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Smith and Morrissey 2008 PolyP study for hemophilia and anticoagulant reversal

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Journal of Thrombosis and Haemostasis, 6: 1750–1756
DOI: 10.1111/j.1538-7836.2008.03104.x
ORIGINAL ARTICLE
Polyphosphate as a general procoagulant agent
S . A . S M I T H and J . H . M O R R I S S E Y
Departments of Internal Medicine and Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA
To cite this article: Smith SA, Morrissey JH. Polyphosphate as a general procoagulant agent. J Thromb Haemost 2008; 6: 1750–6.
Summary. Background: Polyphosphate is secreted by activated
platelets and we recently showed that it accelerates blood
clotting, chiefly by triggering the contact pathway and promoting factor (F) V activation. Results: We now report that
polyphosphate significantly shortened the clotting time of
plasmas from patients with hemophilia A and B and that its
procoagulant effect was additive to that of recombinant FVIIa.
Polyphosphate also significantly shortened the clotting time of
normal plasmas containing a variety of anticoagulant drugs,
including unfractionated heparin, enoxaparin (a low molecular
weight heparin), argatroban (a direct thrombin inhibitor) and
rivaroxaban (a direct FXa inhibitor). Thromboelastography
revealed that polyphosphate normalized the clotting dynamics
of whole blood containing these anticoagulants, as indicated by
changes in clot time, clot formation time, alpha angle, and
maximum clot firmness. Experiments in which preformed FVa
was added to plasma support the notion that polyphosphate
antagonizes the anticoagulant effect of these drugs via accelerating FV activation. Polyphosphate also shortened the clotting
times of plasmas from warfarin patients. Conclusion: These
results suggest that polyphosphate may have utility in reversing
anticoagulation and in treating bleeding episodes in patients
with hemophilia.
Keywords: argatroban, enoxaparin, factor V, hemophilia,
heparin, rivaroxaban, warfarin.
Introduction
Uncontrolled hemorrhage can be a life-threatening problem for
individuals with clotting factor deficiencies such as hemophilia
and may also be a serious complication for patients undergoing
anticoagulant therapy. Even when patients are on stable
anticoagulant therapy, emergent circumstances may necessitate
immediate reversal of anticoagulant status. Rapid normalization of abnormal coagulation generally requires either replacCorrespondence: Stephanie A. Smith or James H. Morrissey, College
of Medicine, University of Illinois at Urbana-Champaign, 417 Med.
Sci. Bldg. MC-714, 506 S. Mathews Ave., Urbana, IL 61801, USA.
Tel.: +1 217 265 4036; fax: +1 217 265 5290.
E-mail: [email protected] (SAS) or [email protected] (JHM)
Received 16 May 2008, accepted 17 July 2008
ing missing clotting factors or administering specific antidotes
[1,2]. For patients receiving warfarin, anticoagulation can be
rapidly reversed via transfusing normal coagulation factors or
more slowly with vitamin K therapy, while heparin can be
rapidly reversed with protamine [2]. However, most newly
approved anticoagulant drugs – and some under development
– lack specific antidotes. Recombinant human factor (F) VIIa
(rFVIIa) is approved for managing hemorrhage in patients
with hemophilia with inhibitors [3,4]. More recently, in vitro
studies as well as experiences with off-label use in patients
suggest that rFVIIa may have general utility in reversing
anticoagulant therapy [5–11], although rFVIIa administration
may sometimes be associated with adverse thromboembolic
events [12,13]. Currently, the primary factors limiting use of
rFVIIa as a universal procoagulant are high cost and potential
liability associated with off-label use.
Polyphosphate (polyP) is a linear polymer of inorganic
phosphate that is present in dense granules of human
platelets [14,15]. PolyP is released from activated platelets
and is cleared from plasma by degradation by plasma
phosphatases [14,16]. We recently reported that polyP is a
potent hemostatic regulator, accelerating blood coagulation
by activating the contact pathway and by promoting FV
activation, which in turn abrogates the anticoagulant
function of tissue factor pathway inhibitor [16]. These
combined effects of polyP shift the timing of thrombin
generation without changing the total amount of thrombin
generated. Most recently, we reported that polyP modulates
fibrin clot structure, resulting in thicker fibrin fibers that are
more resistant to fibrinolysis [17].
Because polyP causes an earlier burst of thrombin
generation during plasma clotting, we hypothesized that
polyP would also exhibit procoagulant effects under circumstances in which coagulation was impaired, including
clotting factor deficiencies or anticoagulant therapy. We
now report that polyP shortened the time to clot formation
in normal plasma to which various anticoagulants were
added in vitro. It also normalized clot dynamics in whole
blood containing these anticoagulant drugs, as measured by
thromboelastography. Furthermore, polyP shortened the
clotting time of plasmas from patients receiving warfarin
and individuals with hemophilia A or B. PolyP was as
effective as adding the missing clotting factors or rFVIIa in
normalizing the clotting times of hemophilia plasmas.
Ó 2008 International Society on Thrombosis and Haemostasis
Polyphosphate as a procoagulant 1751
Materials and methods
Reagents and plasma samples
Enoxaparin (Lovenox) was from Aventis (Bridgewater, NJ,
USA), argatroban was from GlaxoSmithKline (Research
Triangle Park, NC, USA) and rivaroxaban was a gift from
Bayer HealthCare (Berkeley, CA, USA). PolyP (mean
polymer size, 75 phosphate units; sold as Ôsodium phosphate
glassÕ) and unfractionated heparin were from Sigma Aldrich
(St Louis, MO, USA). PolyP concentrations are expressed
herein in terms of phosphate monomer. Factor IX and FVa
were from Enzyme Research Laboratories (South Bend, IN,
USA), rFVIII (Kogenate-FSÒ) was a kind gift of Bayer
HealthCare (Berkeley, CA, USA), and rFVIIa was from
American Diagnostica (Stamford, CT, USA). Hemoliance
RecombiplastinÒ was from Instrumentation Laboratory
(Lexington, MA, USA) and InnovinÒ from Dade Behring
(Newark, DE, USA).
Pooled normal plasma and plasmas congenitally deficient in
FVIII (<1% activity) or FIX (<1% activity) were from
George King Biomedical (Overland Park, KS, USA). Factor
V-immunodepleted plasma was from Haematologic Technologies (Essex Junction, VT, USA). Plasmas from patients stably
anticoagulated with warfarin were from the Carle Foundation
Hospital (Urbana, IL, USA). Plasmas were stored at )70 °C,
thawed at 37 °C for 5 min, and then held at room temperature
for no more than 30 min prior to clotting tests. Fresh whole
blood for thromboelastography was collected from healthy,
adult, non-smoking volunteers not receiving any medication.
This study was approved by the Institutional Review Boards of
the University of Illinois at Urbana-Champaign and the Carle
Foundation Hospital. Written informed consent was obtained
from volunteer blood donors.
Plasma clotting tests
Anticoagulant drugs were added to pooled normal plasma at
amounts selected to span therapeutic and supratherapeutic
concentrations: up to 1 U mL)1 unfractionated heparin,
100 lg mL)1 enoxaparin, 3 lg mL)1 argatroban, or
1 lg mL)1 rivaroxaban. For comparison, therapeutic ranges
for unfractionated heparin and enoxaparin are generally 0.3–
0.7 and 0.6–1.0 U mL)1 (equivalent to 6–10 lg mL)1) by antiFXa activity respectively [18]. Steady-state plasma concentrations for argatroban were reported to be 0.5–0.7 lg mL)1 [19].
Mean peak plasma concentrations for rivaroxaban administered to orthopedic patients were reported to be 0.2 lg mL)1
[20].
In some experiments, clotting times of FV-deficient or
pooled normal plasma spiked with FVa were evaluated with or
without added anticoagulant drugs (0.4 U mL)1 unfractionated heparin, 30 lg mL)1 enoxaparin, 1 lg mL)1 argatroban, or
0.7 lg mL)1 rivaroxaban). In others, clotting times of FVIIIdeficient plasma were evaluated with or without adding up to
0.5 lg mL)1 FVIII or 20 nM FVIIa. Clotting times of FIXÓ 2008 International Society on Thrombosis and Haemostasis
deficient plasmas were evaluated with or without adding up to
4 lg mL)1 FIX or 20 nM FVIIa.
Plasma clotting times were quantified in 96-well polystyrene
microplates (Corning Inc., Corning, NY, USA) by adding
80 lL plasma followed by 160 lL diluted thromboplastin
(Recombiplastin diluted 200-fold for normal plasmas ± anticoagulant, or 8000-fold for hemophilia plasmas ± anticoagulant) in a buffer containing 100 lM sonicated liposomes (20%
phosphatidylserine, 80% phosphatidylcholine), 12.5 mM
CaCl2, 25 mM Tris–HCl pH 7.4, 0.1% bovine serum albumin,
150 mM NaCl. Low concentrations of tissue factor were
employed to obtain significantly prolonged clotting times in the
presence of anticoagulant drugs or with factor-deficient
plasmas. When present, 100 lM polyP was added directly to
the diluted thromboplastin. Clotting was monitored by
turbidity change (A405) for 1 h at room temperature using a
Spectramax microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). Clotting times were calculated
using SigmaPlot 7.101 (SPSS, Inc., Chicago, IL, USA) by
fitting a line to the steepest segment of the absorbance curve
and determining its intersection with the initial baseline A405
(representing the lag phase prior to clot formation). Assays
were repeated five times.
Whole blood thromboelastography
Thromboelastography was performed using the ROTEMÒ
four-channel system (Pentapharm, Munich, Germany) and the
supplied software. Fresh, non-anticoagulated whole blood was
collected via atraumatic venipuncture (discarding the initial
3 mL), then immediately transferred to the supplied disposable
plastic cups (280 lL per cup) and thoroughly mixed with either
20 lL TBS (50 mM Tris–HCl pH 7.4, 150 mM NaCl) or 20 lL
TBS plus the indicated additives (polyP and/or anticoagulant
drugs). Each sample was divided into four cups containing:
TBS only (control), polyP, anticoagulant drug, or polyP plus
anticoagulant drug. Clotting was initiated by adding 20 lL
diluted Innovin (in TBS) within 2 min of blood collection.
Final concentrations were 87.5% whole blood, 1:17 000
dilution of Innovin, 0 or 100 lM polyP, and either no added
anticoagulant drug or one of the following: 0.1 U mL)1
unfractionated heparin, 2.7 lg mL)1 enoxaparin, 1 lg mL)1
argatroban, or 0.2 lg mL)1 rivaroxaban. Measurements were
continued for 2 h and thromboelastography parameters were
recorded using ROTEM software. Effects of each anticoagulant were tested by adding the drug to whole blood from five
different individuals.
Statistical analyses
Statistical analyses were performed using SigmaStat 2.03
(SPSS, Inc.). To account for inter-individual variation, thromboelastography parameters were compared using paired, twotailed t-tests with significance of P < 0.05. Pairwise comparisons for each blood donor were made between results with vs.
without additive. In addition, pairwise comparisons were
1752 S. A. Smith and J. H. Morrissey
performed between blood containing anticoagulant vs. blood
containing anticoagulant plus polyP.
(Fig. 1D), causing an approximately 80% reduction in clotting
time at all rivaroxaban concentrations tested.
Results
PolyP reverses the anticoagulant effect of four drugs in whole
blood thromboelastography
Clot time (min)
B
Clot time (min)
A
20
10
0
30
20
10
0
0.0
0.5
1.0
–1
Heparin (U mL ) in plasma
100
0
50
–1
Enoxaparin (µg mL ) in plasma
Clot time (min)
10
5
Clot time (min)
D 60
C
40
20
0
0
2
3
0
1
0.0
0.5
1.0
–1
Argatroban (µg mL ) in plasma Rivaroxaban (µg mL–1) in plasma
Fig. 1. Polyphosphate (polyP) antagonizes the anticoagulant effect of
heparin, enoxaparin, argatroban, and rivaroxaban. (A) Unfractionated
heparin, (B) enoxaparin, (C) argatroban or (D) rivaroxaban were added at
the indicated concentrations to pooled normal plasma, after which clotting
was initiated by dilute thromboplastin. Clotting reactions contained either
100 lM polyP( ) or no polyP (s). Data are mean ± standard error
(n = 5).
mm
A
60
40
20
0
20
40
60
2
1
4
3
2
4
1
mm
B 60
40
20
0
20
40
60
C 60
40
20
0
20
40
60
D
60
40
20
0
20
40
60
0
3
2
1 4
mm
We examined the ability of polyP of the size secreted by human
platelets (75 phosphate units long) to reverse the anticoagulant
effect of unfractionated heparin, enoxaparin (a low molecular
weight heparin that acts as an indirect FXa inhibitor), argatroban (a direct thrombin inhibitor), or rivaroxaban (a direct FXa
inhibitor). Drugs were added to pooled normal plasma at
concentrations spanning therapeutic and supratherapeutic levels. Clotting was initiated by dilute thromboplastin, and we
compared the clotting times with no added polyP to those
obtained with 100 lM polyP. Each drug prolonged the clotting
time in a dose-dependent manner (Fig. 1).
PolyP antagonized the anticoagulant effect of both unfractionated and low molecular weight heparin, shortening the
clotting time by approximately 50% at all heparin concentrations evaluated (Figs. 1A and B). This is an approximately
50% reversal of the effective heparin dose.
PolyP shortened the clotting time at all argatroban concentrations tested (Fig. 1C). In the presence of polyP, concentrations of argatroban above 1 lg mL)1 failed to further prolong
the clotting time. Consequently, the effects of supratherapeutic
plasma levels of argatroban (1–3 lg mL)1) were blunted by
polyP, resulting in a milder prolongation of clotting time
equivalent to that obtained with argatroban at 0.5 lg mL)1.
Of the four anticoagulant drugs tested, polyP was most
effective at reversing the anticoagulant effect of rivaroxaban
PolyP also reversed the anticoagulant effects of unfractionated
heparin, enoxaparin, argatroban, and rivaroxaban in whole
blood. As can be seen in Fig. 2A and Table 1, thromboelastography showed that adding 0.1 U mL)1 unfractionated
heparin to blood prolonged both the clot time (CT) and clot
formation time (CFT), and it also decreased the a angle and
maximum clot firmness (MCF). PolyP shortened, but did not
completely normalize CT, whereas polyP completely normalized parameters related to the kinetics of increase in clot
firmness (CFT and a angle) and final clot firmness (MCF).
Adding 2.7 lg mL)1 enoxaparin prolonged both CT and
CFT, and it decreased a angle and MCF (Fig. 2B and
Table 1). PolyP essentially normalized all four parameters.
As with the heparins, adding 1 lg mL)1 argatroban prolonged both CT and CFT, and decreased the a angle and MCF
(Fig. 2C and Table 1). Adding polyP normalized CFT, a angle
and MFT, but CT was still prolonged.
mm
PolyP reverses the anticoagulant effect of four drugs in plasma
clotting assays
2
4
10
1
3
3
20
30
Time (min)
40
50
Fig. 2. Whole blood thromboelastography tracings showing that polyphosphate (polyP) reverses the anticoagulant effect of heparin, enoxaparin, argatroban, and rivaroxaban. (A) Unfractionated heparin, (B)
enoxaparin, (C) argatroban or (D) rivaroxaban were added to fresh whole
blood. Each blood sample was divided into four aliquots receiving the
following additions: (1) no additive (red curve); (2) 100 lM polyP (green
curve); (3) anticoagulant (blue curve); or (4) anticoagulant plus 100 lM
polyP (pink curve). Clotting was initiated with dilute thromboplastin.
Ó 2008 International Society on Thrombosis and Haemostasis
Polyphosphate as a procoagulant 1753
Table 1 Thromboelastography parameters
Parameter
No additive
+Polyphosphate
+Anticoagulant
+Anticoagulant and polyphosphate
CT (s)
CFT (s)
a angle (°)
MCF (mm)
8.0
4.4
47.2
52.8
(0.4)
(0.7)
(4.3)
(1.6)
5.1
1.7
69.6
63.0
(0.3)*
(0.1)*
(1.6)*
(1.8)*
Unfractionated heparin
18.8 (1.0)*
14.9 (1.7)*
18.2 (1.7)*
42.4 (2.1)*
11.5
4.5
46.8
52.4
(1.0)*
(0.8)
(4.6)
(2.7)
CT (s)
CFT (s)
a angle (°)
MCF (mm)
6.9
3.5
53.0
57.4
(1.8)
(0.6)
(5.1)
(4.9)
4.8
1.6
71.0
64.8
(0.6)*
(0.1)*
(1.2)*
(2.3) *
Enoxaparin
12.9 (2.2)*
7.6 (1.4)*
31.4 (5.5)*
47.0 (3.2)*
8.0
2.7
60.4
59.8
(0.7)
(1.0)
(7.8)
(2.6)
CT (s)
CFT (s)
a angle (°)
MCF (mm)
8.1
4.4
48.0
55.0
(0.9)
(1.7)
(11.7)
(7.3)
5.1
1.8
69.0
62.2
(0.6)*
(0.5)*
(6.2)*
(4.8)*
Argatroban
14.0 (1.3)*
6.2 (2.4)*
38.8 (9.4)*
49.4 (6.8)*
10.4
3.1
56.4
59.4
(0.9)*
(0.5)
(3.9)
(5.3)*
CT (s)
CFT (s)
a angle (°)
MCF (mm)
7.9
3.9
49.6
55.4
(1.8)
(0.8)
(5.2)
(7.0)
5.4
1.7
70.0
64.0
(0.5)*
(0.2)*
(2.4)*
(4.5)*
Rivaroxaban
11.7 (1.1)*
4.3 (1.3)
47.6 (8.5)
54.8 (8.6)
6.7
1.9
67.4
63.6
(0.8)
(0.5)*
(5.0)*
(4.8)*
*Significantly different from value for blood with no additive. Significantly different from value for blood containing anticoagulant without
polyphosphate. CT, clot time; CFT, clot formation time; MCF, maximum clot firmness.
Our previous work showed that polyP hastens thrombin
generation primarily by accelerating FV activation [16]. We
therefore hypothesized that polyP antagonizes the anticoagulant effects of the drugs tested above by accelerating FV
activation. To test this, we examined the ability of FVa added
to normal plasma to antagonize the anticoagulant effects of
unfractionated heparin, enoxaparin, argatroban, or rivaroxaban. Factor Va antagonized the effects of all four drugs, with
maximum reductions in clotting times equivalent to those
observed with 100 lM polyP in the absence of added FVa
(Fig. 3).
In another test of this hypothesis, we added polyP to
FV-deficient plasma that had been spiked with 1 nM FVa.
This concentration of FVa will only partially abrogate the
Ó 2008 International Society on Thrombosis and Haemostasis
4
2
0
8
6
4
2
0
0
4
8 PP
FVa (nM) in plasma
C 8
8 PP
0
4
FVa (nM) in plasma
D
6
4
2
0
10
Clot time (min)
Clot time (min)
B
6
Clot time (min)
Role of FVa
A
Clot time (min)
Adding 0.2 lg mL)1 rivaroxaban prolonged the CT but did
not significantly alter CFT, a angle or MCF (Fig. 2D and
Table 1). Adding polyP plus rivaroxaban normalized the
prolonged CT value. PolyP actually shifted the other thromboelastography parameters beyond those seen with native
blood: it decreased the CFT in the presence of rivaroxaban
to a value significantly smaller than that of native blood,
and it significantly increased the a angle and MCF relative
to native blood. Thus, the thromboelastography curves
for blood containing polyP plus rivaroxaban were essentially identical to those for blood containing only polyP,
indicating complete reversal of rivaroxaban-dependent
anticoagulation.
0
4
8 PP
FVa (nM) in plasma
30
20
10
0
8 PP
0
4
FVa (nM) in plasma
Fig. 3. Preformed factor (F) Va is equivalent to polyphosphate (polyP)
in antagonizing the anticoagulant effect of heparin, enoxaparin,
argatroban, and rivaroxaban. Clotting was initiated with dilute
thromboplastin using pooled normal plasma containing either the indicated anticoagulant drug (circles) or no drug (squares): (A) 0.4 U mL)1
unfractionated heparin; (B) 30 lg mL)1 enoxaparin; (C) 1 lg mL)1
argatroban; or (D) 0.7 lg mL)1 rivaroxaban. The plasmas also contained
the concentrations of added FVa indicated on the x-axes (open symbols).
For comparison, clotting times are presented for plasma containing
100 lM polyP but no added FVa (closed symbols, indicated as ÔPPÕ).
Data are mean ± standard error (n = 5).
1754 S. A. Smith and J. H. Morrissey
We evaluated the ability of polyP to reverse the prolonged
clotting times of hemophilia A and B plasmas (in clotting assays
using dilute thromboplastin). PolyP decreased the clotting time
of plasmas from five patients with severe hemophilia A and four
patients with severe hemophilia B (Figs. 5A and B). In the
presence of polyP, plasmas from these patients clotted more
rapidly than did normal plasma without polyP.
We also evaluated the ability of polyP to reverse the
prolonged clotting times of plasma from patients receiving
warfarin. Adding polyP shortened, but did not completely
normalize, the clotting time for all patient samples, irrespective
of the patientsÕ International Normalized Ratio (Fig. 5C).
We further evaluated the potential effectiveness of polyP as a
procoagulant agent for hemophilia by comparing the ability of
polyP to shorten the clotting times of hemophilia A or B
plasmas to which the missing clotting factor was replenished, or
to which rFVIIa was added. Adding 100 lM polyP shortened
the plasma clotting times in both FVIII deficiency and FIX
deficiency to a greater degree than did replacement of the
missing clotting factor (Figs. 6A and C). PolyP also shortened
the plasma clotting time to an extent similar to that observed
Clot time (min)
30
20
10
0
N
U
E
A
R
Fig. 4. In the absence of factor (F) V, polyphosphate (polyP) no longer
antagonizes the anticoagulant effect of heparin, enoxaparin, argatroban or
rivaroxaban. Anticoagulants were added to FV-deficient plasma containing 1 nM FVa, after which clotting was initiated by dilute thromboplastin. Anticoagulants were: (U) 0.4 U mL)1 unfractionated heparin; (E)
30 lg mL)1 enoxaparin; (A) 2 lg mL)1 argatroban; (R) 0.7 lg mL)1
rivaroxaban; or (N) no additive. Wells without added polyP (open bars)
were compared to those containing 100 lM polyP (filled bars). Data are
mean ± standard error (n = 5).
Clot time (min)
20
10
0
P1
P2
P3
P4
P5
Hemophilia A patients
N
P7
P8
P9
Hemophilia B patients
N
B
Clot time (min)
PolyP shortens the clotting times of hemophilia plasmas and
plasmas from warfarin patients
A 30
40
20
0
P6
C
Clot time (min)
anticoagulant function of the tested drugs (see Fig. 3).
Furthermore, as this plasma contains no FV, polyP will be
unable to promote any further FVa generation; we therefore
hypothesized that polyP should be without effect on clotting
times. Indeed, we found that adding 100 lM polyP to such
plasma mixtures failed to antagonize the anticoagulant effects
of any of the four drugs tested (Fig. 4). These results argue that
the polyP antagonizes the anticoagulant function of these four
drugs solely by its ability to promote FV activation.
10
8
6
4
2
0
N 1.7 2.3 2.8 3.2 3.7 4.3 4.8
Patients receiving warfarin
(INR value)
Fig. 5. Polyphosphate (polyP) shortens the clotting time of hemophilia
plasmas and plasmas from patients receiving warfarin. Clotting tests were
performed with plasmas from (A) five different patients with hemophilia
A; (B) four different patients with hemophilia B; or (C) seven different
patients receiving warfarin therapy (with the indicated International
Normalized Ratio values). ÔNÕ indicates data from pooled normal plasma.
Clotting reactions without added polyP (open bars) were compared to
those containing 100 lM polyP (filled bars). Data are mean ± standard
error (n = 5).
when rFVIIa was added to the plasmas (Figs. 6B and D).
Thus, in this clotting assay, the effectiveness of 100 lM polyP
was comparable to that of 6 nM rFVIIa in FIX-deficient
plasma or 15 nM rFVIIa in FVIII-deficient plasma. We also
examined the combination of adding polyP along with FIX,
FVIII or rFVIIa and we found that effects of 100 lM polyP
were additive to those of rFVIIa (Fig. 6).
Discussion
We have now demonstrated that polyP of the size secreted by
human platelets partially or completely reversed the anticoagulant effects of four drugs (unfractionated heparin, enoxaparin,
argatroban, and rivaroxaban). PolyP did this in spite of the
differing modes of action of these drugs, and was particularly
Ó 2008 International Society on Thrombosis and Haemostasis
A 30
B
Clot time (min)
Clot time (min)
Polyphosphate as a procoagulant 1755
20
10
0
0.0 0.1 0.2 0.3 0.4 0.5
FVIII (µg mL–1) added to
FVIII deficient plasma
20
10
0
ne fM pM pM pM nM nM
No 200 2 20 200 2 20
FVIIa added to
FVIII deficient plasma
D
Clot time (min)
Clot time (min)
C
30
40
20
0
40
20
0
1
0
2
3
4
FIX (µg mL–1) added to
FIX deficient plasma
ne fM pM pM pM nM nM
No 200 2 20 200 2 20
FVIIa added to
FIX deficient plasma
Fig. 6. Ability of polyphosphate (polyP) to shorten the clotting time of
plasma from patients with hemophilia compared with the effect of
supplementing the plasmas with the missing clotting factor or
recombinant factor (F) VIIa (rFVIIa). (A) Factor VIII or (B) rFVIIa
was added to plasma from a patient with hemophilia A. (C) Factor IX or
(D) rFVIIa was added to plasma from a patient with hemophilia B.
Clotting was initiated by dilute thromboplastin. Clotting reactions without added polyP (s) were compared to those containing 100 lM polyP
( ). Data are mean ± standard error (n = 5). Factor VIII:1IU mL)1
= 0.25 lg mL)1. Factor IX: 1 IU mL)1 = 5.13 lg mL)1. For
comparison, the mean clotting time for pooled normal plasma was
12.2 min.
effective at reversing anticoagulation because of the direct FXa
inhibitor, rivaroxaban.
Thromboelastography confirmed that polyP shortened the
time to initial clot formation (CT) in whole blood. The
anticoagulant effects of unfractionated heparin, enoxaparin, or
argatroban were not entirely reversed by polyP, whereas
adding polyP to blood containing rivaroxaban resulted in a
procoagulant effect similar to that observed in the absence of
the anticoagulant drug. Because the majority of thrombin is
generated after initial clot formation, thromboelastography
provided additional information regarding the dynamics of clot
formation as it occurred after initial clot detection in plasma
clotting assays. Despite the lack of normalization of CT by
polyP in blood anticoagulated with heparin, enoxaparin, and
argatroban, polyP completely reversed the effects of these
anticoagulants on the parameters associated with the dynamics
of clot formation that indicate propagation and rigidity (CFT,
a angle, and MCF).
Interestingly, when these anticoagulant drugs were added to
plasma in which all the FV was already in the active state
(FVa), polyP failed to reverse the anticoagulant effects of any
of the drugs. Furthermore, simply adding FVa to normal
plasma antagonized the anticoagulant effect of these drugs to a
level similar to that observed with polyP. Taken together, these
results argue that polyP abrogates anticoagulant drug activity
by enhancing FVa generation [16]. The ability of polyP to
antagonize argatroban was of particular interest, as a direct
Ó 2008 International Society on Thrombosis and Haemostasis
thrombin inhibitor should act only downstream of the
prothrombinase complex. However, an important function of
thrombin is the feedback activation of FV to FVa, a process
that should be slowed by argatroban (thus delaying clotting),
but we previously showed that polyP accelerates the rate of FV
activation by both FXa and thrombin [16]. We therefore
propose that polyP works to reverse argatrobanÕs anticoagulant function by accelerating FV activation.
We also found that polyP shortened the clotting of
hemophilic plasmas or plasmas from patients receiving warfarin, although it only partially normalized the clotting times of
the latter group. PolyP may have incompletely reversed the
effects of vitamin K antagonists because enhanced FVa
generation was unable to fully augment the assembly of the
prothrombinase complex when undercarboxylated coagulation
factors were present. In contrast, tissue factor-triggered coagulation was completely normalized by polyP when either FVIII
or FIX were deficient, and in fact polyP was as effective at
normalizing the clotting time of hemophilic plasmas as was
replacing the missing clotting factor or adding rFVIIa, both of
which are accepted in vivo therapies for managing hemorrhage
in patients with hemophilia. It is of particular interest that the
procoagulant effect of polyP was additive to that of rFVIIa.
Because polyP acts at a later level in coagulation than does
FVIIa, the two treatments may be complementary. As the costs
associated with treating bleeding episodes in hemophilia with
rFVIIa are so large, even a minor reduction in rFVIIa dose
could profoundly reduce the cost of managing these patients.
Recently, rFVIIa has been proposed as a Ôuniversal procoagulantÕ with the potential to reverse the anticoagulant activity
of a variety of drugs [1,3,21]. In studies in vitro or ex vivo,
rFVIIa partially or completely reversed the anticoagulant
effects of unfractionated heparin [9] enoxaparin [9], fondaparinux [9,10], warfarin [22], argatroban [9], or bivalirudin [9]. In
vivo data regarding reversal of anticoagulation with rFVIIa are
limited, although rFVIIa antagonized the anticoagulant effects
of fondaparinux [11,23] and idraparinux [24] in normal healthy
volunteers. In anecdotal reports and case series, rFVIIa
normalized in vitro clotting and improved or prevented
bleeding in patients receiving unfractionated heparin [25], low
molecular weight heparin [5,25], vitamin K antagonists [7,8,25–
29] or lepirudin [6]. Despite frequent anecdotal reports of using
rFVIIa to reverse anticoagulant-induced hemorrhage, the drug
is not currently approved for this indication. Controlled studies
evaluating its safety and effectiveness in comparison to other
therapies are lacking, and appropriate dose protocols have yet
to be clearly determined [3]. Furthermore, thromboembolic
adverse events following administration of rFVIIa have been
described [12], the majority of which are associated with offlabel use. Lastly, rFVIIa is expensive.
PolyP added to plasma has a half-life of approximately
1.5 h, apparently being degraded by plasma phosphatases [16].
Few data are available for polyP metabolism and clearance in
vivo, although 99mTc-labeled polyP was reported to distribute
to bone [30]. Therapeutic use of polyP for its effects on
coagulation may possibly necessitate continuous or multiple
1756 S. A. Smith and J. H. Morrissey
infusions, but polyP is inexpensive and may therefore represent
a significant cost savings over other available therapies.
The present studies demonstrate that polyP partially or
completely reversed the effects of a variety of anticoagulant
drugs in vitro, and shortened the clotting time of plasmas
deficient in FVIII or FIX. These findings suggest that polyP
might be useful for treatment of bleeding episodes in patients
with compromised hemostatic systems, although in vivo tests
would clearly be needed to establish this.
12
13
14
15
Acknowledgements
We thank Bayer for providing Kogenate and Rivaroxaban,
and Diagnostica Stago-US for generously loaning the ROTEM
system. This work was supported by grants R01 HL47014
from the National Heart, Lung and Blood Institute of the
NIH, and 06-2328 from the Roy J. Carver Charitable Trust.
16
17
18
19
Disclosure of Conflict of Interests
The authors are coinventors on patent applications on the use
of polyphosphate to modulate blood clotting.
20
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Ó 2008 International Society on Thrombosis and Haemostasis
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