Tissue-restricted expression of thrombomodulin in the placenta rescues thrombomodulin-deficient mice from early lethality and reveals a secondary developmental block

Development ◽  
2001 ◽  
Vol 128 (6) ◽  
pp. 827-838 ◽  
Author(s):  
B. Isermann ◽  
S.B. Hendrickson ◽  
K. Hutley ◽  
M. Wing ◽  
H. Weiler
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Embryonic Stem ◽  
Cell Surface Receptor ◽  
Proper Function ◽  
Endothelial Cell Surface ◽  
Consumptive Coagulopathy ◽  
Early Placenta ◽  
Early Lethality ◽  
Developmental Block

The endothelial cell surface receptor thrombomodulin (TM) inhibits blood coagulation by forming a complex with thrombin, which then converts protein C into the natural anticoagulant, activated protein C. In mice, a loss of TM function causes embryonic lethality at day 8.5 p.c. (post coitum) before establishment of a functional cardiovascular system. At this developmental stage, TM is expressed in the developing vasculature of the embryo proper, as well as in non-endothelial cells of the early placenta, giant trophoblast and parietal endoderm. Here, we show that reconstitution of TM expression in extraembryonic tissue by aggregation of tetraploid wild-type embryos with TM-null embryonic stem cells rescues TM-null embryos from early lethality. TM-null tetraploid embryos develop normally during midgestation, but encounter a secondary developmental block between days 12.5 and 16.5 p.c. Embryos lacking TM develop lethal consumptive coagulopathy during this period, and no live embryos are retrieved at term. Morphogenesis of embryonic blood vessels and other organs appears normal before E15. These findings demonstrate a dual role of TM in development, and that a loss of TM function disrupts mouse embryogenesis at two different stages. These two functions of TM are exerted in two distinct tissues: expression of TM in non-endothelial extraembryonic tissues is required for proper function of the early placenta, while the absence of TM from embryonic blood vessel endothelium causes lethal consumptive coagulopathy.

The Suppression of the Coagulation of Nonanticoagulated Whole Blood in vitro by Human Umbilical Endothelial Cells Cultivated on Microcarriers is not Dependent on Protein C Activation

1995 ◽  
Vol 73 (04) ◽  
pp. 719-724 ◽  
Author(s):  
Hans-Peter Kohler ◽  
Michele Müller ◽  
Thomas Bombeli ◽  
P Werner Straub ◽  
André Haeberli
Keyword(s):  
Endothelial Cells ◽  
Whole Blood ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Volume Ratio ◽  
Endothelial Cell Surface ◽  
Direct Measurements ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells (HUVEC) were cultivated on globular microcarriers in order to improve the endothelial cell surface to blood-volume ratio over the conventional flat bed cultures. HUVEC-beads were tested for their modulation of blood coagulation using a combination of two steps: HUVEC-beads were added into the syringe used for the venipuncture, in order to achieve immediate contact between cells and blood, and no anticoagulant was used during the incubation time of HUVEC-beads with whole blood. The coagulation initiation produced by venipuncture was almost completely suppressed as judged by thrombin measurements over a period of 60 min. The activated partial thromboplastin time showed a prolongation by a factor >3. Direct measurements of activated protein C (APC) were negative. Moreover, inhibition of APC-generation with a monoclonal anti-human protein C antibody did not affect the anticoagulant properties of endothelial cells. Therefore the anticoagulant properties exerted by HUVEC-beads are not dependent on APC.

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.

Resistance to activated protein C, the FV: Q506 allele, and venous thrombosis

1998 ◽  
Vol 18 (01) ◽  
pp. 1-10
Author(s):  
A. Hillarp ◽  
S. Rosen ◽  
B. Zöller ◽  
B. Dahlbäck
Keyword(s):  
Venous Thrombosis ◽  
Protein C ◽  
Antithrombin Iii ◽  
Single Point ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Single Point Mutation ◽  
Factor V ◽  
Endothelial Cell Surface

SummaryVitamin K-dependent protein C is an important regulator of blood coagulation. After its activation on the endothelial cell surface by thrombin bound to thrombomodulin, it cleaves and inactivates procoagulant cofactors Va and Villa, protein S and intact factor V working as cofactors. Until recently, genetic defects of protein C or protein S were, together with antithrombin III deficiency, the established major causes of familial venous thromboembolism, but they were found in fewer than 5-10% of patients with thrombosis. In 1993, inherited resistance to activated protein C (APC) was described as a major risk factor for venous thrombosis. It is found in up to 60% of patients with venous thrombosis. In more than 90% of cases, the molecular background for the APC resistance is a single point mutation in the factor V gene, which predicts substitution of an arginine (R) at position 506 by a glutamine (Q). Mutated factor V (FV: Q506) is activated by thrombin or factor Xa in normal way, but impaired inactivation of mutated factor Va by APC results in life-long hypercoagulability. The prevalence of the FV:Q506 allele in the general population of Western countries varies between 2 and 15%, whereas it is not found in several other populations with different ethnic backgrounds. Owing to the high prevalence of FV:Q506 in Western populations, it occasionally occurs in patients with deficiency of protein S, protein C, or antithrombin III. Individuals with combined defects suffer more severely from thrombosis, and often at a younger age, than those with single defects, suggesting severe thrombophilia to be a multigenetic disease.

SIMULTANEOUS PRODUCTION OF ENDOTHELIAL CELL-DERIVED PROTEIN S AND FACTOR V, AND INACTIVATION OF FACTOR Va AT THE ENDOTHELIAL CELL SURFACE

1987 ◽  
Author(s):  
P v d Waart ◽  
K T Preissner ◽  
U Delvos ◽  
G Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Conditioned Medium ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Endothelial Cell Surface ◽  
Factor Va

Several proteins synthesized and expressed by endothelial cells are involved in the regulation of coagulation. The synthesis and expression of factor V and protein S has been demonstrated in independent studies. The present work evaluates the simultaneous synthesis and expression of bovine factor V and protein S and the effect of endothelial protein S on the inactivation of endothelial factor Va by activated protein C. The accumulation of both proteins in conditioned medium was detected by SDS-PAGE followed by immunoblotting, and their activities were tested by functional assays. The synthesis of protein S and factor V per 105 cells over 24 h amounted up to 2 ng protein S and 440 ng factor V, respectively. The addition of thrombin did not increase the yield of synthesized cofactors. Thrombin did neither proteolyse protein S on endothelial cells nor in a purified system in the presence of thrombomodulin and calcium ions. Factor V was secreted partly in its activated form as evidenced by the appearance of active intermediates with M = 220,000-280,000 on immunoblots as well as by only a three-Fold further activation of factor V/Va following addition of thrombin. The rate constant for the inactivation of factor Va by activated protein C was only two-fold higher for factor Va derived from cells cultured in the presence of vitamin K as compared in the presence of warfarin. For the inactivation of comparable factor Va concentrations in conditioned medium a 10-fold higher and on endothelial cells a 40-fold higher concentration of activated protein C was required to obtain similar inactivation rates of factor Va as compared to a purified system. These results suggest that resting endothelial cells contain a factor V activator, and that a regulatory mechanism is operative on the endothelial cell surface that suppresses the inactivation potential of activated protein C/ protein S.

scholarly journals Activation Mechanism of Anticoagulant Protein C in Large Blood Vessels Involving the Endothelial Cell Protein C Receptor

1998 ◽  
Vol 187 (7) ◽  
pp. 1029-1035 ◽  
Author(s):  
Kenji Fukudome ◽  
Xiaofen Ye ◽  
Naoko Tsuneyoshi ◽  
Osamu Tokunaga ◽  
Keishin Sugawara ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Surface ◽  
Blood Coagulation ◽  
Protein C ◽  
Cell Surface Receptor ◽  
Activation Mechanism ◽  
Cell Protein ◽  
Endothelial Cell Surface ◽  
Surface Receptor ◽  
Large Vessels

Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has been identified as an essential component for protein C activation. Although protein C can be activated directly by the thrombin–TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In this study, we demonstrate the constitutive, endothelial cell–specific expression of EPCR in vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity for protein C activation mediated by the thrombin–TM complex. By these mechanisms, EPCR appears to enable significant levels of protein C activation in large vessels. These results suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.

scholarly journals Activated protein C–cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2667-2673 ◽  
Author(s):  
Reto A. Schuepbach ◽  
Clemens Feistritzer ◽  
Lawrence F. Brass ◽  
Matthias Riewald
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Protein C ◽  
Ex Vivo ◽  
Cell Monolayer ◽  
Activated Protein C ◽  
Protective Effects ◽  
Endothelial Cell Surface ◽  
Protease Activated Receptor 1

Activated protein C (APC) signals in endothelial cells ex vivo through protease activated receptor-1 (PAR1). However, it is controversial whether PAR1 can mediate APC's protective effects in sepsis because the inflammatory response results in thrombin generation and thrombin proteolytically activates PAR1 much more efficiently than APC. Here we show that APC can induce powerful barrier protective responses in an endothelial cell monolayer in the presence of thrombin. Using cell surface immunoassays with conformation sensitive monoclonal anti-PAR1 antibodies we analyzed cleavage of endogenous PAR1 on the endothelial cell surface by APC in the absence and presence of thrombin. Incubation with APC caused efficient PAR1 cleavage and upon coincubation with thrombin APC supported additional PAR1 cleavage. Thrombin-cleaved PAR1 rapidly disappeared from the cell surface whereas, unexpectedly, the APC-cleaved PAR1 remained and could be detected on the cell surface, even when thrombin at concentrations of up to 1 nM was also present. Our findings demonstrate for the first time directly that APC can generate a distinct PAR1 population on endothelial cells in the presence of thrombin. The data suggest that different trafficking of activated PAR1 might explain how PAR1 signaling by APC can be relevant when thrombin is present.

Activated protein C: the cure for sepsis - again?

Anaesthesia ◽  
2001 ◽  
Vol 56 (12) ◽  
pp. 1133-1135 ◽  
Author(s):  
Tariq Hoth ◽  
Timothy W. Evans
Keyword(s):  
Protein C ◽  
Activated Protein C

Coagulation Factor V Leiden Mutation Was Detected in the Patients with Activated Protein C Resistance in Thailand

1998 ◽  
Vol 80 (08) ◽  
pp. 344-345 ◽  
Author(s):  
Pasra Arnutti ◽  
Motofumi Hiyoshi ◽  
Wichai Prayoonwiwat ◽  
Oytip Nathalang ◽  
Chamaiporn Suwanasophon ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Factor V ◽  
Activated Protein C Resistance ◽  
Factor V Leiden Mutation ◽  
Coagulation Factor V

High Levels of Circulating Thrombomodulin in Human Foetuses and Children

1999 ◽  
Vol 81 (06) ◽  
pp. 906-909 ◽  
Author(s):  
Marie-Hélène Aurousseau ◽  
Danielle Gozin ◽  
Fernand Daffos ◽  
Armando D’Angelo ◽  
François Forestier ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Protein C ◽  
Endothelial Cell Surface ◽  
A Value ◽  
Median Level ◽  
Group A ◽  
Anticoagulant Function ◽  
Cell Surface Proteoglycan ◽  
Human Foetuses

SummaryThrombomodulin (TM) is an endothelial cell surface proteoglycan with anticoagulant functions, also implicated in cell proliferation, cell-cell adhesion and differentiation. In this study we determined circulating plasma TM (pTM) levels in human foetuses at different stages of pregnancy, at birth and in childhood. TM levels increased with gestational age, the median level reaching a peak of approximately 165 ng/ml between the 23rd and 26th week, thereafter decreasing gradually, reaching a value of 108 ng/ml at birth. pTM continues to decrease progressively during childhood, reaching in the 5-15 years group a median of 56 ng/ml which approaches the adult value. The pTM peak was statistically significant and represents a specific foetal phenomenon as it was independent of the corresponding maternal values. As a whole, the pTM pattern during foetal maturation appears totally different from that of protein C, prothrombin and other coagulation activators and inhibitors and thus, TM may play in the foetus another role in addition to its well-known anticoagulant function.

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