High Levels of Circulating Thrombomodulin in Human Foetuses and Children

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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Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma

Blood ◽

10.1182/blood.v85.7.1815.bloodjournal8571815 ◽

1995 ◽

Vol 85 (7) ◽

pp. 1815-1821 ◽

Cited By ~ 16

Author(s):

C van't Veer ◽

TM Hackeng ◽

D Biesbroeck ◽

JJ Sixma ◽

BN Bouma

Keyword(s):

Endothelial Cell ◽

Tissue Factor ◽

Protein C ◽

Activated Protein C ◽

Coagulation Factor ◽

Protein S ◽

Flow Conditions ◽

Cell Matrix ◽

Anticoagulant Function ◽

Prothrombin Activation

Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin allowing tissue factor-dependent prothrombin activation, was perfused at a wall shear rate of 100 s-1 over tissue factor containing matrices of stimulated endothelial cells placed in a perfusion chamber. Fractions were collected in time at the outlet and prothrombin activation was determined by measuring the activation fragment F1+2 of prothrombin. In normal plasma, a time-dependent prothrombin activation was detected by the generation of fragment1+2. Prothrombin activation had ceased after 12 minutes perfusion, independent of the amount of tissue factor present in the matrix. Depletion of protein S from plasma or inhibition of protein S in plasma by monoclonal antibodies induced a 5- to 25-fold increase of prothrombin activation on the procoagulant endothelial cell matrix. A prolonged prothrombin activation was detected in protein S-depleted plasma up to 20 minutes after onset of the thrombin generation. The increased prothrombin activation in protein S-depleted plasma could not be explained by the absence of the cofactor function of protein S for aPC because depletion of protein C from plasma did not result in increased prothrombin activation. These data provide further evidence for a strong anticoagulant function of protein S in plasma independent from activated protein C.

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Expression and Anticoagulant Function of the Endothelial Cell Protein C Receptor (EPCR) in Cancer Cell Lines

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615692 ◽

2001 ◽

Vol 85 (02) ◽

pp. 356-361 ◽

Cited By ~ 40

Author(s):

Naoko Tsuneyoshi ◽

Shin-ichiro Horiguchi ◽

Xiaofen Ye ◽

Miwako Matsuzaki ◽

Masakazu Toi ◽

...

Keyword(s):

Endothelial Cell ◽

Cancer Cells ◽

Cancer Patients ◽

Cell Lines ◽

Protein C ◽

Negative Regulator ◽

Cell Protein ◽

Breast Cancer Patients ◽

Extremely High Frequency ◽

Anticoagulant Function

SummaryInduction of procoagulant factors in malignant cells is considered to be the major cause of coagulation disorders in cancer. Thrombomodulin (TM), a negative regulator of coagulation was also found to be expressed in cancer cells. We report here evidence for another anticoagulant, the endothelial cell protein C receptor (EPCR), in cancer cells. EPCR was detected in several cell lines derived from various types of cancer. Significant levels of protein C (PC) activation were detected only with cell lines expressed both EPCR and TM. Anti-EPCR monoclonal antibodies (mAbs) specifically inhibited the activation. Thus, EPCR function appears to be important for PC activation by cancer cells. In addition, we detected EPCR expression in tumor cells from breast cancer patients, with an extremely high frequency. EPCR function may contribute to progression or pathogenesis of some types of cancer, and may explain the complexity of coagulopathy in cancer patients.

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The roles of thrombomodulin, protein C, and activated protein C in endothelial cell proliferation

The FASEB Journal ◽

10.1096/fasebj.20.4.a656-d ◽

2006 ◽

Vol 20 (4) ◽

Author(s):

Kristen Bennett ◽

Chris Teigland ◽

Laura Neese Glasscock

Keyword(s):

Cell Proliferation ◽

Endothelial Cell ◽

Protein C ◽

Activated Protein C ◽

Endothelial Cell Proliferation

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Inhibitory effect of adenosine on intimal hyperplasia and proliferation of smooth muscle cells in a carotid arterial anastomosis animal model

Vascular ◽

10.1177/1708538114533962 ◽

2014 ◽

Vol 23 (2) ◽

pp. 124-131 ◽

Cited By ~ 2

Author(s):

Gokhan Albayrak ◽

Erdem Silistreli ◽

Bekir Ergur ◽

Sule Kalkan ◽

Ozalp Karabay ◽

...

Keyword(s):

Cell Proliferation ◽

Smooth Muscle ◽

Endothelial Cell ◽

Neointimal Hyperplasia ◽

Inhibitory Effect ◽

Endothelial Cell Proliferation ◽

Positive Staining ◽

Significant Difference ◽

Group A ◽

Group B

Purpose The effect of adenosine (9-β-0-ribifuranosyladenine) on the endothelial cell proliferation and neointimal hyperplasia is investigated in the rabbit carotid artery anastomosis model. Methods Twenty-eight New Zealand white rabbits were arranged in four groups of seven animals each. The right carotid arteries of each animal were transsected and re-anastomosed. The left sides remained as control. In Group A, no medication was used. In Group B, subcutaneous Adenosine was applied for 3 days. In Group C, the same dose was applied for 7 days, and in Group D for 21 days. After 28 days, the luminal diameters, luminal areas, intima/media ratios were all measured by using histopathological evaluation. Findings The mean luminal diameters and areas of the four groups were smaller than the control ones. Massive thickening of smooth muscle cell proliferation and dense intensifying in the connecting tissues were observed most prominently in Group A, in decreasing degrees within other groups. Intima/media ratio was highest in Group A. Scoring the quantity of e-NOS positive staining also revealed a significant difference between the experimental groups and their control associates. Conclusion The process of endothelial cell proliferation and neointimal hyperplasia can be significantly reduced by the use of adenosine.

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ENDOTHELIUM AND PROTEIN S: SYNTHESIS, RELEASE AND REGULATION OF ANTICOAGULANT ACTIVITY

10.1055/s-0038-1642962 ◽

1987 ◽

Author(s):

Peter P Nawroth ◽

Jerry Brett ◽

Susan Steinberg ◽

Charles T Esmon ◽

David M Stern

Keyword(s):

Endothelial Cell ◽

Protein C ◽

Specific Binding ◽

Activated Protein C ◽

Protein S ◽

Intracellular Protein ◽

C Protein ◽

Factor Va ◽

Intracellular Vesicles ◽

Anticoagulant Function

The protein C-protein S pathway is closely linked to the vessel wall. In terms of protein C, endothelium has been shown to provide the receptor thrombomodulin, which promotes thrombin-mediated formation of activated protein C. Optimal anticoagulant function of activated protein C requires protein S and a cellular surface. Recent studies have indicated that endothelium can facilitate assembly of the activated protein C-protein S complex and that bovine endothelium expresses specific binding site(s) for protein S which promote its anticoagulant function. Expression of protein S binding sites is subject to down-regulation by Tumor Necrosis Factor (TNF) . Exposure of cultured bovine endothelium to TNF results in decreased 125I-protein s binding and attenuated rates of Factor Va inactivation after 2 hrs followed by negligible 125I-protein S binding and Factor Va inactivation by 10 hrs. These changes persist for over 48 hrs, in contrast to the more transient rise in endothelial cell tissue factor induced by TNF which returns to baseline by 24 hrs.In addition to providing binding sites for protein S, endothelium constitutively synthesizes and releases this vitamin K-dependent anticoagulant cofactor. Release of protein S is blocked by addition of warfarin, indicating that y-carboxylation facilitates the release of intracellular protein S. Morphologic studies, at the level of electron microscope, have shown protein S antigen to be present in cisternae of rough endoplasmic reticulum, the trans face of the golgi and a population of intracellular vesicles which appear to be distributed at the cellular periphery. By immunofluorescence, the distribution of protein S is distinct from that of von Willebrand Factor. The intracellular vesicles containing protein S constitute a storage pool potentially available for rapid release. Treatment of endothelium with norepinephrine results in release of protein S over the next 20 min. Release is half-maximal at a norepinephrine concentration of about 0.1 uM and is not observed with the biologically inactive entantiomer (+) norepinephrine. Norepinephrine-induced release of intracellular protein S can be blocked by prazosine (10-7 7 M), but not by propranolol (10-6 M) or yohimbine (10-5 M). These data are consistent with release of protein S being a receptor-mediated process dependent on an endothelial cell alpha 1 adrenergic receptor. Blockade of norepinephrine-induced release of protein S by pertussis toxin treatment of endothelium further defines the intracellular pathway of protein S and implicates regulatory G proteins in the stimulus-response coupling. Electron microscopic studies have shown that following exposure of endothelium to norepinephrine the intracellular vesicles containing protein S undergo exocytosis at the plasma membrane. These data define a new relationship between the autonomic nervous system and the coagulation mechanism.Protein S is clearly an endothelial cell-associated anticoagulant protein. A specific binding site on the endothelial cell surface can regulate its anticoagulant function on the vessel wall. Endothelial cell synthesis and release of protein S defines a new level of participation of endothelium in the protein C-protein S pathway.

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

10.1055/s-0038-1644737 ◽

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.

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Activation of Protein C by a Complex between Thrombin and Endothelial Cell Surface Protein

Pathobiology of the Endothelial Cell ◽

10.1016/b978-0-12-521980-8.50013-4 ◽

1982 ◽

pp. 121-136 ◽

Cited By ~ 7

Author(s):

CHARLES T. ESMON ◽

NAOMI L. ESMON ◽

JULIE SAUGSTAD ◽

WHYTE G. OWEN

Keyword(s):

Endothelial Cell ◽

Cell Surface ◽

Protein C ◽

Surface Protein ◽

Cell Surface Protein ◽

Endothelial Cell Surface

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

Journal of Experimental Medicine ◽

10.1084/jem.187.7.1029 ◽

1998 ◽

Vol 187 (7) ◽

pp. 1029-1035 ◽

Cited By ~ 71

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.

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

Blood ◽

10.1182/blood-2007-09-113076 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2667-2673 ◽

Cited By ~ 48

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.

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