(5):783-786. Epub 2020 Mar 30.

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

Exomic rare variant polymorphisms (c. 300 000) were analysed in the Scripps Venous Thrombosis (VTE) registry (subjects aged <55 years). Besides coagulation factor V (F5) single nucleotide polymorphisms (SNPs), family with sequence similarity 134, member B (FAM134B; rs78314670, Arg127Cys) and myosin heavy chain 8 (MYH8; rs111567318, Glu1838Ala) SNPs were associated with recurrent VTE (n = 34 cases) (false discovery rate-adjusted P < 0·05). FAM134B (rs78314670) was associated with low plasma levels of anticoagulant glucosylceramide. Analysis of 50 chr17p13.1 MYH rare SNPs (clustered skeletal myosin heavy chain genes) using collapsing methods was associated with recurrent VTE (P = 2·70 ×10 ). When intravenously injected, skeletal muscle myosin was pro-coagulant in a haemophilia mouse tail bleeding model. Thus, FAM134B and MYH genetic variants are plausibly linked to VTE risk.
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http://dx.doi.org/10.1111/bjh.16613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732677PMC
September 2020

FVIIa (Factor VIIa) Induces Biased Cytoprotective Signaling in Mice Through the Cleavage of PAR (Protease-Activated Receptor)-1 at Canonical Arg41 (Arginine41) Site.

Arterioscler Thromb Vasc Biol 2020 05 26;40(5):1275-1288. Epub 2020 Mar 26.

From the Department of Cellular and Molecular Biology, The University of Texas Health Science Center, Tyler (V.K., U.R.P., L.V.M.R.).

Objective: Recent studies showed that FVIIa (factor VIIa), upon binding to EPCR (endothelial cell protein C receptor), elicits endothelial barrier stabilization and anti-inflammatory effects via activation of PAR (protease-activated receptor)-1-mediated signaling. It is unknown whether FVIIa induces PAR1-dependent cytoprotective signaling through cleavage of PAR1 at the canonical site or a noncanonical site, similar to that of APC (activated protein C). Approach and Results: Mouse strains carrying homozygous R41Q (canonical site) or R46Q (noncanonical site) point mutations in PAR1 (QQ41-PAR1 and QQ46-PAR1 mice) were used to investigate in vivo mechanism of PAR1-dependent pharmacological beneficial effects of FVIIa. Administration of FVIIa reduced lipopolysaccharide-induced inflammation, barrier permeability, and VEGF (vascular endothelial cell growth factor)-induced barrier disruption in wild-type (WT) and QQ46-PAR1 mice but not in QQ41-PAR1 mice. In vitro signaling studies performed with brain endothelial cells isolated from WT, QQ41-PAR1, and QQ46-PAR1 mice showed that FVIIa activation of Akt (protein kinase B) in endothelial cells required R41 cleavage site in PAR1. Our studies showed that FVIIa cleaved endogenous PAR1 in endothelial cells, and FVIIa-cleaved PAR1 was readily internalized, unlike APC-cleaved PAR1 that remained on the cell surface. Additional studies showed that pretreatment of endothelial cells with FVIIa reduced subsequent thrombin-induced signaling. This process was dependent on β-arrestin1.

Conclusions: Our results indicate that in vivo pharmacological benefits of FVIIa in mice arise from PAR1-dependent biased signaling following the cleavage of PAR1 at the canonical R41 site. The mechanism of FVIIa-induced cytoprotective signaling is distinctly different from that of APC. Our data provide another layer of complexity of biased agonism of PAR1 and signaling diversity.
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http://dx.doi.org/10.1161/ATVBAHA.120.314244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405933PMC
May 2020

Plasma skeletal muscle myosin phenotypes identified by immunoblotting are associated with pulmonary embolism occurrence in young adults.

Thromb Res 2020 05 27;189:88-92. Epub 2020 Feb 27.

Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, CA, USA; Division of Hematology, Department of Medicine, University of California, San Diego, CA, USA.

Background: Purified skeletal muscle myosin (SkM) binds factor Xa and is procoagulant. The molecular forms of SkM in human plasma have not been characterized.

Method: Human plasma SkM heavy chain (HC) isoforms of different molecular weights were detected by a newly developed immunoblotting protocol. In this pilot study, the distribution of SkM HC antigen isoforms in plasmas of healthy subjects and young adult patients with venous thrombosis was analyzed.

Results: Multiple SkM HC antigen bands were detected in human plasmas, corresponding to full-length SkM HC, heavy meromyosin, or the S1 fragment. Plasma immunoblots of healthy subjects displayed three major phenotypes: Type I with two primary bands for full-length SkM and heavy meromyosin, and two lesser bands including S1 fragment (54%); Type II with bands primarily for full-length SkM HC (34%); and Type III with only a band for the S1 fragment (12%). Plasma SkM HC antigen Type II phenotype was associated with an increased occurrence of isolated pulmonary embolism in younger patients, respectively (≤50 years old).

Conclusions: Three SkM HC antigen phenotypes were identified in human plasma by immunoblotting, and Type II phenotype was correlated with the occurrence of isolated pulmonary embolisms in younger patients.
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http://dx.doi.org/10.1016/j.thromres.2020.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213899PMC
May 2020

Serum amyloid A4 is a procoagulant apolipoprotein that it is elevated in venous thrombosis patients.

Res Pract Thromb Haemost 2020 Feb 29;4(2):217-223. Epub 2019 Dec 29.

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

Background: Serum amyloid A4 (SAA4) is an apolipoprotein that is in the SAA family and it is constitutively translated. Previously, acute-phase SAA1 and SAA2 levels were associated with venous thromboembolism (VTE).

Objective: We investigated the association of plasma SAA4 with VTE and the role of SAA4 in coagulation.

Patients And Methods: The association of SAA4 with VTE in a case-control study of adult VTE subjects (N = 113 each group) and the effects of recombinant SAA4 on plasma blood coagulation assays and prothrombin activation initiated by factor Xa were evaluated.

Results: Plasma SAA4 levels in VTE subjects were higher vs. controls (48.1 vs. 38.4 µg/mL;  < .001). Elevated plasma SAA4 level (above the 90th percentile of controls) was associated with increased VTE occurrence (odds ratio, 3.8; 95% confidence interval, 1.8-8.0). This association remained significant after the adjustment for acute-phase SAA level, suggesting that SAA4 associated with VTE is independent of acute-phase SAA. Two isoforms of SAA4, that is, glycosylated and nonglycosylated SAA4 isoforms, were each higher in VTE patients. When recombinant SAA4 was added to plasma, it shortened factor Xa-1-stage clotting times, showing that it enhances clotting in plasma. In reaction mixtures containing purified factors Xa and Va and prothrombin, recombinant SAA4 increased prothrombin activation, showing that it enhances prothrombinase activity.

Conclusion: Elevated plasma constitutive SAA4 levels were linked to VTE in adults, and SAA4 can enhance thrombin generation in plasma. Our data highlight a previously unknown procoagulant activity of SAA4 that appears to be related to risk of venous thrombotic events.
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http://dx.doi.org/10.1002/rth2.12291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040552PMC
February 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

Cardiac and Skeletal Muscle Myosin Exert Procoagulant Effects.

Shock 2019 11;52(5):554-555

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

Introduction: Trauma-induced coagulopathy (TIC) and the tissue injury-provoked procoagulant profile are prevalent in severely injured patients, but their mechanisms remain unclear. Myosin, exposed by or released from tissue injury, may play a role in promoting thrombin generation and attenuating fibrinolysis. The objective of the study is to examine the effects of cardiac and skeletal muscle myosins on coagulation in whole blood using thrombelastography (TEG).

Materials And Methods: Whole blood was collected from healthy adult volunteers (n=8) and native TEGs were performed to evaluate the global coagulation response in the presence of cardiac or skeletal muscle myosin by measuring reaction (R) time (minutes), clot angle (), and maximum amplitude (MA, mm). TEG measurements were compared using paired t tests.

Results: Cardiac and skeletal muscle myosins decreased R, from 10.8 min to 8.0 min (P<0.0001) and 6.9 min (P =0.0007), respectively. There were no effects observed on clot propagation (angle) or clot strength (MA) with myosin addition. In the presence of tPA, both cardiac and skeletal muscle myosins shortened R from 11.1 min to 8.62 min (P=0.0245) and 7.75 min (P =0.0027), respectively), with no changes on angle or MA.

Conclusions: Cardiac and skeletal muscle myosins exhibit procoagulant effects in TEG assays. These whole blood TEG results support the hypothesis that cardiac and skeletal muscle myosins may be either pro-hemostatic or prothrombotic depending on context.
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http://dx.doi.org/10.1097/SHK.0000000000001426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989346PMC
November 2019

Molecular interaction site on procoagulant myosin for factor Xa-dependent prothrombin activation.

J Biol Chem 2019 10 3;294(41):15176-15181. Epub 2019 Sep 3.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037

Skeletal muscle myosin has potent procoagulant activity that is based on its ability to enhance thrombin generation due to binding coagulation factors Xa and Va and accelerating prothrombin activation. A well-studied myosin inhibitor that binds to myosin's neck region inhibits myosin-dependent prothrombin activation. Hence, to identify a potential binding site(s) on skeletal muscle myosin for factor Xa, 19 peptides (25-40 residues) representing the neck region, which consists of a regulatory light chain, an essential light chain, and a heavy chain (HC), were screened for inhibition of myosin-supported prothrombin activation. Peptide HC796-835 comprising residues 796-835 of the heavy chain strongly inhibited myosin-enhanced prothrombin activation by factors Xa and Va (50% inhibition at 1.2 μm), but it did not inhibit phospholipid vesicle-enhanced prothrombin activation. Peptide inhibition studies also implicated several myosin light chain sequences located near HC796-835 as potential procoagulant sites. A peptide comprising HC796-835's C-terminal half, but not a peptide comprising its N-terminal half, inhibited myosin-enhanced prothrombin activation (50% inhibition at 1.2 μm). This inhibitory peptide (HC816-837) did not inhibit phospholipid-enhanced prothrombin activation, indicating its specificity for inhibition of myosin-dependent procoagulant mechanisms. Binding studies showed that purified factor Xa was bound to immobilized peptides HC796-835 and HC816-837 with apparent values of 0.78 and 1.3 μm, respectively. In summary, these studies imply that HC residues 816-835 in the neck region of the skeletal muscle myosin directly bind factor Xa and, with contributions from light chain residues in this neck region, contribute to provision of myosin's procoagulant surface.
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http://dx.doi.org/10.1074/jbc.AC119.010236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791324PMC
October 2019

Tissue factor pathway inhibitor primes monocytes for antiphospholipid antibody-induced thrombosis.

Blood 2019 10 21;134(14):1119-1131. Epub 2019 Aug 21.

Center for Thrombosis and Hemostasis and.

Antiphospholipid antibodies (aPLs) with complex lipid and/or protein reactivities cause complement-dependent thrombosis and pregnancy complications. Although cross-reactivities with coagulation regulatory proteins contribute to the risk for developing thrombosis in patients with antiphospholipid syndrome, the majority of pathogenic aPLs retain reactivity with membrane lipid components and rapidly induce reactive oxygen species-dependent proinflammatory signaling and tissue factor (TF) procoagulant activation. Here, we show that lipid-reactive aPLs activate a common species-conserved TF signaling pathway. aPLs dissociate an inhibited TF coagulation initiation complex on the cell surface of monocytes, thereby liberating factor Xa for thrombin generation and protease activated receptor 1/2 heterodimer signaling. In addition to proteolytic signaling, aPLs promote complement- and protein disulfide isomerase-dependent TF-integrin β1 trafficking that translocates aPLs and NADPH oxidase to the endosome. Cell surface TF pathway inhibitor (TFPI) synthesized by monocytes is required for TF inhibition, and disabling TFPI prevents aPL signaling, indicating a paradoxical prothrombotic role for TFPI. Myeloid cell-specific TFPI inactivation has no effect on models of arterial or venous thrombus development, but remarkably prevents experimental aPL-induced thrombosis in mice. Thus, the physiological control of TF primes monocytes for rapid aPL pathogenic signaling and thrombosis amplification in an unexpected crosstalk between complement activation and coagulation signaling.
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http://dx.doi.org/10.1182/blood.2019001530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776793PMC
October 2019

Activated protein C ameliorates chronic graft-versus-host disease by PAR1-dependent biased cell signaling on T cells.

Blood 2019 08 26;134(9):776-781. Epub 2019 Jun 26.

Division of Blood and Marrow Transplantation, Masonic Cancer Center and.

Soluble thrombomodulin plasma concentrations are elevated in steroid-resistant graft-versus-host disease (GVHD), implying endothelial hypofunctioning for thrombomodulin-dependent generation of activated protein C's (APC) anticoagulant, anti-inflammatory, and antiapoptotic functions. Recombinant thrombomodulin or APC administration decreases acute GVHD, manifested by intense inflammation and tissue destruction. Here, we administered recombinant murine wild-type (WT) APC to mice with established chronic GVHD (cGVHD), a less-inflammatory autoimmune-like disease. WT APC normalized bronchiolitis obliterans-induced pulmonary dysfunction. Signaling-selective APC variants (3A-APC [APC with lysine 191-193 replaced with 3 alanines] or 5A-APC [APC with lysine 191-193 replaced with 3 alanines and arginine 229/230 replaced with 2 alanines]) with normal cytoprotective properties, but greatly reduced anticoagulant activity, provided similar results. Mechanistically, WT APC and signaling-selective variants reduced T follicular helper cells, germinal center formation, immunoglobulin, and collagen deposition. WT APC can potentially cleave protease-activated receptor 1 (PAR1) at Arg41 or Arg46, the latter causing anti-inflammatory signaling. cGVHD was reduced in recipients of T cells from WT PAR1 or mutated Gln41-PAR1 donors but not from mutated Gln46-PAR1 donors. These data implicate donor T-cell APC-induced noncanonical cleavage at Arg46-PAR1, which is known to confer cytoprotective and anti-inflammatory activities. Together, these data indicate that APC anticoagulant activity is dispensable, whereas anti-inflammatory signaling and cytoprotective cell signaling by APC are essential. Because a phase 2 ischemic stroke clinical trial did not raise any safety issues for 3A-APC treatment, our studies provide a foundational platform for testing in clinical cGVHD therapy.
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http://dx.doi.org/10.1182/blood.2019001259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716074PMC
August 2019

Neuroprotection and vasculoprotection using genetically targeted protease-ligands.

Brain Res 2019 07 14;1715:13-20. Epub 2019 Mar 14.

Department of Neurology, Cedars Sinai Medical Center, 127 S San Vicente Blvd, Los Angeles, CA 90048, United States. Electronic address:

Thrombin and activated protein C (APC) are known coagulation factors that exhibit profound effects in brain by acting on the protease activated receptor (PAR). The wild type (WT) proteases appear to impact cell survival powerfully, and therapeutic forms of APC are under development. Engineered recombinant thrombin or APC were designed to separate their procoagulant or anticoagulant effects from their cytoprotective properties. We measured vascular disruption and neuronal degeneration after a standard rodent filament stroke model. For comparison to a robust anticoagulant, we used a GpIIb/IIIa inhibitor, GR144053. During 2 h MCAo both WT murine APC and its mutant, 5A-APC, significantly decreased neuronal death 30 min after reperfusion. During 4 h MCAo, only 5A-APC significantly protected neurons but both WT-APC and 5A-APC exacerbated vascular disruption during 4 h MCAo. Human APC mutants appeared to reduce 24 h neuronal injury significantly when given after 2 h delay after MCAo. In contrast, 24 h vascular damage was worsened by high doses of WT and mutant APCs, although only statistically significantly for high dose 3K3A-APC. Mutated thrombin worsened vascular damage significantly without affecting neuron damage. GR144053 failed to ameliorate vascular disruption or neuronal injury despite significant anticoagulation. Differential effects on neurons and the vasculature were demonstrated using wild-type and mutated proteases. The mutants murine 3K3A-APC and 5A-APC protected neurons in this rodent model but in high doses worsened vascular leakage. Cytoactive effects of plasma proteases may be separated from their coagulation effects. Further studies should explore impact of dose and timing on cytoactive and vasculoactive properties of these drugs.
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http://dx.doi.org/10.1016/j.brainres.2019.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6500472PMC
July 2019

3K3A-activated protein C blocks amyloidogenic BACE1 pathway and improves functional outcome in mice.

J Exp Med 2019 02 15;216(2):279-293. Epub 2019 Jan 15.

Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA

3K3A-activated protein C (APC), a cell-signaling analogue of endogenous blood serine protease APC, exerts vasculoprotective, neuroprotective, and anti-inflammatory activities in rodent models of stroke, brain injury, and neurodegenerative disorders. 3K3A-APC is currently in development as a neuroprotectant in patients with ischemic stroke. Here, we report that 3K3A-APC inhibits BACE1 amyloidogenic pathway in a mouse model of Alzheimer's disease (AD). We show that a 4-mo daily treatment of 3-mo-old 5XFAD mice with murine recombinant 3K3A-APC (100 µg/kg/d i.p.) prevents development of parenchymal and cerebrovascular amyloid-β (Aβ) deposits by 40-50%, which is mediated through NFκB-dependent transcriptional inhibition of BACE1, resulting in blockade of Aβ generation in neurons overexpressing human Aβ-precursor protein. Consistent with reduced Aβ deposition, 3K3A-APC normalized hippocampus-dependent behavioral deficits and cerebral blood flow responses, improved cerebrovascular integrity, and diminished neuroinflammatory responses. Our data suggest that 3K3A-APC holds potential as an effective anti-Aβ prevention therapy for early-stage AD.
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http://dx.doi.org/10.1084/jem.20181035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363429PMC
February 2019

Final Results of the RHAPSODY Trial: A Multi-Center, Phase 2 Trial Using a Continual Reassessment Method to Determine the Safety and Tolerability of 3K3A-APC, A Recombinant Variant of Human Activated Protein C, in Combination with Tissue Plasminogen Activator, Mechanical Thrombectomy or both in Moderate to Severe Acute Ischemic Stroke.

Ann Neurol 2019 01 7;85(1):125-136. Epub 2019 Jan 7.

The Scripps Research Institute, La Jolla, CA.

Objective: Agonism of protease-activated receptor (PAR) 1 by activated protein C (APC) provides neuro- and vasculoprotection in experimental neuroinjury models. The pleiotropic PAR1 agonist, 3K3A-APC, reduces neurological injury and promotes vascular integrity; 3K3A-APC proved safe in human volunteers. We performed a randomized, controlled, blinded trial to determine the maximally tolerated dose (MTD) of 3K3A-APC in ischemic stroke patients.

Methods: The NeuroNEXT trial, RHAPSODY, used a novel continual reassessment method to determine the MTD using tiers of 120, 240, 360, and 540 μg/kg of 3K3A-APC. After intravenous tissue plasminogen activator, intra-arterial mechanical thrombectomy, or both, patients were randomized to 1 of the 4 doses or placebo. Vasculoprotection was assessed as microbleed and intracranial hemorrhage (ICH) rates.

Results: Between January 2015 and July 2017, we treated 110 patients. Demographics resembled a typical stroke population. The MTD was the highest-dose 3K3A-APC tested, 540 μg/kg, with an estimated toxicity rate of 7%. There was no difference in prespecified ICH rates. In exploratory analyses, 3K3A-APC reduced ICH rates compared to placebo from 86.5% to 67.4% in the combined treatment arms (p = 0.046) and total hemorrhage volume from an average of 2.1 ± 5.8 ml in placebo to 0.8 ± 2.1 ml in the combined treatment arms (p = 0.066).

Interpretation: RHAPSODY is the first trial of a neuroprotectant for acute ischemic stroke in a trial design allowing thrombectomy, thrombolysis, or both. The MTD was 540 μg/kg for the PAR1 active cytoprotectant, 3K3A-APC. A trend toward lower hemorrhage rate in an exploratory analysis requires confirmation.

Clinical Trial Registration: Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT02222714. ANN NEUROL 2019;85:125-136.
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http://dx.doi.org/10.1002/ana.25383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342508PMC
January 2019
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