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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Cardiovascular Biology Research Program,
Oklahoma Medical Research Foundation; Departments of Pathology and
Biochemistry and Molecular Biology, University of Oklahoma Health
Sciences Center; and Howard Hughes Medical Institute, Oklahoma City,
Oklahoma.
Endothelial cell protein C receptor (EPCR) augments protein C
activation by the thrombin-thrombomodulin complex about 5-fold in
vitro. Augmentation is EPCR concentration dependent even when the EPCR
concentration is in excess of the thrombomodulin. EPCR is expressed
preferentially on large blood vessel endothelium, raising questions
about the importance of protein C-EPCR interaction for augmenting
systemic protein C activation. In these studies, this question was
addressed directly by infusing thrombin into baboons in the presence or
absence of a monoclonal antibody to EPCR that blocks protein C binding.
Activated protein C levels were then measured directly by capturing the
enzyme on a monoclonal antibody and assaying with chromogenic
substrate. Blocking protein C-EPCR interaction resulted in about an
88% decrease in circulating activated protein C levels generated in
response to thrombin infusion. Leukocyte changes, fibrinogen
consumption, fibrin degradation products, and vital signs were similar
between the animals infused with thrombin alone and those infused with
thrombin and the anti-EPCR antibody. The results indicate that EPCR
plays a major role in protein C activation and suggest that defects in
the EPCR gene might contribute to increased risk of thrombosis.
(Blood. 2001;97:1685-1688) The endothelial cell protein C receptor (EPCR) is a
type 1 transmembrane protein1 and is homologous to the
major histocompatibility complex class 1/CDI family of proteins. EPCR
demonstrates a relatively endothelial cell-specific expression pattern
with the expression levels much higher on large vessel endothelium,
especially large arteries, and low to absent on capillaries. Cell
cultures of human endothelium2 have demonstrated that EPCR
augments the activation of protein C by the thrombin-thrombomodulin
(TM) complex at least 5-fold.3-5 Reconstitution of EPCR
and TM into phospholipid vesicles revealed that the rate of protein C
activation was dependent on the EPCR concentration even when EPCR was
far in excess of the TM,4 which, when combined with the
expression profile of EPCR, suggests that the major impact of the
receptor would be in large blood vessels. Because, due to
surface-to-volume arguments, the TM concentration is so much higher in
the microcirculaton than in the large vessels, it could be assumed, as
we have done previously, that the majority of protein C activation
occurs in the microcirculation. The influence of EPCR on protein C
activation kinetics is primarily on the Km for
the reaction,4 which is probably physiologically significant because the Km of the thrombin-TM
complex for protein C in the absence of EPCR is far below the plasma
concentration of protein C.
The EPCR appears to be physiologically significant in the control of
thrombosis and inflammation and in the host response to gram-negative
sepsis. Inhibition of protein C binding to EPCR exacerbates the baboon
response to sublethal Escherichia coli, converting it into a
lethal response characterized by disseminated intravascular coagulation
(DIC), microvascular thrombosis, capillary leak, leukocyte
infiltration, congestion in the tissues, and increased cytokine
elaboration.6 In animals challenged with lethal levels of E coli, infusion of activated protein C (APC) was able to
block the microvascular thrombosis and leukocyte
activation,7 raising the question of which of the above
physiologic changes were due to impaired APC generation.
In addition, clinical studies have identified patients with mutations
in the EPCR gene. The frequency of the mutation in patients with venous thrombosis appears to be higher than in the control groups.8 This result is entirely consistent with the
concept that EPCR mutation might be roughly equivalent to heterozygous protein C deficiency if one assumes several things. First, the rate of
protein C activation is dependent on protein C and EPCR concentrations.
The former is supported by analysis of APC levels in humans with
variable levels of protein C.9 These studies showed that
the levels of APC were proportional to protein C concentration. With
respect to EPCR, there is no direct clinical data, but the in vitro
data discussed above strongly indicate that protein C activation rates
in vivo might be directly tied with the EPCR concentration. It is not
possible to assess the impact of EPCR on protein C activation in vivo
because we cannot accurately assess the EPCR concentration, the
cellular distribution of EPCR and TM, or the impact blood flow and
regional variations thereof have on the participation of EPCR in
protein C activation.
Because of the potential clinical significance of EPCR deficiency in
thrombotic disease and the uncertainty of the role of EPCR in systemic
protein C activation, in the present study we analyzed the impact of
inhibiting protein C interaction with EPCR on the extent of APC
formation in response to thrombin infusion. This is the simplest in
vivo model in which these questions can be addressed because the
previous studies with inflammatory models of thrombosis, sepsis, and
DIC revealed that blocking protein C binding to EPCR resulted in a
spectrum of changes among which included a large increase in thrombin generation.
Reagents
Pre-experimentation and experimentation procedures
Infusion procedures Experiments were performed on 6 baboons. Animals were fasted overnight before each experiment, but were allowed water ad libitum. Each animal was sedated with ketamine hydrochloride (14 mg/kg, intramuscularly) on the morning of the study, and then, using a percutaneous catheter in the cephalic vein, anesthetized with sodium pentobarbital (2 mg/kg initially and with additional amounts approximately every 20 minutes for 8 hours to maintain a light level of surgical anesthesia).11 Animals were intubated orally and allowed to breathe spontaneously. The femoral artery and vein were cannulated aseptically and used for measuring arterial pressure and obtaining blood samples, respectively. A percutaneous catheter was placed in the saphenous vein and used to infuse thrombin.11 Each anesthetized baboon was positioned on its side in contact with controlled temperature heating pads.Experimental groups Six animals were studied. The first 2 were infused with thrombin alone. The last 4 were studied twice. Each animal first was infused with thrombin alone (2 U/kg per minute for 60 minutes). This was followed 2 to 3 days later by a bolus infusion of inhibitory anti-EPCR mAb 1494 (5 mg/kg) at T 30 minutes followed by a 60-minute infusion of thrombin as described above. Each animal thus served as its own control. After the 60-minute infusion, the animals were observed for an additional 60 minutes out to T +120 minutes.In the control experiments, 2 baboons were infused with the noninhibitory mAb 1510 (5 mg/kg) as described above before infusing. Sampling Mean systemic arterial pressure (MSAP) and heart rate were monitored with a Strathem pressure transducer and Hewlett Packard (Avondale, PA) recorder. Rectal temperature was measured with a telethermometer (Yellow Springs Instrument, Yellow Springs, OH). The above measurements were made, and blood samples were collected at T = 30, 0, +20, 40, 60, 80, 100, and 120 minutes, where T = 0
designates when the infusion of thrombin was begun. Less than 10% of
the animals' calculated blood volume (70 mL/kg) was withdrawn over the
8-hour monitoring period.
Assays Plasma APC levels were measured by a minor modification of the enzyme capture assay described by Gruber and Griffin.12 The modification involved using a different mAb (HPC 1241) to trap the APC in the baboon plasma. Fibrin degradation products (FDP) were measured by latex agglutination assay.12 Fibrinogen concentration was determined based on the thrombin clotting time.13 Activated partial thromboplastin time (aPTT) was determined based on the one-stage assay for plasma thromboplastin antecedent.14 Platelet and white cell counts were determined in a Coulter counter (Brea, CA).Statistical analysis The data were analyzed using analysis of variance with the Duncan multicomparison test to determine significant differences for a given variable between groups at given times. An analysis of variance was also used to determine significant differences (P < .05) between time 0 (T 0) and baseline and subsequent times for a given variable and a given group.
To assess the impact of EPCR on thrombin-dependent protein C
activation in vivo, we infused thrombin into baboons that were either
untreated or pretreated with the anti-EPCR mAb 1494 that blocks protein
C binding to EPCR. Because these studies require comparison of protein
C activation with and without the blocking antibody, we were concerned
about intra-animal variations in protein C activation. To circumvent
this problem, we infused baboons first with thrombin alone and then 2 to 3 days later repeated the infusion either with or without
pretreatment with mAb 1494. As can be seen in Figure
1, when the thrombin infusions were
repeated in the same animal on different days, the circulating levels
of APC were similar in both cases. This allows each animal to serve as
its own control. In contrast, when the infusion was repeated after mAb
1494 had been administered, there was a dramatic decrease in the
circulating levels of APC compared to the first infusion. The peak
circulating levels of APC were 137 ± 13 ng/mL to 17 ± 4 ng/mL and
therefore approximately an 86% decrease in APC levels (Figure
2A). The differences in circulating APC
levels in animals infused with thrombin with and without the blocking
antibody present were also reflected in a much greater anticoagulant
response to thrombin infusion in the absence of the antibody than in
its presence (Figure 2B). Without the antibody present, the maximum
increase in the aPTT was 46.3 ± 12.8 seconds compared to a maximum
increase of 12.3 ± 4.7 seconds when the antibody was also present.
In both cases, more than 60% of the increase in the aPTT could be
blocked by the in vitro addition of a polyclonal antibody to
protein C.
To test the possibility that impact of the antibody was not directly related to blocking protein C binding to EPCR, we infused an mAb to EPCR that binds to EPCR without blocking protein C binding. When thrombin was infused in 2 baboons treated with this nonblocking antibody, the peak levels of circulating APC rose to approximately those seen in the animals infused with thrombin alone (average 164 ng/mL in the nonblocking and 124 ng/mL in the blocking antibody group). Thus, the dramatic inhibition of protein C activation seen with the blocking antibody was not due to a nonspecific antibody effect. Although the above results suggest that the decrease in circulating APC
caused by the anti-EPCR mAb is due directly to impaired protein C
activation, it is possible that the impact is secondary to other major
physiologic changes or increased fibrin deposition. Similar changes
were observed when the fibrinogen and FDP levels were measured with or
without the blocking antibody (Figure 3). The plasma fibrinogen levels fell to a nadir of 62% ± 5% in
animals infused with thrombin without the antibody and 48% ± 5% in
animals infused with thrombin in the presence of the antibody. Peak FDP rose to 190 µg/dL in the animals infused with thrombin in the absence
of the antibody and 116 µg/dL in animals infused with thrombin in the
presence of the antibody. Although the value in the animals infused
with thrombin alone is somewhat higher at its peak, it should be noted
that except for this peak time, the other samples had much more similar
values.
Infusion of thrombin alone or anti-EPCR mAb plus thrombin produced no significant changes in the vital signs. At the end of the 2-hour period of observation, the values for all animals were: MSAP, 95 ± 5 mm Hg; heart rate, 131 ± 6/min; respiration rate, 24 ± 1/min; and temperature 36.9°C ± 0.2°C. The white cell counts rose from 7.7 at T 0 to 11.0 at T +120 minutes for the thrombin alone group, and 6.1 at T 0 to 12.4 at T +120 minutes for the thrombin plus anti-EPCR group.
The present study reveals that EPCR makes a major contribution to the activation of protein C initiated by thrombin infusion in primates. The extent of the stimulation was much greater than we had anticipated based on the cell culture data. In cell culture, blocking protein C-EPCR interaction reduces the protein C activation rate about 5-fold.3 However, because the levels of EPCR are much higher in larger vessels than in the capillaries2 and the rate of protein C activation in vitro is dependent on EPCR concentration,3-5 we would have anticipated that the acceleration in the capillaries would be less than on the cultured cells. Several factors could contribute to the unexpectedly high contribution of EPCR to protein C activation in vivo. The fact that EPCR decreases the Km for protein C activation will result in protection of the thrombin-TM complex from inhibition by circulating inhibitors such as antithrombin15 or protein C inhibitor.16 In vitro studies have indicated that in the presence of these inhibitors but in the absence of protein C and EPCR, the half life of thrombin bound to TM is on the order of 2 seconds.16 In addition, the increase in local protein C concentration near the thrombin-TM complex would augment protein C activation by limiting access to other good substrates for the thrombin-TM complex such as the thrombin-dependent fibrinolysis inhibitor (TAFI).17 Because TAFI and protein C are both relatively good substrates for the thrombin-TM complex, TAFI would serve as a competitive inhibitor of protein C activation in vivo, a phenomenon that could be partially overcome by elevating the local protein C concentration through interactions with EPCR. It is also possible that the EPCR levels are higher in the microcirculation than we infer from the qualitative immunohistochemistry. In favor of this possibility, one study has reported detectable EPCR expression in the microcirculation.18 In addition, at least in cell culture, a relatively high percentage of EPCR is intracellular. Under flow conditions, it is possible that some of this EPCR redistributes to the cell surface. Finally, under flow conditions, EPCR could be important for maintaining relatively high levels of protein C on the cell surface while rapid protein C activation is occurring. By so doing, EPCR could protect against local substrate depletion. The decreased levels of APC generated following thrombin infusion in the presence of the inhibitory antibody to EPCR are most likely a direct effect of inhibition of protein C activation rather than major physiologic changes. The lack of major changes in blood pressure and heart rate would argue against the inhibition of protein C binding to EPCR causing decreased protein C activation due to microvascular occlusion or major changes in flow. The lack of difference in the leukocyte response to thrombin infusion with or without the antibody also argues against microvascular occlusion or leukocyte-mediated down-regulation of thrombomodulin in this system. The observation that the fibrinogen consumption is similar in the animals infused with thrombin either in the presence or absence of the anti-EPCR mAb is of interest. Given that the APC levels are decreased by 88% in the presence of the antibody, one might expect that any thrombin-mediated augmentation of coagulation would result in more thrombin generation in animals given the anti-EPCR antibody. The observation that the decrease in fibrinogen is similar suggests that fibrinogen consumption is due almost exclusively to the exogenous thrombin that was infused. This would imply that the production of thrombin initiated by factor XI activation,19,20 which would require factors V and VIII, plays little role in thrombin formation in this system. The major implication of the present work is that EPCR plays a major role in protein C activation contributing to the systemic levels of APC in response to thrombin. This implies that defects in EPCR would result in a major increase in the risk for thrombosis. Consistent with this hypothesis is our recent observation that blocking EPCR-protein C interactions increases fibrin deposition in tissues in response to low levels of E coli.6 The present findings are also consistent with the report of gene abnormalities in EPCR being associated with an increased risk of venous and possibly arterial thrombosis.8
Submitted August 9, 2000; accepted November 6, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Charles T. Esmon, Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th St, Oklahoma City, OK 73104; e-mail: charles-esmon{at}omrf.ouhsc.edu.
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© 2001 by The American Society of Hematology.
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  J. H. Finigan, A. Boueiz, E. Wilkinson, R. Damico, J. Skirball, H. H. Pae, M. Damarla, E. Hasan, D. B. Pearse, S. P. Reddy, et al. Activated protein C protects against ventilator-induced pulmonary capillary leak Am J Physiol Lung Cell Mol Physiol, June 1, 2009; 296(6): L1002 - L1011. [Abstract] [Full Text] [PDF]
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B. Saposnik, E. Lesteven, A. Lokajczyk, C. T. Esmon, M. Aiach, and S. Gandrille Alternative mRNA is favored by the A3 haplotype of the EPCR gene PROCR and generates a novel soluble form of EPCR in plasma Blood, April 1, 2008; 111(7): 3442 - 3451. [Abstract] [Full Text] [PDF]
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A. Gruber, U. M. Marzec, L. Bush, E. Di Cera, J. A. Fernandez, M. A. Berny, E. I. Tucker, O. J. T. McCarty, J. H. Griffin, and S. R. Hanson Relative antithrombotic and antihemostatic effects of protein C activator versus low-molecular-weight heparin in primates Blood, April 1, 2007; 109(9): 3733 - 3740. [Abstract] [Full Text] [PDF]
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L. R. Mollica, J. T. B. Crawley, K. Liu, J. B. Rance, P. N. Cockerill, G. A. Follows, J.-R. Landry, D. J. Wells, and D. A. Lane Role of a 5'-enhancer in the transcriptional regulation of the human endothelial cell protein C receptor gene Blood, August 15, 2006; 108(4): 1251 - 1259. [Abstract] [Full Text] [PDF]
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 D. Brenner, J. Labreuche, P.-J. Touboul, K. Schmidt-Petersen, O. Poirier, C. Perret, J. Schonfelder, C. Combadiere, M. Lathrop, F. Cambien, et al. Cytokine Polymorphisms Associated With Carotid Intima-Media Thickness in Stroke Patients Stroke, July 1, 2006; 37(7): 1691 - 1696. [Abstract] [Full Text] [PDF]
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W. Li, X. Zheng, J.-M. Gu, G. L. Ferrell, M. Brady, N. L. Esmon, and C. T. Esmon Extraembryonic expression of EPCR is essential for embryonic viability Blood, October 15, 2005; 106(8): 2716 - 2722. [Abstract] [Full Text] [PDF]
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 J. H. Finigan, S. M. Dudek, P. A. Singleton, E. T. Chiang, J. R. Jacobson, S. M. Camp, S. Q. Ye, and J. G. N. Garcia Activated Protein C Mediates Novel Lung Endothelial Barrier Enhancement: ROLE OF SPHINGOSINE 1-PHOSPHATE RECEPTOR TRANSACTIVATION J. Biol. Chem., April 29, 2005; 280(17): 17286 - 17293. [Abstract] [Full Text] [PDF]
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B. Saposnik, J.-L. Reny, P. Gaussem, J. Emmerich, M. Aiach, and S. Gandrille A haplotype of the EPCR gene is associated with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis Blood, February 15, 2004; 103(4): 1311 - 1318. [Abstract] [Full Text] [PDF]
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B. A. Kerlin, S. B. Yan, B. H. Isermann, J. T. Brandt, R. Sood, B. R. Basson, D. E. Joyce, H. Weiler, and J.-F. Dhainaut Survival advantage associated with heterozygous factor V Leiden mutation in patients with severe sepsis and in mouse endotoxemia Blood, November 1, 2003; 102(9): 3085 - 3092. [Abstract] [Full Text] [PDF]
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 C. T. Esmon The Protein C Pathway Chest, September 1, 2003; 124(3_suppl): 26S - 32S. [Abstract] [Full Text] [PDF]
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A. Slungaard, J. A. Fernandez, J. H. Griffin, N. S. Key, J. R. Long, D. J. Piegors, and S. R. Lentz Platelet factor 4 enhances generation of activated protein C in vitro and in vivo Blood, July 1, 2003; 102(1): 146 - 151. [Abstract] [Full Text] [PDF]
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J. B. Rance, G. A. Follows, P. N. Cockerill, C. Bonifer, D. A. Lane, and R. E. Simmonds Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites Blood, June 1, 2003; 101(11): 4393 - 4401. [Abstract] [Full Text] [PDF]
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 S. R. Lentz Thrombosis of Vein Grafts: Wall Tension Restrains Thrombomodulin Expression Circ. Res., January 10, 2003; 92(1): 12 - 13. [Full Text] [PDF]
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M. J. A. Simmelink, P. G. de Groot, R. H. W. M. Derksen, J. A. Fernandez, and J. H. Griffin Oral anticoagulation reduces activated protein C less than protein C and other vitamin K-dependent clotting factors Blood, December 1, 2002; 100(12): 4232 - 4233. [Abstract] [Full Text] [PDF]
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J.-M. Gu, J. T. B. Crawley, G. Ferrell, F. Zhang, W. Li, N. L. Esmon, and C. T. Esmon Disruption of the Endothelial Cell Protein C Receptor Gene in Mice Causes Placental Thrombosis and Early Embryonic Lethality J. Biol. Chem., November 1, 2002; 277(45): 43335 - 43343. [Abstract] [Full Text] [PDF]
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S. R. Lentz, F. J. Miller Jr, D. J. Piegors, R. A. Erger, J. A. Fernandez, J. H. Griffin, and D. D. Heistad Anticoagulant Responses to Thrombin Are Enhanced During Regression of Atherosclerosis in Monkeys Circulation, August 13, 2002; 106(7): 842 - 846. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) to the infusion 2 days later (
).
).
Two to 3 days later the same animals were infused with 5 mg/kg
anti-EPCR mAb 1494 30 minutes before repeating the thrombin infusion
described above (
). The circulating APC concentrations were then
determined at the times indicated. (B) Plasma samples were collected
from the animals infused with thrombin alone or thrombin plus the
anti-EPCR antibody at the same time the samples were taken for APC
level determination. The aPTT assays were then performed on
these samples.
enzyme levels are proportional to total protein C levels.
Thromb Haemost.
1996;75:56-61