Abstract 75: Characterization of Knock-in Mice Harboring a Variant of Endothelial Protein C Receptor with Impaired Ability to Bind Protein C: Impact on Coagulation and Hematopoiesis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Laura Pepler ◽  
Dhruva J Dwivedi ◽  
Patricia C Liaw
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Flow Cytometric Analysis ◽  
Activated Protein C ◽  
Extramedullary Hematopoiesis ◽  
Biological Role ◽  
Endothelial Protein C Receptor ◽  
Hematopoietic Stem ◽  
Endothelial Cell Surface ◽  
Inflammatory Phenotype

The endothelial protein C receptor (EPCR) binds to protein C (PC) and increases the rate of activated protein C (APC) generation by the thrombin-thrombomodulin complex at the endothelial cell surface. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects, many of which are EPCR-dependent. The physiologic importance of EPCR is also demonstrated in EPCR knockout mice, which show placental thrombosis and early embryonic lethality. EPCR is also highly expressed on hematopoietic stem cells (HSC), however there is no known biological role for EPCR in this cell type. Currently, there are no animal models to study the biological role of EPCR independent of its interaction with PC/APC. In this study, we generated a knock-in mouse model harboring a variant of EPCR (R84A) which lacks ability to bind to PC/APC. We hypothesize that loss of PC/APC binding to EPCR will result in a procoagulant and pro-inflammatory phenotype. EPCR R84A/R84A mice are viable, have a normal lifespan and show no evidence of spontaneous thrombosis. Histological analysis of EPCR R84A/R84A mice identified a splenic disorder, characterized by splenomegaly and extramedullary hematopoiesis. Flow cytometric analysis of the spleen from EPCR R84A/R84A mice revealed a significant increase in the percentage of CD34+ cells, representing HSCs (2.4 ± 0.1% for EPCR R84A/R84A mice and 1.4 ± 0.2% for WT mice; P<0.05). To initiate thrombin generation in mice, an intravenous injection of FXa was administered. Plasma APC levels of FXa challenged EPCR R84A/R84A mice were reduced by 150% and thrombin-antithrombin levels (TAT) (an indicator of thrombin generation) were increased by 100% when compared to WT mice. The elevated TAT levels in FXa challenged EPCR R84A/R84A mice were accompanied by an increase in the size and number of fibrin clots in the lungs. EPCR R84A/R84A mice are viable suggesting that defects in EPCR that impair PC binding do not affect embryogenesis or development. Introduction of the R84A mutation in EPCR results in impaired PC activation, and a procoagulant phenotype upon thrombotic challenge. Enlargement of the spleen in EPCR R84A/R84A mice suggests that EPCR may play a biological role in the regulation of hematopoiesis.

Endothelial protein C receptor–expressing hematopoietic stem cells reside in the perisinusoidal niche in fetal liver

Blood ◽  
2010 ◽  
Vol 116 (4) ◽  
pp. 544-553 ◽  
Author(s):  
Hiroko Iwasaki ◽  
Fumio Arai ◽  
Yoshiaki Kubota ◽  
Maria Dahl ◽  
Toshio Suda
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Protein C ◽  
Fetal Liver ◽  
Activated Protein C ◽  
Endothelial Protein C Receptor ◽  
Hematopoietic Stem ◽  
Protease Activated Receptor 1 ◽  
Slow Cycling

Abstract Hematopoietic stem cells (HSCs) are maintained in specialized niches in adult bone marrow. However, niche and HSC maintenance mechanism in fetal liver (FL) still remains unclear. Here, we investigated the niche and the molecular mechanism of HSC maintenance in mouse FL using HSCs expressing endothelial protein C receptor (EPCR). The antiapoptotic effect of activated protein C (APC) on EPCR+ HSCs and the expression of protease-activated receptor 1 (Par-1) mRNA in these cells suggested the involvement of the cytoprotective APC/EPCR/Par-1 pathway in HSC maintenance. Immunohistochemistry revealed that EPCR+ cells were localized adjacent to, or integrated in, the Lyve-1+ sinusoidal network, where APC and extracellular matrix (ECM) are abundant, suggesting that HSCs in FL were maintained in the APC- and ECM-rich perisinusoidal niche. EPCR+ HSCs were in a relatively slow cycling state, consistent with their high expression levels of p57 and p18. Furthermore, the long-term reconstitution activity of EPCR+ HSCs decreased significantly after short culture but not when cocultured with feeder layer of FL-derived Lyve-1+ cells, which suggests that the maintenance of the self-renewal activity of FL HSCs largely depended on the interaction with the perisinusoidal niche. In conclusion, EPCR+ HSCs resided in the perisinusoidal niche in mouse FL.

Regulation Of Hematopoietic Stem Cell Trafficking By The Coagulation Pathway

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 456-456
Author(s):  
Shiri Gur Cohen ◽  
Tomer Itkin ◽  
Orit Kollet ◽  
Sagarika Chakrabarty ◽  
Aya Ludin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Hematopoietic Stem

Hematopoeitic stem and progenitor cells (HSPC) dynamically switch between a quiescent, non-motile mode in the bone marrow (BM), to an active state, in which they proliferate, differentiate and egress to the circulation. Injection of the coagulation protease thrombin induced rapid HSPC mobilization to the blood via activation of its major receptor, protease activated receptor 1 (PAR1) on BM hematopoietic and stromal cells. We hypothesized that coagulation factors control stem cells fate in the BM. We examined if thrombin is generated in the murine BM and found by immunohistochemistry prothrombin associated with bone lining osteoblasts in the endosteum region. These cells also highly express osteopontin which induces stem cell quiescence and retention. Cleavage of osteopontin by thrombin or by osteoclast derived cathepsin K induces stem cell mobilization. In addition, a unique structure of multinucleated CD45+ cell clusters in the trabecular-rich area of the murine femoral metaphysis express the cell surface receptor Tissue Factor (TF), a potent initiator of the coagulation cascade leading to thrombin generation. These clusters were found adjacent to multinucleated TRAP (tatrate resistant acid phosphate) positive active osteoclasts. In vitro, we found that immature osteoclasts expressed TF in cell fusion areas, suggesting that osteoclast maturation also activates the coagulation thrombin/PAR1 axis, thus mediating HSPC recruitment to the circulation. Supporting this notion, bleeding which prompts a hemostatic response and thrombin production, is a strong inducer of osteoclasts activation and HSPC mobilization. In addition, injection of bacterial lipopolysaccharides (LPS) is known to activate osteoclasts and induce HSPC mobilization (Kollet et al Nat Med 06). We found that LPS injection upregulated TF expression by CD45+ myeloid cells in the murine BM. LPS treatment provoked massive HSPC mobilization, which was attenuated by PAR1 inhibition. To further address the role of thrombin in stem cell maintenance, we targeted prothrombin in vivo by applying Antisense Oligonucleotides (ASO) knockdown technology, previously shown to induce a dose- and time-dependent up to 90% reduction of prothrombin mRNA levels in the murine liver (Monia et al Blood 2010). Prothrombin depletion altered the BM niche microenvironment by expanding the mesenchymal stem and progenitor (MSPC) population and the long-term repopulating CD34-/ROSlow/LSK HSPC population in the BM. In untreated mice, TF was also expressed by a small MSPC population, suggesting that the bone stomal compartment may also contribute to the regulation of HSPC mobilization upon demand. To further asses the role of thrombin generation in HSPC development, we examined the involvement of the endothelial cell receptor Thrombomodulin (TM) that is pivotal for the anticoagulant pathway which mediates activation of protein C. TM protein is expressed by BM small blood vessels resembling sinusoids and by neighboring MSPC. By immunohistochemistry, we also detected activated protein C on the same blood vessels. A mouse model with a mutation in the TM gene (TMPro/Pro) is characterized by reduced capacity for activated protein C generation which in turn increases thrombin levels in these mice. We found increased circulating hematopoietic stem cells in TMPro/Pro mice, suggesting that chronically increased basal levels of thrombin generation can promote HSC egress. Conversely, short term (5 day) intermittent treatment of mice with low dose thrombin that mainly causes activated protein C formation in vivo, display higher levels of CD34-/ROSlow/LSK and EPCR+LSK stem cells in the BM, indicating additional roles for the anticoagulant pathway in BM stem cell pool maintenance. In summary, our results provide evidence that the activator of the coagulation cascade, TF, and coagulation factors Thrombin and activated protein C are present in the BM and regulate and integrate functions of hematopoietic stem and progenitor cells and BM stromal progenitor cells. Disclosures: Crosby: Isis pharmaceuticals: the ASO for prothrombin was obtained from Isis pharmaceuticals Other. Monia:Isis pharmaceuticals: the ASO for prothrombin was obtained from Isis pharmaceuticals Other.

scholarly journals Extravascular coagulation in hematopoietic stem and progenitor cell regulation

Blood ◽  
2018 ◽  
Vol 132 (2) ◽  
pp. 123-131 ◽  
Author(s):  
T. Son Nguyen ◽  
Tsvee Lapidot ◽  
Wolfram Ruf
Keyword(s):  
Protein C ◽  
Chemotherapy Resistance ◽  
Activated Protein C ◽  
Coagulation Factors ◽  
Endothelial Protein C Receptor ◽  
Protective Mechanisms ◽  
Hematopoietic Stem ◽  
Biased Signaling ◽  
Integrin Α4 ◽  
Protease Activated Receptor 1

Abstract The hemostatic system plays pivotal roles in injury repair, innate immunity, and adaptation to inflammatory challenges. We review the evidence that these vascular-protective mechanisms have nontraditional roles in hematopoietic stem cell (HSC) maintenance in their physiological bone marrow (BM) niches at steady-state and under stress. Expression of coagulation factors and the extrinsic coagulation initiator tissue factor by osteoblasts, tissue-resident macrophages, and megakaryocytes suggests that endosteal and vascular HSC niches are functionally regulated by extravascular coagulation. The anticoagulant endothelial protein C receptor (EPCR; Procr) is highly expressed by primitive BM HSCs and endothelial cells. EPCR is associated with its major ligand, activated protein C (aPC), in proximity to thrombomodulin-positive blood vessels, enforcing HSC integrin α4 adhesion and chemotherapy resistance in the context of CXCL12-CXCR4 niche retention signals. Protease-activated receptor 1–biased signaling by EPCR-aPC also maintains HSC retention, whereas thrombin signaling activates HSC motility and BM egress. Furthermore, HSC mobilization under stress is enhanced by the fibrinolytic and complement cascades that target HSCs and their BM niches. In addition, coagulation, fibrinolysis, and HSC-derived progeny, including megakaryocytes, synergize to reestablish functional perivascular HSC niches during BM stress. Therapeutic restoration of the anticoagulant pathway has preclinical efficacy in reversing BM failure following radiation injury, but questions remain about how antithrombotic therapy influences extravascular coagulation in HSC maintenance and hematopoiesis.

Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets

1993 ◽  
Vol 69 (02) ◽  
pp. 124-129 ◽  
Author(s):  
Susan Solymoss ◽  
Kim Thi Phu Nguyen
Keyword(s):  
Western Blotting ◽  
Protein C ◽  
Thrombin Generation ◽  
Blood Platelets ◽  
Activated Protein C ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Two Stage ◽  
One Stage ◽  
Plasma Factor

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.

Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging

2021 ◽  
Author(s):  
Di Ren ◽  
Julia Fedorova ◽  
Kayla Davitt ◽  
Tran Ngoc Van Le ◽  
John H Griffin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein C ◽  
Ex Vivo ◽  
Activated Protein C ◽  
Wild Type ◽  
Endothelial Protein C Receptor ◽  
Cardiac Damage ◽  
Downstream Signaling ◽  
Cardioprotective Effects ◽  
Epcr Shedding

Background: Activated protein C (APC) is a plasma serine protease with anticoagulant and anti-inflammatory activities. Endothelial protein C receptor (EPCR) is associated with APC's activity and mediates its downstream signaling events. APC exerts cardioprotective effects during ischemia and reperfusion (I/R). This study aims to characterize the role of the APC-EPCR axis in ischemic insults in aging. Methods: Young (3-4 months) and aged (24-26 months) wild type C57BL/6J mice, as well as EPCR point mutation (EPCR R84A/R84A ) knock-in C57BL/6J mice incapable of interaction with APC and its wild type of littermate C57BL/6J mice, were subjected to I/R. Wild type APC, signaling-selective APC-2Cys, or anticoagulant-selective APC-E170A were administrated before reperfusion. Results: The results demonstrated that cardiac I/R reduces APC activity, and the APC activity was impaired in the aged versus young hearts possibly attributable to the declined EPCR level with aging. Serum EPCR measurement showed that I/R triggered the shedding of membrane EPCR into circulation, while administration of APC attenuated the I/R-induced EPCR shedding in both young and aged hearts. Subsequent echocardiography showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction during I/R in both young and aged mice. Importantly, APC elevated the resistance of the aged heart to ischemic insults through stabilizing EPCR. However, all these cardioprotective effects of APC were blunted in the EPCR R84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMP-activated protein kinase (AMPK) mediates acute adaptive response while protein kinase B (AKT) is involved in chronic metabolic programming in the hearts with APC treatment. Conclusions: I/R stress causes shedding of the membrane EPCR in the heart, and administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

Abstract 34: Endogenous Superoxide Dismutase Protects From Impaired Generation of Activated Protein C and Enhanced Susceptibility to Experimental Thrombosis in Mice

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Sanjana Dayal ◽  
Sean X Gu ◽  
Katinan M Wilson ◽  
Ryan Hutchins ◽  
Steven R Lentz
Keyword(s):  
Superoxide Dismutase ◽  
Protein C ◽  
Activated Protein C ◽  
Vena Cava ◽  
Oxidative Modification ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Experimental Thrombosis

In vitro studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo . We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that Sod1-/- mice formed stable occlusions significantly faster than Sod1+/+ mice (P<0.05). In an inferior vena cava (IVC) stasis model Sod1- /- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs. Sod1+/+ mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from Sod1-/- and Sod1+/+ mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in Sod1-/- and Sod1 +/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in Sod1 -/- mice (P<0.05 vs. Sod1 +/+ mice). Further, in vivo generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in Sod1-/- mice (P<0.05 vs. Sod1+/+ mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1 -/- mice vs. Sod1 +/+ mice, suggesting that altered generation of activated protein C in Sod1-/- mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.

Phospholipid-bound Tissue Factor Modulates both Thrombin Generation and APC-mediated Factor Va Inactivation

1999 ◽  
Vol 82 (12) ◽  
pp. 1673-1679 ◽  
Author(s):  
Katalin Váradi ◽  
Jürgen Siekmann ◽  
Peter Turecek ◽  
H. Peter Schwarz ◽  
Victor Marder
Keyword(s):  
Tissue Factor ◽  
Surface Density ◽  
Protein C ◽  
Thrombin Generation ◽  
Feedback Inhibition ◽  
Activated Protein C ◽  
Reaction System ◽  
Inverse Correlation ◽  
High Concentration ◽  
Factor Va

SummaryHemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

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