© 1995 American Heart Association, Inc.
Articles |
Antithrombotic Effects of Activated Protein C and Protein S in a Rabbit Model of Microarterial Thrombosis
From the Departments of Plastic and Reconstructive Surgery (B.A.), Experimental Research (B.A.), and Clinical Chemistry and Coagulation Research (B.D.), University of Lund, Malmö General Hospital, Malmö, Sweden.
Correspondence to Björn Arnljots, Department of Plastic and Reconstructive Surgery, Malmö General Hospital, S-214 01 Malmö, Sweden.
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Abstract |
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Abstract The antithrombotic properties of activated protein C (APC) and protein S were investigated in a rabbit model of microarterial thrombosis. The study focused on the ability of intact and thrombin-cleaved bovine protein S to potentiate the biological effects of bovine APC in vivo. Segments of the central arteries of the ears were subjected to arteriotomy, deep-vessel wall trauma, and arteriotomy suture. Five minutes before vascular reperfusion, groups of rabbits were infused with boluses of 0.1 mg/kg bovine APC alone or combined with different doses of intact (0.5, 0.1, or 0.05 mg/kg) or thrombin-cleaved (0.5 mg/kg) protein S. APC in combination with the two higher doses of protein S produced a potent antithrombotic response, as judged by assessment of vessel patency rates, while only the group receiving APC+0.5 mg/kg protein S showed significant reduction of thrombus weights as well. The biological effect depended on the active site in APC, as the antithrombotic effect was lost on pretreatment of APC with the serine protease inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone. The potentiation of the APC response by protein S depended on the structural integrity of the protein, and cleavage of the thrombin-sensitive region in protein S by thrombin resulted in a loss of biological response. No hemorrhagic side effects were noted by the APC–protein S combination, and the anticoagulant response was mild, even to the highest doses of APC and protein S. The study demonstrates the specificity of the APC–protein S interaction in vivo and shows both the necessity of an active catalytic site in APC and the physiological importance of the APC–protein S interaction. APC appears to be a suitable antithrombotic agent when fully potentiated by its cofactor protein S, as it produces only mild plasma anticoagulation and no hemorrhagic side effects.
Key Words: protein C • protein S • thrombosis • animal model
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Introduction |
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The vitamin K–dependent plasma proteins C and S are key components of an endogenous anticoagulation system that is crucial for the maintenance of a normal hemostatic balance.1 2 Deficiencies of protein C, protein S, and resistance to the anticoagulant function of activated protein C (APC), a condition caused by a point mutation in the gene for factor V, are associated with thrombophilia.3 Antithrombotic properties of APC have been demonstrated in animal models that use endotoxin shock challenge4 5 as well as in different models of thrombosis.6 7 8 9 10 11 12 13 In pilot cases, protein C has successfully been used in the treatment of human thrombotic disease,14 15 and site-activated mutant forms of human protein C with promising anticoagulant profiles have been engineered.16
Protein C is a 60-kD multidomain protein that circulates in plasma as a
zymogen of a serine protease (concentration, 3 to 5 mg/L). It is
converted to an active serine protease by thrombin that is bound to the
endothelial cell membrane protein
thrombomodulin.1 The protein C molecule consists of an
amino-terminal 
-carboxyglutamic acid–containing module, two
epidermal growth factor–like modules, and a serine protease module,
which contains the catalytic site. APC demonstrates a high degree of
substrate specificity, and the only physiologically
important substrates are the activated forms of coagulation factors V
and VIII.1 By degrading factors Va and VIIIa, APC inhibits
the coagulation processes.
The anticoagulant action of APC is enhanced by a 75-kD
nonenzymatic cofactor, protein S.1 2 Protein S consists of
an amino-terminal -carboxyglutamic acid–rich module, a
thrombin-sensitive region, four epidermal growth factor–like
modules, and a carboxy-terminal region that is homologous to sex
hormone–binding globulin. The concentration of protein S in plasma is
20 to 25 mg/L. In human plasma, 60% of protein S occurs in complex
with C4b-binding protein, a regulator of the complement
pathway.2 Only the free form of protein S acts as a
cofactor to APC. Rabbit plasma, unlike its human counterpart, contains
only free protein S.17 18 Protein S, besides potentiating
the proteolytic effect of APC, also abolishes the protective effects
that factors IXa and Xa provide against APC-mediated degradation of
factors VIIIa and Va, respectively.19 20 Protein S
increases the affinity of APC for negatively charged phospholipids,
where the two proteins seem to form 1:1 stoichiometric
complexes.1 2 The cofactor function of protein S is to a
certain degree species specific, eg, the effects of bovine APC are not
potentiated by rabbit protein S to any major extent.21
This species specificity provides an opportunity to specifically assess
the in vivo effects of APC and protein S.13
The functions of APC and protein S depend on the structural integrity of the proteins; eg, the anticoagulant function of APC is lost when its serine protease domain is blocked.22 In protein S the thrombin-sensitive region and the first epidermal growth factor–like module are important for the expression of APC-cofactor activity.23 The anticoagulant properties of protein S are lost upon cleavage of protein S by thrombin.2 24
In previous studies using this model, we have described antithrombotic effects of bovine APC and a powerful potentiation of this effect by its cofactor protein S, which demonstrates the importance of protein S as an in vivo cofactor to APC.12 13 Based on these data, the present study was designed to further characterize the in vivo interaction between APC and protein S by assessing the effects of intact and modified proteins in the same model. The present study also extends previous work by evaluating the antithrombotic action of APC and protein S as a function of their molar ratios. The necessity of an active catalytic site in APC and the in vivo importance of the APC–protein S interaction are demonstrated as well as the possibility of APC combined with protein S as an antithrombotic intervention.
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Methods |
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Thrombosis Model
Rabbits (Swedish loop) of either sex (mean weight, 3.0 kg) were fed a standard pellet diet and were anesthetized with intravenous pentobarbital. The experimental procedure has been described in detail.12 In brief, segments of the central arteries of both ears (outer diameter,
1 mm) were prepared,
and blood flow was interrupted by placing vascular clamps 7 mm apart.
Longitudinal arteriotomies (7 mm) were performed, vascular lumina were
everted and flattened, and 5 mm of vascular lumina was denuded with a
scalpel blade that created a standardized vessel trauma and exposed
deep layers of media to the bloodstream.25 The
arteriotomies were closed with running sutures. After reperfusion
(opening of the vascular clamps), arteriotomy bleeding was
recorded. Vessel patency was assessed at 30 and 120 minutes after
release of the clamps by using a standard microsurgical empty/refill
test,12 26 and vessels were classified as being patent
(including the subdivision "reduced patency") or occluded. After
the final patency test, the thrombotic material was removed and
weighed, and the animals were killed by injection of concentrated
sodium pentobarbital and alcohol.
Proteins
Bovine protein C was purified and activated by
thrombin.12 27 The purity of the protein, as judged by
sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (reduced and unreduced protein), exceeded 95% (not
shown). Active-site–blocked APC was prepared by incubating APC (3.1
g/L in Tris-HCl 50 mmol/L and NaCl 0.15 mol/L, pH 7.5, containing 1 g/L
bovine serum albumin [BSA]) with 200 µmol/L of
D-phenylalanyl-L-prolyl-L-arginine
chloromethylketone (PPACK; Calbiochem) for 1 hour at 37°C. Free PPACK
was removed by dialysis against Tris-HCl 50 mmol/L and NaCl 0.15 mol/L,
pH 7.5.
Bovine protein S was purified.28 Thrombin-cleaved protein S was obtained by incubating protein S (0.8 g/L in Tris-HCl 50 mmol/L, NaCl 0.15 mol/L, and EDTA 2 mmol/L, pH 7.5) with bovine thrombin (10 U/mL) at 37°C for 2 hours, a procedure that led to complete cleavage of the thrombin-sensitive region (results not shown). Thrombin was removed from the protein S solution by passage through a 0.9x10-cm SP-Sephadex column equilibrated in Tris-HCl 50 mmol/L and NaCl 0.15 mol/L, pH 7.5. Thrombin bound to the column, and the cleaved protein S was recovered in the pass-through fractions. Any remaining thrombin was inhibited by addition of PPACK, after which the protein S was extensively dialyzed against Tris-HCl 50 mmol/L and NaCl 0.15 mol/L, pH 7.5.
In Vitro Tests of APC and Protein S Functions
The anticoagulant function of intact and
active-site–blocked APC was assessed with a clotting assay by using
human plasma essentially as described.12 Human plasma (100
µL) was incubated with 100 µL activated partial thromboplastin time
(APTT) reagent (Automated APTT from Organon Teknika) for 5 minutes,
after which clotting was started by addition of 100 µL of
CaCl2 30 mmol/L in Tris 50 mmol/L and NaCl 0.15 mol/L, pH
7.5, containing 1 g/L BSA. Various combinations of intact or
thrombin-cleaved bovine protein S (0 or 10 mg/L) and intact or
active-site–inhibited bovine APC (0, 0.5, or 1 mg/L) were included in
the CaCl2 solution. Intact bovine APC but not
active-site–blocked APC prolonged the clotting time twofold to
threefold, but only in the presence of intact bovine protein
S.12 17 The anticoagulant effects of bovine APC, with and
without protein S, in a rabbit plasma system have been
described.12 17
Experimental Protocol
Seven groups of rabbits were treated with bovine APC (0.1
mg/kg), APC (0.1 mg/kg) and protein S (0.5, 0.1, or 0.05 mg/kg),
active-site–blocked APC (0.1 mg/kg) and protein S (0.5 mg/kg), APC
(0.1 mg/kg) and thrombin-cleaved protein S (0.5 mg/kg), or vehicle
(Tris-HCl 50 mmol/L and NaCl 0.15 mol/L, pH 7.5, containing 1 g/L BSA).
There were 10 rabbits in each group except for one group of 11 animals
that received APC and protein S 0.5 mg/kg (data from 20 and 22 vessels,
respectively, were obtained from each group). The animals were treated
in a blinded, random fashion, except for the APC and protein S 0.05
mg/kg group, which was added after completion of the randomized series.
The substances were given as bolus injections into one marginal ear
vein 5 minutes before vascular reperfusion. Data concerning plasma
levels and elimination of infused bovine APC and protein S are
available.13
Coagulation Analyses
Blood was collected through an indwelling aortic cannula placed
through a femoral artery, and coagulation analyses and
platelet counts were performed.12 Samples for APTT
analyses, drawn just before and at 10, 60, and 120 minutes
after administration of substances, were immediately
centrifuged (2000g for 15 minutes), and the plasma
was frozen at -70°C until analyzed (MLA Electra 900
automatic analyzer). Blood samples for platelet counts were
drawn just before and 60 minutes after administration of the substances
and were analyzed in an STKS Coulter automatic
analyzer.
Statistical Methods
Statistical testing was performed12 by using
STATXACT software (CYTEL Software Corp), taking into
consideration that pairwise, and thus interdependent, observations were
obtained from each animal. Arteriotomy bleeding times and thrombus
weights were compared by using the Mann-Whitney U test and
vessel patency by using the Jonckheere-Terpstra test.29
Two-sided probability values are given, and the data are
presented as medians and appropriate percentiles to facilitate
the demonstration of asymmetry. Probability values <.05 were regarded
as significant.
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Results |
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Bovine APC (0.1 mg/kg), when given together with an approximately equimolar amount of bovine protein S (0.1 mg/kg), produced a powerful antithrombotic response that was reflected by significantly increased vessel patency rates at 30 and 120 minutes after reperfusion (Fig 1
). Increasing the protein S dose fivefold did not
improve the patency rates. However, thrombus weights were significantly
lower in the group of animals that received the highest protein S dose
than in the vehicle group (Fig 2). Reduction of the
protein S concentration to 0.05 mg/kg led to a reduced antithrombotic
effect. The structural integrity of protein S was shown to be crucially
important because the combination of APC and thrombin-cleaved protein S
demonstrated no antithrombotic effect. The powerful antithrombotic
effect exerted by the combination of APC (0.1 mg/kg) and protein S (0.5
mg/kg) depended on the active site in APC because active-site–blocked
APC had no antithrombotic effect.
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, occluded vessels; and 
, patent vessels. Other
abbreviations as in Fig 1
None of the infused protein combinations produced any hemorrhagic side
effects as judged by arteriotomy bleeding times (Fig 3).
Moreover, platelet counts were not changed by any of the
substances.
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In contrast to the powerful antithrombotic response, the ex vivo
anticoagulant response, as measured by prolongation of APTT, was mild
after infusion of intact APC and the highest dose of intact protein S.
The APTT was prolonged to a maximum of around 1.2 times baseline at 10
minutes after injection. Even though the anticoagulant effect was weak,
it was found to depend on the active site of APC and on the structural
integrity of the thrombin-sensitive region of protein S (Fig 4).
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, APC+protein S; 
, APC+thrombin-degraded
protein S. |
Discussion |
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In this study, structurally modified APC and protein S were used to investigate the specificity of the APC–protein S interaction; the results are the in vivo correlates to earlier in vitro data. Anticoagulant and antithrombotic properties of APC were lost upon treatment of APC with the synthetic serine protease inhibitor PPACK. This shows that the active site of APC, which is known to be responsible for its in vitro anticoagulant properties, is also crucial for expression of antithrombotic actions in vivo. Furthermore, we demonstrated the structural intergrity of protein S to be important, because the potentiation of the anticoagulant and antithrombotic effects of APC seen with intact protein S were not observed when using thrombin-cleaved protein S. Thus, the thrombin-sensitive region of protein S is critical for both the in vivo and in vitro functions. At high Ca2+ concentrations (>5 mmol/L) both intact and thrombin-cleaved protein S have high affinities for negatively charged phospholipid membranes, while at physiological Ca2+ concentrations the phospholipid-binding ability of protein S is partially lost upon thrombin cleavage.2 For reasons that are not yet clear, APC together with thrombin-cleaved protein S gave a lower patency rate than APC alone. However, since bovine APC and rabbit protein S are interactive to a low but still detectable degree,17 an explanation may be that thrombin-cleaved bovine protein S competitively interferes with rabbit protein S.
In agreement with previous data obtained with this model,12 13 the present study demonstrated an antithrombotic effect of APC that is comparable to hirudin and superior to heparin.30 Whereas APC alone did not significantly affect the antithrombotic score, a powerful antithrombotic effect was obtained by the same dose of APC (0.1 mg/kg) in combination with a molar excess of protein S.13 We have now investigated the interaction of APC and protein S in different molar ratios. The antithrombotic effect of APC was manifest in combination with an excess or an approximately equimolar dose of protein S but was lost when reduced to a 1:2 ratio relative to APC. These data are compatible with in vitro findings that have suggested a 1:1 molar ratio of the interaction between APC and protein S and are the first in vivo indications supporting this hypothesis.
Notably, the powerful antithrombotic effects in vivo were achieved without hemorrhagic side effects or any significant plasma anticoagulation. APC is theoretically highly attractive as an antithrombotic agent because of its endogenous origin and specific mode of action, and the present findings clearly suggest a therapeutic potential for APC in the prevention and treatment of thrombotic disease, with the reservation that it might not be effective in individuals suffering from APC resistance.3 Obviously, interspecies differences may prove confounding in the extrapolation of these results to clinical practice in humans. On the other hand, there is a high degree of coagulation-factor homology among the species used, and cross-species antithrombotic effects have been demonstrated concordantly among several groups by using human or bovine APC in diverse species.6 7 8 9 10 11 12 13
APC has a half-life in human plasma of around 25 minutes,16 which is a relatively short time for an antithrombotic agent. On the other hand, the administration of the biologically stable zymogen protein C will be ineffective in practically all thrombotic events because the available amount of surface thrombomodulin is limited, and protein C activation depends on the generation of thrombin. To this end, mutant forms of protein C with increased substrate utilization by thrombin independent of thrombomodulin have been genetically engineered.16 Such mutants may be administered as stable profactors to circulate with a long half-life and then be activated at sites where significant thrombin generation occurs. This concept of antithrombotic intervention is intriguing, but it remains to be evaluated in vivo.
A reduction of the APC dose from 0.1 to 0.01 mg/kg in this model is accompanied by a loss of antithrombotic effect even when the APC is fully potentiated by protein S,13 which shows that quantities close to the physiological content of protein C are needed to prevent thrombosis following a powerful thrombotic challenge. Considering the present data describing the molar ratio of the APC–protein S interaction, this also seems to highlight the necessity of an optimal protein S function. Therefore, in considering the potential use of APC as an antithrombotic agent in humans, our findings would suggest coadministration of protein S to be beneficial, particularly in situations associated with low protein S levels or with thrombin cleavage of protein S, a condition that definitely occurs in disseminated intravascular coagulation.31
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Acknowledgments |
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This work was supported by the Swedish Medical Council (grants No. 07143 and 10827) and by grants from the King Gustaf V's 80th Birthday Trust, the King Gustav V and Queen Victoria Trust, the Magnus Bergwall Trust, the Albert Påhlsson Trust, the Johan Kock Trust, Malmö General Hospital research funds, Anna-Lisa and Sven Eric Lundgrens Trust, and the Crafoord Trust. We gratefully acknowledge Ulf Strömberg, PhD, for help with the statistical analyses and Bergisa Hildebrand, Lise Borge, and the staff at the Department for Coagulation Disorders, Malmö General Hospital, for skillful technical assistance.
Received February 21, 1995; accepted April 10, 1995.
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