Blood, 1 December 2002, Vol. 100, No. 12, pp. 4232-4233
BRIEF REPORT
Oral anticoagulation reduces activated protein C less than
protein C and other vitamin K-dependent clotting
factors
Marleen J. A. Simmelink,
Philip G. de Groot,
Ronald H. W. M. Derksen,
José A. Fernández, and
John H. Griffin
From the Thrombosis and Haemostasis Laboratory,
Department of Haematology, and Department of Rheumatology & Clinical
Immunology, University Medical Center, Utrecht, The Netherlands; the
Institute of Biomembranes, Utrecht University, Utrecht, The
Netherlands; and the Department of Molecular and Experimental Medicine,
The Scripps Research Institute, La Jolla, CA.
 |
Abstract |
Oral anticoagulant therapy, which is used for prophylaxis and
management of thrombotic disorders, causes similar reductions in plasma
levels of vitamin K-dependent procoagulant and anticoagulant clotting
factor zymogens. When we measured levels of circulating activated
protein C, a physiologically important anticoagulant and
anti-inflammatory agent, in patients on oral anticoagulant therapy,
the results unexpectedly showed that such therapy decreases levels of
activated protein C substantially less than levels of protein
C, prothrombin, and factor X, especially at lower levels of
prothrombin and factor X. Thus, we suggest that oral anticoagulant therapy results in a relatively increased expression of the protein C
pathway compared with procoagulant pathways not only because there is
less prothrombin to inhibit activated protein C anticoagulant activity,
but also because there is a disproportionately higher level of
circulating activated protein C.
(Blood. 2002;100:4232-4233)
© 2002 by The American Society of Hematology.
| |
Introduction |
Although unfractionated or low-molecular-weight
heparins are often used for immediate management of thrombotic risks or
manifestations, oral anticoagulants (OACs) are generally used for
prolonged therapy.1 OACs act as antagonists of the normal
biosynthesis of vitamin K-dependent proteins. Vitamin K is required
for the synthesis of biologically active procoagulant factors II, VII,
IX, and X, as well as the anticoagulant factors protein C, protein S,
and protein Z. These plasma proteins participate in calcium-dependent interactions with negatively charged phospholipids through
-carboxyglutamyl (Gla) residues in their amino-terminal region.
Vitamin K is required for the postribosomal conversion of glutamyl
residues in precursor proteins to Gla residues in the mature plasma
proteins. OACs block Gla formation, resulting in dysfunctional proteins
that lack 8 to 11 Gla residues per protein molecule. Partially
carboxylated clotting factors are dysfunctional because of impaired
interactions with phospholipid membranes.2 During
sustained oral anticoagulation, procoagulant activity levels of vitamin
K-dependent clotting factors average 15% to 25%, whereas antigen
levels average approximately 40% to 60%, and the various vitamin
K-dependent proteins decrease in parallel to each
other.3-7
Activated protein C (APC) is a normal circulating component of
plasma8 whose formation from zymogen protein C is thought to be effected by the thrombin/thrombomodulin complex in a reaction that is enhanced by the endothelial protein C
receptor.9,10 Oral anticoagulant therapy causes a decrease
in circulating soluble endothelial protein C receptor.11
APC is a physiologic anticoagulant and anti-inflammatory agent that was
recently approved by the Food and Drug Administration (FDA) for therapy
of severe sepsis because it substantially reduces sepsis-related
mortality.12 Low levels of circulating APC were recently
suggested to be a risk factor for venous thrombosis.13
Smirnov et al14 reported that prothrombin inhibits APC
anticoagulant activity and hypothesized that during anticoagulant
therapy the reduction of prothrombin levels would result in an
increased effectiveness of APC. To shed light on the physiology of APC,
we have an interest to quantitate APC in various human subjects.
Recently, we measured circulating APC in plasma of patients with
systemic lupus erythematosus (SLE) with antiphospholipid antibodies who
did not use OACs.15 We extended our studies to a group of
SLE patients with antiphospholipid antibodies who used OACs and also to
a group of patients who used OACs for cardiac diseases. Here we report
the unexpected finding that OACs reduce levels of circulating APC less
than those of protein C, prothrombin (FII), and factor X (FX).
| |
Study design |
Blood samples were collected by venipuncture using plastic tubes
containing 3.8% trisodium citrate (0.129 M; 9:1 vol/vol) from 84 patients with SLE (28 of these were using OACs) and from 31 cardiac
patients on OAC therapy. Protein C, FII, and FX levels were measured in
platelet-poor plasma. Protein C levels were measured by a coagulometric
assay (Protein C Reagent, coagulometric; Dade Behring Marburg GmbH,
Germany) and by a chromogenic assay (Berichrom protein C; Dade
Behring) that measures all forms of protein C in plasma. Levels of
factors II and X were measured with a coagulometric method that
determines levels of procoagulantly active factors (Thromborel S, Dade
Behring). Protein C, FII, and FX levels were expressed as the
percentage of the level in pooled normal plasma (40 healthy blood
donors). Circulating APC levels were measured by an enzyme capture
assay that measures all forms of APC.8
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Results and discussion |
The mean values for APC, protein C, FII, and FX in subjects on
OACs were 69%, 22% (coagulometric protein C), 54% (chromogenic protein C), 35%, and 24%, respectively, whereas the values for SLE
patients not receiving OACs were 94%, 123% (coagulometric protein C),
119% (chromogenic protein C), 102%, and 111%, respectively. For all
subjects on OACs, levels of FII, FX, and protein C similarly and
proportionally decreased during OAC treatment (data not shown) as
previously reported3-7 and as illustrated in Figure
1A for FII and protein C (chromogenic)
where the ratio of FII/protein C did not vary much over the range of
protein C observed. In contrast, when the ratio of APC to protein C
(chromogenic), FII, and FX were calculated and graphed as a function of
protein C (chromogenic), FII, and FX, respectively, each of these
ratios increased as the concentration of level of each zymogen factor
decreased because of OACs (Figure 1B-D). Compared with data for
subjects not taking OACs (Figure 1B-D), there was a strong and inverse
relationship between the APC/protein C ratio and protein C (B), the
APC/FII ratio and FII (C), and the APC/FX ratio and FX (D). This
finding indicates that during OAC treatment the decrease in APC levels is much less than the respective decreases in protein C, FII, and FX.
Notably for FII and FX levels below 25%, the APC/FII and APC/FX ratios
increase from 2 to 5 and 2 to 8, respectively.
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| Figure 1.
Comparisons of effects of oral anticoagulant therapy on
circulating APC, protein C, FII, and FX.
 represents patients using oral anticoagulants.  indicates
SLE patients without oral anticoagulant treatment. (A) Correlation
between FII/protein C ratio and protein C level (chromogenic).
(B) Correlation between APC/protein C ratio and protein C level
(chromogenic). (C) Correlation between APC/FII ratio and FII level. (D)
Correlation between APC/FX ratio and FX level.
|
|
The APC immunocapture assay used in our studies captures both normally
carboxylated and partially carboxylated forms of APC. The protein C
chromogenic assay used here also reflects both normally and partially
carboxylated forms of protein C. Thus, the increase of the ratio of
APC/protein C at low concentrations of protein C indicates that all
forms of APC are disproportionately higher than all forms of protein C
zymogen. This finding is consistent with the increased ratios of
APC/FII and APC/FX at low FII and FX levels.
Elevated plasma prothrombin levels, often because of the 20210A
prothrombin gene polymorphism, are a significant risk factor for venous
thrombosis.16 Because prothrombin inhibits APC
anticoagulant activity in vitro and because this inhibition is reduced
at low concentrations of prothrombin, it was hypothesized that during anticoagulant therapy the reduction of prothrombin levels would result
in an increased effectiveness of whatever APC is
available.14 Furthermore, it was assumed, without
experimental data, that the level of APC would be proportional to the
level of protein C in patients using OACs.14 On the basis
of the results here, we suggest that OAC therapy results in a
relatively increased expression of the protein C pathway compared with
procoagulant pathways not only because there is less prothrombin to
inhibit activated protein C anticoagulant activity but also because
there is a disproportionately higher level of circulating APC. As both
plasmin and factor Xa are capable of activating protein C to give
APC,17,18 it can be speculated that the contribution of
these (or other) proteases in the generation of basal levels of APC
during OAC therapy explains why APC levels are not reduced
proportionally to prothrombin.
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Footnotes |
Submitted February 1, 2002; accepted July 13, 2002.
Prepublished online
as Blood First Edition Paper, August 8, 2002; DOI
10.1182/blood-2002-01-0329.
Supported by grants from The Dutch League against Rheumatism (NR
97.1.401) and the National Institutes of Health (R37HL52246).
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: M. J. A. Simmelink,
Thrombosis and Haemostasis Laboratory, Department of Haematology
(G03.647), University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; e-mail:
m.simmelink{at}lab.azu.nl.
| |
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Abstract
Introduction
