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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1617-1625
Phosphorothioate Oligonucleotides Inhibit the Intrinsic Tenase
Complex
By
John P. Sheehan and
Hao-Chang Lan
From the University of Texas Health Science Center at San Antonio,
Department of Medicine/Hematology, San Antonio, TX.
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ABSTRACT |
Systemic administration of ISIS 2302, a 20-mer antisense
phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both
monkey and human studies. The anticoagulant effects of ISIS 2302 were
investigated with both in vitro coagulation assays in human plasma and
purified enzyme systems. At high oligonucleotide plasma concentrations
(>100 µg/mL), prolongation of the prothrombin and
thrombin times was observed. In a thrombin time assay using purified
components, high concentrations of ISIS 2302 inhibited thrombin
clotting activity both by stimulating inhibition by heparin cofactor II
and directly competing with fibrinogen for binding to anion binding
exosite I. In contrast, low concentrations of ISIS 2302 (<100
µg/mL) showed a selective, linear prolongation of the activated
partial thromboplastin time (PTT). The rate limiting effect of 50 µg/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was
identified by sequential modification of the clotting assay. Delaying
addition of oligonucleotide until after contact activation failed to
correct prolongation of the PTT. The calcium-dependent steps of the
intrinsic pathway were individually assessed by adding sufficient
activated coagulation factor to correct the PTT in plasma deficient in
that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed
to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor
X-deficient plasma with or without oligonucleotide present. ISIS 2302 (50 µg/mL) did not prolong a modified Russel viper venom time,
suggesting no significant inhibition of prothrombinase. Thus, 50 µg/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic
tenase activity. ISIS 2302 showed partial inhibition of intrinsic
tenase activity (to approximately 35% of control) at clinically
relevant oligonucleotide concentrations in a chromogenic assay. This
activity was oligonucleotide sequence-independent but required the
phosphorothioate backbone, suggesting that inhibition of intrinsic
tenase is a general property of this class of oligonucleotides. These
results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ANTISENSE THERAPY targets specific mRNA
sequences in an attempt to selectively suppress expression of the
corresponding protein and inhibit disease processes. This approach
requires knowledge of the disease process and the relevant target gene
sequence but promises theoretical specificity in the targeting of
pathophysiologic mechanisms.1 Multiple antisense compounds
are currently in phase I or II trials for indications in infectious
disease, vascular restenosis, cancer, and inflammation.2
Intercellular adhesion molecule-1 (ICAM-1), a receptor for the
 2-integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18),
participates in leukocyte emigration in response to inflammatory
stimuli. ICAM-1 expression is induced by inflammatory mediators (tumor
necrosis factor, interleukin-1, interferon- ) and upregulated in a
number of inflammatory disease states.3,4 Antisense
oligonucleotides that inhibit ICAM-1 expression are efficacious in
murine models of carrageenan-induced inflammation, collagen-induced
arthritis, dextran sulfate-induced inflammatory bowel disease, and
cardiac allograft rejection.5 ISIS 2302, an antisense
oligonucleotide that inhibits human ICAM-1 expression, is currently in
phase II trials for Crohn's disease, ulcerative colitis, rheumatoid
arthritis, psoriasis, and renal transplant rejection.2,6
Most first-generation antisense compounds (including ISIS 2302) are
phosphorothioate oligonucleotides in which a nonbridging oxygen in the
phosphodiester backbone is replaced with sulfur. This modification
results in increased resistance to exonucleases while preserving
high-affinity binding to the complementary RNA sequence. The
phosphorothioate backbone supports RNAase H-dependent cleavage of
oligo-RNA duplexes (an important antisense mechanism) and heavily
influences the pharmacological behavior of these oligonucleotides. This
modification also increases binding to a variety of protein targets,
which may result in hybridization-independent effects.1 These hybridization-independent effects may result in important side
effects of therapy. Second- and third-generation antisense compounds
contain alternative backbone or 2 sugar modifications that may
show less protein binding (and fewer hybridization-independent effects)
than the phosphorothioates. However, these oligonucleotide modifications do not appear to support RNAase H-dependent degradation of target mRNA.7-9 The in vivo efficacy of these
alternative modifications and their antisense mechanisms awaits
confirmation.
Prolongation of the activated partial thromboplastin time (PTT) is
commonly observed following administration of ISIS 2302 in both
cynomolgus monkeys and humans.6,10 In the monkeys, prolongation of the thrombin time and complement activation have also
been observed with doses greater than 3 mg/kg administered by 2-hour
intravenous infusion. Prolongation of the PTT correlates directly with
plasma concentrations of phosphorothioate oligonucleotides in the
cynomolgus monkeys.1,11,12 The basis for the anticoagulant effects of these oligonucleotides was previously unknown.
Phosphorothioate oligonucleotides and the anticoagulant drug heparin
are both linear polyanionic polymers, suggesting the potential for
binding to similar sites on protein surfaces. Several examples of
phosphorothioate oligonucleotides interacting with heparin-binding
proteins already exist, including basic fibroblast growth factor and
platelet-derived growth factor.1,13 Likewise,
numerous coagulation proteases (thrombin, factors Xa, IXa, XIa),
cofactors (factors VIII and V), and serpin inhibitors (antithrombin and
heparin cofactor II) can bind to heparin or heparan sulfate. Thus,
these proteins represent potential targets for the interaction of
phosphorothioate oligonucleotides with the coagulation cascade.
This investigation examines the anticoagulant effects of the
phosphorothioate oligonucleotide ISIS 2302. The effects of this oligonucleotide were analyzed in both plasma-based clotting assays and
purified enzyme systems to define potential anticoagulant mechanisms.
These investigations show that ISIS 2302 selectively inhibits the
intrinsic tenase complex (factor IXaB, factor VIIIa, phospholipid, and calcium) at plasma oligonucleotide concentrations that prolong the PTT to an extent similar to that observed in vivo (1.5 times control).6 This activity is independent of oligonucleotide sequence but requires the phosphorothioate backbone, suggesting inhibition of intrinsic tenase is a general property of this
class of oligonucleotides. Higher plasma concentrations of ISIS 2302 also inhibit thrombin by stimulating heparin cofactor II (HCII)
activity or directly competing with fibrinogen for binding. These
findings are relevant to both the therapeutic use of systemically administered phosphorothioate oligonucleotides and the design of
potential inhibitors of the intrinsic tenase complex, a novel target
for anticoagulant therapy.
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MATERIALS AND METHODS |
Reagents.
Lyophilized, pooled normal human plasma (Dade Ci-trol I), rabbit brain
partial thromboplastin containing ellagic acid (Dade Actin), and rabbit
brain thromboplastin (Dade Thromboplastin C Plus) were obtained from
Baxter Diagnostics, Inc (Deerfield, IL). Rabbit brain
cephalin, recombinant hirudin, and reptilase (Atroxin) were purchased
from Sigma (St Louis, MO). Factor-deficient plasmas (V, VIII, IX, X,
and XI) were purchased from George King Biomedical, Inc (Overland Park,
KS). HCII-immunodepleted plasma was obtained from Affinity Biologicals
Inc (Hamilton, Ontario, Canada). Human factors XIa, IXaB,
X, Xa, prothrombin, fibrinogen, HCII, and purified Russel viper venom
Xa activator (RVV-Xa) were purchased from Enzyme Research (South Bend,
IN). Human thrombin was purified from prothrombin activated with
Oxyuranus scutellatus venom preabsorbed with Amberlite CG-50
resin (Sigma) as previously described.14 Human factor V was
obtained from Hematologic Technologies, Inc (Essex Junction, VT).
Albumin-free factor VIII (Alphanate) was generously provided by the
Alpha Therapeutic Corporation (Los Angeles, CA). Human antithrombin
(ATIII) was obtained from Doug Tollefsen (Washington University, St
Louis, MO). The chromogenic substrates S-2765 and S-2238 were purchased
from Kabi-Pharmacia (Franklin, OH). Unfractionated, clinical-grade
porcine heparin was purchased from Elkins-Sinn (Cherry Hill, NJ). All
other chemicals were at least reagent grade and purchased from major
suppliers.
Oligonucleotides.
All oligonucleotides were provided by ISIS Pharmaceuticals (Carlsbad,
CA). ISIS 2302 (antisense) is a 20-mer phosphorothioate oligodeoxyribonucleotide molecule with the base sequence
(5-GCCCAAGCTGGCATCCGTCA-3). Additional oligonucleotides
include the phosphorothioate sense (5-TGACGGATGCCAGCTTGGGC-3) and scrambled versions
(5-GACGCATCGCGCCTACATCG-3) of ISIS 2302, and a
phosphodiester analogue of ISIS 2302.
Molecular masses (kD) and extinction coefficients
( 0.1%).
These values are: human factor XIa, 160,000 and 1.34; factor
IXaB, 46,000 and 1.43; factor X, 58,900 and 1.16;
factor Xa, 46,000 and 1.40; factor V, 330,000 and 0.96; thrombin 36,700 and 1.83; ATIII, 58,000 and 0.62; HCII, 65,600 and 0.59; and Russel viper venom factor X activator, 79,000 and 1.34.
Clotting times in human plasma.
All clotting times were performed in polystyrene cuvettes with a
BBL Fibro System fibrometer (Becton Dickinson, Lincoln
Park, NJ) warmed to 37°C. Dilutions of ISIS 2302 were made in
phosphate-buffered saline. Prothrombin times (PT) were performed by
incubating ISIS 2302 (0.05 mL) with normal pooled human plasma (0.1 mL)
for one minute at 37°C, adding rabbit brain thromboplastin (0.15 mL), and determining the clotting time. The rabbit brain thromboplastin was resuspended in 75% of the standard volume to compensate for the
reduced volume used in the PT assay. Activated PTTs were performed by
incubating ISIS 2302 (0.05 mL), human plasma (0.1 mL), and rabbit brain
partial thromboplastin (0.1 mL) for 3 minutes at 37°C; adding 0.04 mol/L CaCl2 (0.05 mL); and determining the clotting time.
To assess the effect of ISIS 2302 on contact activation, the PTT was
performed as above with or without 50 µg/mL of plasma oligonucleotide
present, extending the first stage incubation from 3 to 10 minutes.
Alternatively, addition of ISIS 2302 was delayed until just before
recalcification. To assess the effect of ISIS 2302 on the
calcium-dependent steps, the PTT was performed in specific
factor-deficient plasmas (factor XI, IX, VIII, or V) as above with or
without 50 µg/mL of plasma ISIS 2302. The minimal amount of activated
coagulation factor (factor XIa, IXa, VIIIa, or Va) to correct the PTT
to control levels in the absence of ISIS 2302 was added during
recalcification, and the clotting time was determined. A modified
Russel viper venom time was performed by incubating ISIS 2302 (0.05 mL), pooled normal or factor V-deficient human plasma (0.1 mL), 10%
(vol/vol) rabbit brain cephalin (0.05 mL), and 1.2 nmol/L RVV-Xa (0.05 mL) for 30 seconds at 37°C; adding 0.04 mol/L CaCl2
(0.05 mL); and determining the clotting time. Thrombin or reptilase
times in human plasma were performed by incubating ISIS 2302 (0.05 mL)
with human plasma (0.2 mL) for 3 minutes at 37°C. Human thrombin
(0.05 mL, final concentration approximately 15 nmol/L) or reptilase
(0.05 mL, 10 µg/mL) was added and the clotting time determined.
Factor V and VIII activation for PTT assays.
Factor V was activated with 0.2 nmol/L thrombin for 10 minutes at room
temperature in 0.15 mol/L NaCl, 20 mmol/L HEPES, pH 7.4, 5 mmol/L
CaCl2, and 0.01% Tween 20. Hirudin (0.4 nmol/L) was added
to neutralize thrombin, and the activation mix was added to plasma at
the time of recalcification. Factor VIII activity was quantitated by
constructing a standard curve for clotting activity with serial
dilutions of normal plasma into factor VIII-deficient plasma.
A clotting activity of 1 U/mL was assumed equivalent to 0.7 nmol/L
plasma factor VIII concentration.15 Factor VIII was activated with 40 nmol/L thrombin for 30 seconds at room temperature in
the same buffer as above. Hirudin (80 nmol/L) was added to neutralize
thrombin, and the activation mix was added immediately to plasma with
recalcification. Controls without hirudin gave equivalent clotting
times.
Purified thrombin time.
ISIS 2302 was incubated in 180 µL of clotting buffer (150 mmol/L
NaCl, 10 mmol/L CaCl2, 10 mmol/L imidazole-HCl, pH 7.4, 6.6% PEG-8000) for 3 minutes at 37°C, followed by addition of
20 µL plasma-derived or recombinant thrombin (approximately 20 nmol/L final concentration). The assay was initiated by addition of
human fibrinogen (50 µL of a 2 mg/mL solution) and the clotting time determined. To assess for potential cofactor activity, either 500 nmol/L ATIII or 250 nmol/L HCII was included in the buffer, fibrinogen
was added as above, and the clotting time was initiated by adding
plasma-derived thrombin. Controls without ATIII or HCII gave identical
clotting times when either fibrinogen or thrombin was added last.
Expression and purification of recombinant thrombins.
The plasmid constructs pCMVPT (wild-type), pR70E, and pR89E, stable
transfection into CV-1 (wild-type, R70E) or BHK (R89E) cell lines and
purification from conditioned media, were described previously.14,16 Thrombin amino acid residues are numbered sequentially from the first residue of the B chain. This corresponds to
the alternative chymotrypsin numbering system (in parentheses) as
follows: R70 (75) and R89 (93).
Chromogenic assay for intrinsic tenase activity.
A chromogenic assay for intrinsic tenase complex activity was performed
under conditions of limiting factor VIIIa concentration, as previously
described, with minor modifications.17 Albumin-free human
factor VIII (4.2 nmol/L) was activated with 40 nmol/L thrombin in 0.15 mol/L NaCl, 20 mmol/L HEPES, pH 7.4, 5 mmol/L CaCl2, and 0.01% Tween for 30 seconds at room temperature. Thrombin was
neutralized with recombinant hirudin (60 nmol/L), and the activation
mixture was diluted 50-fold into 0.15 mol/L NaCl, 20 mmol/L HEPES, pH 7.4, 2 mmol/L CaCl2, and 0.1% PEG-8000 buffer containing
84 pmol/L human factor VIIIa, 3 nmol/L human factor IXaB,
and 2% (vol/vol) rabbit brain cephalin. Human factor X was immediately
added to 300 nmol/L, and the reaction was sampled (50 µL) at 15, 30, 45, and 60 seconds into 10 µL of 0.25 mol/L EDTA, pH 7.4. The
chromogenic substrate S-2765 (100 µL) was added to 300 µmol/L with
polybrene (70 µg/mL) present, and the amount of factor Xa was
determined by comparing the rate of substrate hydrolysis (change in
absorbance at 405 nm over 2 minutes) in a kinetic microtiter
plate reader (Vmax, Molecular Devices Corp, Menlo Park,
CA) to a standard curve constructed with purified
factor Xa. The initial rate of intrinsic tenase complex activity
(factor Xa generation) was determined by plotting factor Xa
concentration versus time under conditions where less than 10% total
substrate cleavage occurred. The rate of factor Xa generation
(nmol/L/min) was linear over the time course of the assay (0 to 60 seconds), and with respect to factor VIIIa concentration (0 to 140 pmol/L).
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RESULTS |
Effect of ISIS 2302 on global tests of coagulation.
Significant prolongation of the PTT (to 1.5 times control) is observed
during in vivo administration of ISIS 2302 (2 mg/kg over 2 hours) to
humans subjects.6 This prolongation of the PTT correlates
with the effects observed at similar plasma oligonucleotide concentrations in cynomolgus monkeys.11 Thus, the effect of increasing concentrations of ISIS 2302 on the PT, activated PTT, and
thrombin time was evaluated in pooled normal human plasma (Fig 1A and B). ISIS 2302 was expressed as
the equivalent plasma oligonucleotide concentration (not assay volume
concentration). At low plasma concentrations of oligonucleotide (0 to
100 µg/mL), the predominant effect was a rapid, linear prolongation
of the PTT (Fig 1B). Gradual prolongation of the PT (100 to 1,000 µg/mL) and thrombin time (>500 µg/mL) was observed at higher
plasma concentrations of ISIS 2302 (Fig 1A). The selective prolongation
of the PTT at low concentrations of ISIS 2302 (<100 µg/mL) suggests
inhibition of factor(s) within the intrinsic coagulation pathway.
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| Fig 1.
(A) Effect of ISIS 2302 on the prothrombin ( ),
activated partial thromboplastin (PTT) ( ), and thrombin times ( )
in normal pooled human plasma. (B) Detail of the plasma oligonucleotide
concentration range that shows selective prolongation of the PTT (see
text). Clotting times are expressed as the mean of triplicate
determinations with error bars representing ±2 SD.
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Effect of ISIS 2302 on thrombin, fibrinogen, and inhibition by ATIII
and HCII.
The effect of ISIS 2302 on fibrinogen cleavage was examined by
comparing thrombin and reptilase clotting times in normal pooled human
plasma. The amount of human thrombin added was reduced to match control
values with the reptilase clotting time (18 to 20 seconds). Increasing
amounts of oligonucleotide did not prolong the reptilase time, even at
concentrations that significantly prolonged the thrombin time
(Fig 2). Reptilase and thrombin cleave the
identical peptide bond in fibrinogen (arg16-gly17) to release fibrinopeptide A.18 The failure of ISIS 2302 to prolong the reptilase time suggests that the oligonucleotide does not affect the
ability of fibrinogen to be cleaved but interacts directly with
thrombin to inhibit substrate recognition.
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| Fig 2.
Comparison of the effect of ISIS 2302 on the reptilase
() and dilute thrombin times (). The thrombin concentration was
reduced to give control values equivalent to the reptilase assay (18 to
19 seconds). Clotting times are expressed as the mean of triplicate
determinations with error bars representing ±2 SD.
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The mechanism for the inhibition of fibrinogen clotting by ISIS 2302 was examined in a purified thrombin time assay using selected
recombinant thrombins with arginine to glutamate substitutions in anion
binding exosite I (R70E) or exosite II (R89E). ISIS 2302 showed nearly
identical dose-dependent prolongation of the thrombin time with both
wild-type (Fig 3) and plasma-derived
thrombin (Fig 4). This result indicates
that ISIS 2302 can directly inhibit fibrinogen cleavage by thrombin and
does not require a plasma cofactor. A similar prolongation of the
thrombin time by ISIS 2302 was observed with thrombin R89E (exosite II)
at slightly higher oligonucleotide concentrations. In contrast, the
dose response to ISIS 2302 showed a marked shift to the right for
thrombin R70E (exosite I), indicating resistance to oligonucleotide
effects. Residue Arg 70 in anion binding exosite I of thrombin is not
required for fibrinogen cleavage but is located in close proximity to
the fibrinogen binding site.16 The resistance of thrombin
R70E to inhibition by the oligonucleotide suggests that the mutation
disrupts a binding site for ISIS 2302 in exosite I of thrombin. The
comparative lack of effect of an identical mutation in exosite II (R89E
in the heparin binding site) suggests this is not simply due to a change in total protein charge. Thus, ISIS 2302 can inhibit clotting by
directly competing with fibrinogen for binding to exosite I of
thrombin. This mechanism for thrombin inhibition is observed with other
polyanions, including a 15-mer phosphodiester oligonucleotide aptamer
that binds specifically to exosite I.19
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| Fig 3.
Effect of ISIS 2302 on fibrinogen clotting in a purified
thrombin time assay. Purified recombinant wild-type thrombin (),
thrombin R70E (exosite I mutant) (), or thrombin R89E (exosite II
mutant) () was added to clotting buffer (see Materials and Methods)
containing increasing amounts of ISIS 2302 (200 µL). The assay was
initiated by addition of 50 µL of 2 mg/mL human fibrinogen and the
time to clot formation determined. Clotting times are expressed as the
mean of triplicate determinations with error bars representing ±2
SD.
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| Fig 4.
Effect of ATIII and HCII on inhibition of clotting
activity by ISIS 2302 in a purified thrombin time assay. The clotting
time for plasma-derived thrombin was determined as described in the
legend of Fig 3, except that thrombin was added last to initiate the
clotting time. The ISIS 2302 dose response was compared in the absence
of inhibitor (), with 500 nmol/L ATIII (), or 250 nmol/L HCII
(). Clotting times are expressed as the mean of triplicate
determinations with error bars representing ±2 SD.
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Phosphorothioate oligonucleotides are linear, polyanionic polymers,
suggesting the possibility that their anticoagulant effects may involve
mechanisms similar to glycosaminoglycans. The ability of ATIII and HCII
to act as plasma cofactors for ISIS 2302 was addressed by addition of
these serpins to the purified thrombin time. Addition of ATIII (500 nmol/L) slightly prolonged the baseline clotting time but had no
significant effect on the dose response to ISIS 2302 (Fig 4). Likewise,
ISIS 2302 failed to accelerate the inhibition of thrombin or factor Xa
by ATIII with purified components under pseudo-first-order conditions
(data not shown). These results suggest that ATIII is not a plasma
cofactor for ISIS 2302. In contrast, HCII (250 nmol/L) did not affect
the baseline clotting time but markedly enhanced the ISIS 2302 dose
response (Fig 4). This shift in the oligonucleotide dose response
suggests that inhibition of thrombin clotting activity by ISIS 2302 in plasma depends largely on the presence of HCII. Likewise,
immunodepletion of HCII from plasma markedly reduced the ability of the
oligonucleotide to prolong the thrombin time, confirming the role of
HCII as a plasma cofactor for ISIS 2302 (Fig 5).
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| Fig 5.
Effect of ISIS 2302 on the plasma thrombin time in parent
() and HCII immunodepleted () plasma. Clotting times are
expressed as the mean of triplicate determinations with error bars
representing ±2 SD.
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Effect of low doses of ISIS 2302 on the PTT.
Prolongation of the PTT is the predominant effect of ISIS 2302 at
plasma concentrations below 100 µg/mL, suggesting selective inhibition of the intrinsic coagulation pathway (Fig 1B). To further evaluate effects on the intrinsic pathway, a plasma concentration of 50 µg/mL ISIS 2302 was selected. This concentration prolongs the PTT to
a degree similar to that observed in phase II studies (1.5 times
control).6 The rate-limiting step for clot formation in the
presence of ISIS 2302 was identified by sequential modification of the
PTT to bypass potential inhibitory effects of oligonucleotide. The
effect of ISIS 2302 on contact activation was assessed by prolonging
the incubation with contact activator (ellagic acid) or delaying
addition of ISIS 2302 until after that incubation was complete
(Table 1). Neither prolonged incubation
with ellagic acid nor delayed addition of the oligonucleotide corrected
prolongation of the PTT by ISIS 2302. Failure of these modifications to
correct the PTT to control levels suggests that the rate-limiting
defect induced by the oligonucleotide is distal to the contact
activation system. This result also infers that ISIS 2302 inhibits a
portion of the intrinsic pathway that is relevant to in vivo
coagulation.
The PTT was further modified to identify the rate-limiting step for
oligonucleotide inhibition of the calcium-dependent portion of the
intrinsic pathway. Briefly, sufficient activated coagulation factor
(factor XIa, IXa, VIIIa, Xa, or Va) was added to plasma deficient in
that factor (during recalcification) to correct the PTT to control
levels in the absence of oligonucleotide. Correction of the PTT with
activated factor in the presence of 50 µg/mL ISIS 2302 suggests that
the oligonucleotide acts proximally to that factor activity. Likewise,
failure to correct the PTT with activated factor in the presence of
oligonucleotide suggests that the rate-limiting defect is distal to the
activation of that factor. The addition of sufficient factor XIa, IXa,
or VIIIa to correct the PTT to control levels in the absence of the
oligonucleotide failed to correct the clotting time in the presence of
50 µg/mL ISIS 2302 (Table 2). The failure
of these activated factors to correct the PTT suggests that the
rate-limiting step in inhibition by the oligonucleotide is at or below
the level of the intrinsic tenase complex (factors IXa, VIIIa,
phospholipid, and calcium). Furthermore, addition of maximal amounts of
plasma factor XIa (30 nmol/L) or IXa (90 nmol/L; equivalent to complete
activation) cannot correct the PTT to control values, further
indicating that the defect is distal to the activation of these factors
(data not shown). In contrast, addition of 0.2 nmol/L factor Xa
corrects prolongation of the PTT in both the absence and presence of
ISIS 2302 (Table 2). The ability of subnanomolar amounts of factor Xa
(<1% plasma factor X levels) to bypass the inhibitory effect of ISIS
2302 suggests that the rate-limiting step lies proximal to generation
of factor Xa (ie, the intrinsic tenase complex). Thus, 50 µg/mL ISIS
2302 prolongs the PTT via inhibition of intrinsic tenase complex
activity.
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Table 2.
Effect of ISIS 2302 on the Correction of the PTT by
Activated Coagulation Factor in Factor-Deficient Plasma
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The determination that intrinsic tenase activity is rate limiting in
the presence of ISIS 2302 does not rule out additional effects on
downstream enzyme complexes (ie, the prothrombinase complex). Addition
of sufficient factor Va to correct the PTT in factor V-deficient
plasma to control levels failed to do so in the presence of the
oligonucleotide (Table 2). In principle, this may result from
insufficient generation of factor Xa by the intrinsic tenase complex or
from additional inhibitory effects of the oligonucleotide on
prothrombinase complex activity. To assess the effect of ISIS 2302 on
plasma prothrombinase activity, a modified Russel viper venom time was
performed. Purified factor X activator from Russel viper venom (RVV-Xa)
and rabbit brain cephalin were added to pooled normal human plasma in
the presence and absence of 50 µg/mL ISIS 2302, followed by
recalcification (Table 3). No difference in
the clotting times was observed, suggesting that 50 µg/mL ISIS 2302 does not significantly inhibit the prothrombinase complex. The clotting
time was markedly prolonged when performed in factor V-deficient
plasma, indicating that the assay is dependent on formation of the
prothrombinase complex (factors Xa, Va, phospholipid, and
calcium) rather than excess factor Xa activation by the venom. Thus,
the failure of factor Va to correct the PTT to control levels in the
presence of oligonucleotide is secondary to inadequate generation of
factor Xa by the intrinsic tenase complex. The lack of effect of 50 µg/mL ISIS 2302 on plasma prothrombinase activity (Table 3), and
minimal effect on plasma thrombin time (Fig 1B), confirms that
prolongation of the PTT is primarily due to inhibition of intrinsic
tenase complex activity.
Effect of ISIS 2302 on intrinsic tenase activity.
To further characterize the inhibitory effects of ISIS 2302, a
chromogenic assay designed to measure the rate of factor Xa generation
by the intrinsic tenase complex was used.17 A limiting amount of thrombin-activated human factor VIIIa was added to a reaction
mix containing human factor IXaB, rabbit brain cephalin, and buffer containing 2 mmol/L CaCl2. Human factor X was
immediately added and the reaction mixture sampled over time to
determine the rate of factor Xa generation by chromogenic substrate
cleavage (see Materials and Methods). ISIS 2302 markedly inhibited
intrinsic tenase activity (factor Xa generation), reaching near maximal effect at 10 to 15 µg/mL oligonucleotide
(Fig 6). Further increases in ISIS 2302 concentration showed approximately 35% residual activity, consistent
with partial inhibition of the enzyme complex. This marked inhibition
of intrinsic tenase activity at low oligonucleotide concentrations is
consistent with results of the clotting assays, confirming that this
enzyme complex is the major molecular target for the anticoagulant
effects of ISIS 2302.
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| Fig 6.
Effect of oligonucleotides on intrinsic tenase complex
activity. Increasing concentrations of oligonucleotide were added to an
intrinsic tenase reaction containing final concentrations of 84 pmol/L
human factor VIIIa, 3 nmol/L human factor IXaB, 2%
(vol/vol) rabbit brain cephalin, in 0.15 mol/L NaCl, 20 mmol/L HEPES,
pH 7.4, 2 mmol/L CaCl2, and 0.1% PEG-8000 buffer. Human
factor X was added to 300 nmol/L and the reaction sampled (50 µL) at
15, 30, 45, and 60 seconds into 10 µL of 0.25 mol/L EDTA, pH 8.0. The
chromogenic substrate S-2765 was then added to 300 µmol/L with
polybrene 70 µg/mL and the amount of factor Xa generated determined
by comparison of the rate of cleavage with a standard curve (see
Materials and Methods). The dose response for ISIS 2302 (antisense)
(), sense (), scrambled (), and phosphodiester
oligonucleotides ( ) is shown. The rate of factor Xa generation
(nmol/L/min) in the presence of oligonucleotide was expressed as a
percentage of the control value without oligonucleotide present. Data
points represent the average of two determinations.
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Effect of oligonucleotide structure on inhibition of intrinsic tenase
complex activity.
The specific oligonucleotide structure required for inhibition of
intrinsic tenase activity was also evaluated in the chromogenic assay.
The role of oligonucleotide sequence specificity was addressed by
comparison of the dose response for antisense, sense, and scrambled versions of ISIS 2302 (Fig 6). Each of these phosphorothioate oligonucleotides showed a similar dose response for inhibition of
intrinsic tenase activity, indicating that a specific oligonucleotide base sequence was not required. The role of the oligonucleotide backbone was addressed by comparison of ISIS 2302 with the identical phosphodiester oligonucleotide (Fig 6). The phosphodiester
analogue of ISIS 2302 did not show any significant inhibitory
activity, suggesting that the phosphorothioate backbone was required
for inhibition of intrinsic tenase. These results suggest that
inhibition of the intrinsic tenase complex activity is a general
property of phosphorothioate oligonucleotides. Consistent with these
conclusions, prolongation of the PTT has been reported for a number of
oligonucleotides of the phosphorothioate class.11
| |
DISCUSSION |
Phosphorothioate oligonucleotides exhibit a number of
hybridization-independent effects modulated by protein-oligonucleotide interactions. Inhibition of coagulation is an important
hybridization-independent effect of this class of
oligonucleotides.1 ISIS 2302 showed two major anticoagulant
effects, the predominant mechanism depending on the plasma
oligonucleotide concentration. High concentrations of ISIS 2302 (>100
µg/mL) resulted in significant inhibition of fibrin clot formation by
thrombin, shown by prolongation of the thrombin time (Fig 1). In part,
the oligonucleotide inhibits clotting by direct interaction with
exosite I of thrombin (the fibrinogen recognition site), as evidenced
by a lack of effect on the reptilase time (Fig 2), dose-dependent
prolongation of the purified thrombin time, and the relative resistance
to inhibition of thrombin R70E (Fig 3). However, HCII appears to be a
major plasma cofactor for ISIS 2302, based on the marked enhancement of
the oligonucleotide dose response with addition of this serpin to the
purified thrombin time (Fig 4) and the significantly reduced potency of
ISIS 2302 in plasma immunodepleted of HCII (Fig 5). Glycosaminoglycans
accelerate the inhibition of thrombin by HCII through an allosteric (or
conformational) mechanism.20 Numerous polyanions, including
polyphosphates and phosphodiester oligonucleotides, accelerate this
inhibition in purified systems.21 Stimulation of HCII
activity by phosphorothioate oligonucleotides in normal human plasma
has not been previously reported. In contrast, ATIII is not a plasma
cofactor for ISIS 2302, as it shows minimal effects on the dose
response in the purified thrombin time. Unlike heparin, ISIS 2302 failed to accelerate protease inhibition by ATIII through either a
conformational (factor Xa) or template mechanism (thrombin; data not
shown).14,22 Thus, the predominant mechanism for
prolongation of the thrombin time was stimulation of HCII activity by
the oligonucleotide. However, this effect of the oligonucleotide
requires plasma oligonucleotide concentrations that are at least
several fold higher than commonly observed in either animal or human
studies.6,11 Likewise, prolongation of the PT by ISIS 2302 suggests that additional inhibitory effects may occur at high plasma
oligonucleotide concentrations (>100 µg/mL).
In contrast, low plasma concentrations of ISIS 2302 (<100 µg/mL)
selectively prolonged the PTT, suggesting inhibition of the intrinsic
coagulation pathway (Fig 1B). Systemic administration of ISIS 2302 (2 mg/kg over 2 hours) in humans results in transient prolongation of the
PTT to approximately 1.5 times control, similar to the therapeutic
target range for heparin.6 A plasma concentration of ISIS
2302 (50 µg/mL) that prolonged the PTT to 1.5 times control in vitro
was analyzed in a series of modified clotting assays. The
oligonucleotide did not prolong the PTT by inhibiting contact activation (Table 1), but it inhibited the calcium-dependent portion of
the intrinsic pathway. Analysis of the calcium-dependent factors
indicated that the rate-limiting effect of the oligonucleotide was at
the level of the intrinsic tenase complex activity. This conclusion is
supported by the ability to bypass the effect of ISIS 2302 with
subnanomolar factor Xa and the failure of proximally acting factors
(factors XIa, IXa, or VIIIa) to correct prolongation of the PTT (Table
2). This concentration of factor Xa (0.2 nmol/L, <1% plasma factor
X) is similar to that observed during the clotting of minimally
altered whole blood, suggesting this mechanism may exist in vivo as
well.23 The failure of factor VIIIa to correct the
inhibition is consistent with a defect in intrinsic tenase complex
activity or assembly but does not address additional potential defects
in cofactor activation. Intrinsic tenase complex activity is
selectively inhibited at this oligonucleotide concentration, as shown
by the lack of effect on prothrombinase (Table 3) and thrombin clotting
times (Fig 1B). Thus, prolongation of the PTT by 50 µg/mL ISIS 2302 is secondary to inhibition of the intrinsic coagulation pathway at the
level of intrinsic tenase activity.
The effect of ISIS 2302 on intrinsic tenase complex activity in plasma
was confirmed in a purified assay system under conditions of limiting
factor VIIIa concentration. ISIS 2302 showed a marked partial
inhibition of intrinsic tenase activity with near maximal inhibition at
10 to 15 µg/mL oligonucleotide and approximately 35% (of control)
residual intrinsic tenase activity at higher concentrations (Fig 6).
This pattern of inhibition is very similar to antithrombin-independent
inhibition of intrinsic tenase activity by heparin.24
Furthermore, inhibition of intrinsic tenase complex appears to be a
general property of phosphorothioate oligonucleotides, based on the
lack of sequence specificity and requirement for the phosphorothioate
backbone (Fig 6). Prolongation of the PTT by unrelated phosphorothioate
oligonucleotides has also been observed, suggesting that inhibition of
the intrinsic tenase may be relevant to all first-generation antisense
compounds.11 In principle, this inhibition may represent an
effect on assembly, catalytic rate, or stability of the enzyme complex.
Defining the specific mechanism of inhibition will require determining
the effects of ISIS 2302 on factor VIII activation, binding
interactions of the individual intrinsic tenase components, steady
state kinetic constants for factor X activation, and stability of
factor VIIIa.
Inhibition of the intrinsic tenase complex by phosphorothioate
oligonucleotides has important implications for the therapeutic use of
these first-generation antisense compounds. More than 10 antisense
phosphorothioate oligonucleotides are currently in clinical trials in
the United States and Europe.2 ISIS 2302 has recently advanced to a large phase IIb trial (300 patients) for
steroid-dependent Crohn's disease. In a smaller phase II trial (20 patients), intravenous administration of ISIS 2302 (2 mg/kg over 2 hours) was associated with transient prolongation of the PTT to
approximately 1.5 times control.6 In the cynomolgus monkey
studies, prolongation of the PTT is dose related, correlates with
plasma oligonucleotide levels, and is observed with a number of
phosphorothioate oligonucleotides.11 The results of the
present investigation indicate that prolongation of the PTT by ISIS
2302 is secondary to inhibition of the intrinsic tenase complex and
that this inhibition is a potential side effect of all systemically
administered phosphorothioate oligonucleotides. The risk of bleeding
complications due to these coagulation abnormalities is unknown. The
plasma half-life of phosphorothioate oligonucleotides is relatively
short (30 to 60 minutes), and adjustment of dosing regimens to reduce
peak plasma oligonucleotide levels may further reduce the theoretical
risk of hemorrhagic complications. This potential toxicity may be more
problematic if concurrent surgical or invasive procedures are
contemplated. However, because of uncertainties in extrapolating from
in vitro to in vivo coagulation, the ultimate risk assessment must come
from clinical trials. In this regard, it should be noted that serious
hemorrhagic complications have not been recorded in any clinical trials
performed by ISIS Pharmaceuticals to date, including studies in
patients with Crohn's disease and a spectrum of malignancies
(personal communication, December 1997, J. Tami, ISIS
Pharmaceuticals).
These findings may impact on the design of future antisense
oligonucleotide modifications. The effect of alternative backbone or
2-O sugar modifications on intrinsic tenase activity will need to be
addressed for second- and third-generation antisense oligonucleotides. Furthermore, RNA-DNA duplexes containing these alternative backbones or
2-O sugar modifications (in either strand) appear to be resistant to
RNAase H cleavage.25 If efficient inhibition of a
particular mRNA target requires an RNAase H-dependent mechanism, then
hybrid oligonucleotides may be required in which a phosphorothioate
cleavage site is flanked by alternative oligonucleotide modifications. The rate of RNAase H-dependent cleavage decreases with reduction in
the deoxynucleotide portion of chimeric oligonucleotides, and gaps of  4 nucleotides do not support enzyme activity.25 The minimal phosphorothioate structure that inhibits intrinsic tenase activity has not been defined. Rational design of hybrid
oligonucleotides to maximize RNAase H cleavage rates and minimize
effects on coagulation will require determination of this minimal
inhibitory oligonucleotide structure.
Inhibition of the intrinsic tenase complex may also have potential
advantages for dissociating the hemorrhagic and antithrombotic effects
of anticoagulant therapy. Unfractionated, low molecular weight, and
low-affinity (for antithrombin) forms of heparin inhibit the intrinsic
tenase complex by a similar antithrombin-independent, partial
inhibition mechanism. This inhibition occurs at concentrations within
the therapeutic range of both unfractionated and low molecular weight
heparin, suggesting it may contribute to the antithrombotic efficacy of
these drugs.24 Low-affinity heparin shows antithrombotic activity in a rabbit thrombosis model, further suggesting the importance of antithrombin-independent mechanisms.26
Analysis of tissue factor (TF)-initiated clotting in minimally altered whole blood suggests that factor Xa is the limiting factor in thrombin
generation, which does not reach maximal rates until well after initial
clot formation occurs.23 In a purified system, TF-factor
VIIa concentration affects the lag phase (initiation) for thrombin
generation but does not affect the ultimate rate of thrombin generation
in the propagation phase (amplification). Thus, the intrinsic tenase
complex is primarily responsible for activating sufficient factor Xa to
result in an explosive increase in thrombin generation during clot
formation.27 Partial inhibition of intrinsic tenase would
thus be expected to dampen thrombin generation during the propagation
phase without inhibiting initiation of coagulation. Selective dampening
of the propagation phase may disproportionately increase the
antithrombotic effect relative to hemorrhagic risk, improving the risk
to benefit ratio of anticoagulant therapy.24 Development of
specific intrinsic tenase inhibitors will facilitate testing of this
hypothesis. Inhibition of intrinsic tenase by phosphorothioate
oligonucleotides shows that this anticoagulant effect can be achieved
with diverse chemical structures. Thus, determining the minimal
inhibitory oligonucleotide structure may facilitate rational design of
synthetic intrinsic tenase inhibitors.
| |
FOOTNOTES |
Submitted December 29, 1997;
accepted April 22, 1998.
Supported in part by National Institutes of Health Grant 1K08 HL 02923 and a grant from ISIS Pharmaceuticals (to J.P.S.).
Address reprint requests to John P. Sheehan, MD, Department of
Medicine/Hematology, 7703 Floyd Curl Dr, San Antonio, TX 78284; e-mail:
sheehan{at}uthscsa.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Pete Lollar (Emory University, Atlanta, GA) for helpful
discussions regarding the chromogenic assay for intrinsic tenase
activity and Dr P. Bhattacharya (Alpha Therapeutic
Corp, Los Angeles, CA) for providing human factor VIII.
| |
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Abstract
Introduction
Abstract