(Received for publication, August 3, 1994; and in revised form, November 18, 1994)
From the
Coagulation Factor IX/IXa has been shown to bind to cellular
surfaces, and Factor IXa expresses its procoagulant activity by
assembling into the intrinsic Factor X activating complex (Factors
IXa/VIIIa/X), which also forms on membrane surfaces. This led us to
identify cellular proteins which bind Factor IX/IXa; an 55-kDa
polypeptide was purified to homogeneity from bovine lung extracts based
on its capacity to bind
I-Factor IX in a dose-dependent
and saturable manner. From protein sequence data of the amino terminus
and internal peptides, the
55-kDa polypeptide was identified as
calreticulin, a previously identified intracellular calcium-binding
protein. Recombinant calreticulin bound vitamin K-dependent coagulation
factors,
I-Factor IX,
I-Factor X, and
I-prothrombin (K
values of
2.7, 3.2, and 8.3 nM, respectively), via interaction with
its C-domain, although it did not affect the coagulant properties of
these proteins.
I-Calreticulin also bound to endothelial
cells in vitro (K
7.4
nM), and mouse infusion studies showed an initial rapid phase
of clearance in which calreticulin could be localized on the vascular
endothelium. Exposure of endothelial cells to calreticulin led to
dose-dependent, immediate, and sustained increase in the production of
nitric oxide, as measured using a porphyrinic microsensor. In a canine
electrically induced thrombosis model, intracoronary infusion of
calreticulin (n = 7) prevented occlusion of the left
circumflex coronary artery in a dose-dependent manner compared with
vehicle-treated controls (n = 5). These results
indicate that calreticulin interacts with the endothelium to stimulate
release of nitric oxide and inhibit clot formation.
The vitamin K-dependent coagulation Factor IX/IXa has an
important role in hemostasis and can contribute to the pathogenesis of
thrombosis (1, 2, 3, 4) . In view of
the well-known rapid clearance of Factor IX from the intravascular
space, the association of infused and endogenous Factor IX with the
vessel wall(5, 6) , and in vitro studies
demonstrating Factor IX binding to endothelium and
platelets(7, 8, 9) , studies have been
performed to characterize the molecular basis of this coagulation
protein-cell surface interaction. At the level of the ligand, the
amino-terminal -carboxyglutamic acid-containing domain of Factor
IX has been shown to be essential for cell surface binding (10, 11, 12, 13, 14) . At
the level of the cell surface site, previous studies have suggested
that Factor IX binding involves a protease-sensitive polypeptide, which
on endothelial cells had a predicted molecular mass of
150
kDa(15) , and on platelets appeared to involve proteins with
molecular masses of
150 and
250 kDa(16) .
To further
characterize polypeptides which interact with Factor IX, we employed
bovine lung extract as a starting material and isolated the major
species which bind Factor IX. An 55-kDa polypeptide was purified to
homogeneity and proved to be identical to calreticulin, based on
protein sequence analysis of the amino terminus and trypsin-cleaved
internal peptides. Calreticulin demonstrated specific binding to Factor
IX and also interacted similarly with Factors X and prothrombin,
although calreticulin was distinct from the endothelial binding site.
Interaction of calreticulin with these vitamin K-dependent coagulation
factors did not change their coagulant properties. In contrast,
calreticulin bound to endothelial cells in vitro, localized to
the vessel wall after intravenous infusion, and stimulated production
of nitric oxide. Intracoronary administration of calreticulin prevented
coronary thrombosis in a canine model. These results suggest that
calreticulin has a novel, potentially clinically applicable
antithrombotic function.
Recombinant rabbit calreticulin and discrete domains
from calreticulin were expressed in Escherichia coli using the
glutathione S-transferase (GST) fusion protein system with
pGEX-3X plasmid(22) , as described previously(23) .
Plasmids containing intact calreticulin or C-domain (amino acids
330-401) were expressed in BNN103 E. coli host(23) , and GST fusion proteins were purified
to homogeneity by one-step glutathione-Sepharose 4B affinity
chromatography (Pharmacia). Calreticulin or C-domain fusion protein was
cleaved with Factor Xa, separated from Factor Xa using monoQ FPLC
chromatography, reapplied to glutathione-Sepharose 4B, and the
pass-through contained homogeneous recombinant calreticulin. Plasmids
containing the N-domain (amino acids 1-182), the P-domain (amino
acids 182-290), and C-domain were also expressed as fusion
proteins. GSH and the GSH fusion proteins of the individual domains
were purified as above, immobilized on polyvinylchloride wells (see
polyvinylchloride plate binding assay, below), and used only in an
experiment to compare the relative binding of I-Factor
IX. Native rabbit calreticulin was isolated by a selective ammonium
sulfate precipitation procedure in the presence of protease inhibitors
followed by FPLC monoQ as described(24) . Calreticulin was
radiolabeled using the same method for preparing
I-Factor
IX. Following radiolabeling,
I-calreticulin was separated
from free iodine by gel filtration, and the final tracer had a specific
radioactivity of 3.0
10
cpm/ng, was >90%
precipitable in trichloroacetic acid (20%), and migrated as a single
band of
55 kDa on SDS-PAGE (10%).
Equilibrium
data were analyzed according to the equation of Klotz and Hunston (25) (B = nKA/[1 + K], where B = specifically
bound ligand (total binding, wells incubated with tracer alone, minus
nonspecific binding, wells incubated with tracer in the presence of
excess unlabeled material), n = sites/well, K = the dissociation constant, and A = free
ligand concentration) using nonlinear least-squares analysis
(Enzfitter). Data showing inhibition of
I-Factor IX
binding to calreticulin by unlabeled Factors IX, X, and prothrombin
were fit to the equation(26) : b = B
[A]K
/(Ka;K
+ K
[X] + K
[A]), where b = bound radioligand (
I-Factor IX);
[A] = concentration of radioligand; K
= K
in the absence
of inhibitor; [X] = concentration of
inhibitor (unlabeled Factor IX, X, or prothrombin); B
= maximal binding of radioligand in the absence of
inhibitor.
Binding of I-calreticulin to endothelium
was studied using confluent endothelial monolayers (0.32
cm
/well). Bovine aortic endothelial cells were isolated and
cultured as described previously(21) . Wells were incubated
with binding buffer (0.05 ml/well) containing
I-calreticulin alone (total binding) or in the presence
of a 100-fold molar excess of unlabeled calreticulin (nonspecific
binding). Following a 2-h incubation at 4 °C, wells were washed six
times rapidly with ice-cold washing buffer as above. Where indicated,
binding buffer was replaced by washing buffer, except that the amount
of calcium chloride or EDTA was varied as stated. Dissociation was
studied by the method of infinite dilution (27) . Following a
binding assay, performed as described above, wells were washed and
fresh binding buffer was added for the indicated time. Wells were then
washed once and residual radioactivity determined.
For
sequence analysis, 55-kDa polypeptide with peak Factor IX binding
activity was subjected to SDS-PAGE (10%), transferred to polyvinylidene
difluoride (PVDF) membranes, and the
55 kDa was subjected to
amino-terminal sequencing. Proteolytic digestion of protein adsorbed to
PVDF pieces was performed by treating strips with 0.2%
polyvinylpyrrolidine solution followed by trypsin in Tris-HCl, pH 8.5,
as described(29) . The resultant peptide fragments were
isolated by reversed-phase HPLC on a C
column (2.1
5 cm, YMC Inc., Morris Plain, NJ). Blotted protein samples on PVDF and
peptide fragments recovered by HPLC peptide mapping were sequenced
using an Applied Biosystems model 470A gas-phase sequencer with an
``on-line'' phenylthiohydantoin amino acid
analyzer(30, 31) .
To assess tissue deposition
of I-calreticulin/
I-albumin, the animals
were sacrificed, organs removed, rinsed in phosphate-buffered saline,
and dried overnight (80 °C). Subsequently, the weight and
radioactivity (cpm) were measured(39) . The method of Spady and
Dietschy (40) was used to calculate the tissue spaces
employing the following formula: tissue space (microliters plasma/g dry
tissue weight) = [cpm in tissue/(g dry weight
cpm/µl plasma)]. To correct for nonspecific tissue trapping of
calreticulin, a tissue space for
I-albumin was
calculated. The specific tissue space of calreticulin was determined by
subtracting the
I-albumin tissue space from the
I-calreticulin tissue space(39) .
Figure 1:
Binding of I-Factor IX to
lung extract immobilized in PVC wells. A, dependence on
extract concentration. PVC wells were incubated with dilutions of lung
extract (protein concentration as indicated) for 15 h at 4 °C,
additional sites on wells were blocked by exposure to blocking buffer,
and then a binding assay was performed by addition of
I-Factor IX alone (6.3 nM; total binding) or in
the presence of a 100-fold molar excess of unlabeled Factor IX
(nonspecific binding) for 2 h at 4 °C. Wells were then washed,
bound
I-Factor IX was eluted by exposure to
EDTA-containing buffer, and specific binding (total minus nonspecific
binding) was determined (mean ± S.E. of triplicate
determinations). B, dependence on
I-Factor IX
concentration. The experiment was performed as in A above,
except that the concentration of lung extract was fixed (4 µg/ml),
and the concentration of
I-Factor IX was varied as
indicated. Specific binding is plotted versus the
concentration of
I-Factor IX added to the wells, and the inset shows Scatchard analysis of the same data. Parameters of
binding were: K
= 1.4 ±
0.13 nM and n = 4.32 ± 0.17 fmol/well. C, competition study with unlabeled Factors IX, X, and
prothrombin. Lung extract (4 µg/ml) was adsorbed to PVC wells as
above, excess sites on the wells were blocked with albumin, and then a
binding assay was performed by adding
I-Factor IX alone
(6.3 nM) or in the presence of a 100-fold molar excess of
unlabeled Factors IX, X, or prothrombin (II) for 2 h at 4 °C.
Unbound tracer was removed by washing, bound
I-Factor IX
was eluted by exposure to EDTA-containing buffer, and binding (mean
± S.E. of triplicate determinations) is
shown.
Lung extract was subjected to
chromatography on hydroxylapatite, and Factor IX binding activity was
recovered with the major protein peak step-eluted with 0.5 M NaPO. Fractions with Factor IX binding activity were
pooled and applied to FPLC MonoQ, and Factor IX binding activity was
detected in fractions corresponding to a small protein peak eluted as
the column was developed with an ascending salt gradient at
0.4-0.5 M NaCl. Fractions with peak Factor IX
binding activity were pooled and subjected to nonreduced SDS-PAGE (Fig. 2, lane 1), and a complex pattern of bands was
observed by Coomassie Blue staining. When material in slices of the gel
was eluted and tested in the PVC assay, Factor IX binding activity was
observed only in fractions which comigrated with a protein band
corresponding to
55 kDa (Fig. 2, lane 2). The
latter eluted material was subjected to reduced or non-reduced SDS-PAGE (Fig. 2, lanes 3 and 4, respectively), and in
each case a single band,
55 kDa, was observed. Elution of this
protein from the non-reduced gel (Fig. 2, lane 4)
demonstrated the presence of Factor IX binding activity in the PVC
assay (Fig. 2, lane 5).
Figure 2:
SDS-PAGE and gel elution of 55-kDa
polypeptide derived from lung extract which binds Factor IX. Lane
1, nonreduced SDS-PAGE (10%) of the pool from MonoQ with Factor IX
binding activity visualized by Coomassie Blue staining. Lane
2, activity profile of material eluted from the indicated slice in lane 1. A nonstained lane on the gel otherwise identical to lane 1 was sliced (
4-mm pieces), subjected to elution as
described in the text, and used to coat PVC wells for a binding assay
with
I-Factor IX alone (6.3 nM) or in the
presence of an 100-fold excess of unlabeled Factor IX. Factor IX
binding activity is seen to be maximal in the slices (nos. 5 and 6) corresponding to
55 kDa. Data are expressed as
counts/min bound/sample of extract. Lanes 3 and 4,
the material eluted from slices nos. 5-6 in lane 2 was
subjected to reduced (lane 3) or nonreduced (lane 4)
SDS-PAGE (10%), and material on the gel was visualized by silver
staining. Lane 5, activity profile of the material in lane
4; the material eluted from slices no. 5 and 6 in lane 4 was subjected to nonreduced SDS-PAGE (10%) and again the gel was
sliced and proteins eluted. The latter eluted material was tested in
the PVC assay for its ability to bind
I-Factor IX.
Details of methods are described in the text, and for activity assays (lanes 2 and 5), the mean of duplicate determinations
is shown. Apparent molecular weights, shown by the arrows,
were interpolated from semilogarithmic plots based on the migration of
standard proteins run simultaneously (phosphorylase B, 97.4 kDa; bovine
serum albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa;
trypsin inhibitor, 21.5 kDa; lysozyme, 14.3
kDa).
The polypeptide in the
55-kDa polypeptide band was then subjected to protein sequencing. A
portion of the amino-terminal sequence aligned closely with that
previously reported for rabbit (46) calreticulin, as well as
the human, dog, rat, and pig
counterparts(24, 47, 48, 49) . In
addition, the band was cleaved with trypsin, fragments were separated
on reversed phase HPLC and subjected to sequence analysis, and found to
match those in rabbit calreticulin: no. 1 residues 262-265; no. 2
residues 32-36 and 39-43, and no. 3, residues
306-322(46) .
Figure 3:
SDS-PAGE of 55 kDa polypeptide and
purified, recombinant rabbit calreticulin. The
55-kDa polypeptide
purified from bovine lung extract (1 µg/lane; lanes 1 and 3) or purified recombinant rabbit calreticulin prepared in E. coli (1 µg/lane; lanes 2 and 4) was
subjected to nonreduced (lanes 1 and 2) or reduced (lanes 3 and 4) SDS-PAGE (10%). Gels were stained
with Coomassie Blue. Migration of molecular mass markers is shown
(numbers indicate molecular masses in kDa).
Figure 4:
Binding of Factors IX, X, and
prothrombin to recombinant calreticulin. A, dependence on
calreticulin concentration. PVC wells were incubated with the indicated
concentration of recombinant calreticulin, excess sites were blocked
with albumin-containing buffer, and then a binding assay was performed
by adding I-Factor IX (6.3 nM; A) alone
or in the presence of a 100-fold excess of the respective unlabeled
protein. Specific binding is shown (mean ± S.E. of triplicate
determinations). B-D, dependence on concentration of Factors
IX, X, and prothrombin. PVC wells were incubated with recombinant
calreticulin (9.1 pmol/well), excess sites were blocked with blocking
buffer, and then a binding assay was performed by adding the indicated
concentration of either
I-Factor IX (B),
I-Factor X (C), or
I-prothrombin (D) alone or in the presence of a 100-fold excess of the
respective unlabeled protein. Specific binding is plotted versus free/added
I-labeled clotting factor (either Factor
IX, X, or prothrombin). Data were analyzed by the nonlinear
least-squares program, and the curve indicates the best fit line.
Parameters of binding were for panel B, K
= 2.73 ± 0.36 nM and n = 21.8 ± 1.13 fmol/well; for panel C, K
= 3.24 ± 0.69 nM and n = 32.9 ± 2.44 fmol/well; and for panel D, K
= 8.26
± 0.52 nM and n = 27.6 ± 0.91
fmol/well. The inset shows Scatchard analysis of the same
data.
In competition
experiments performed to determine if each of these vitamin K-dependent
coagulation factors interacted with identical/overlapping sites on
calreticulin, dose-dependent inhibition of I-Factor IX
binding to calreticulin was observed on addition of unlabeled Factors
IX, X, and prothrombin (Fig. 5, A-C, respectively).
Although the relationship fits closely to a model of competitive
inhibition (26) showing K
values of
6,
5, and 12 nM for studies with unlabeled Factors IX, X, and
prothrombin, respectively (Fig. 5, A-C, insets), these values are viewed as approximate. These data
suggest that the K-dependent coagulation factors interacted with
similar/overlapping sites on calreticulin.
Figure 5:
Competitive binding study: effect of
unlabeled Factors IX, X, and prothrombin on the binding of I-Factor IX to recombinant calreticulin. Wells were
incubated with calreticulin (9.1 pmol/well) for 15 h at 4 °C,
excess sites in the wells were blocked with albumin-containing buffer,
and then a binding assay was performed by adding three different
concentrations of
I-Factor IX (circle = 6
nM; square = 3 nM; triangle = 1.5 nM) in the presence of the indicated
concentrations of either unlabeled Factor IX (A), unlabeled
Factor X (B), or unlabeled prothrombin (C). Inset, Dixon plot showing 1/bound (1/B,
fmol
) versus protein added
(nM).
The interaction of
calreticulin with I-Factor IX was calcium dependent, with
maximal effect by an added calcium concentration between 2-5
mM (data not shown). Since calreticulin has two
calcium-binding domains, a high affinity, low capacity site (P-domain)
and a low affinity, high capacity site (C-domain), experiments were
performed with each of these domains, as well as amino-terminal
N-domain (50; for this experiment only, calreticulin domains were
expressed and studied as glutathione S-transferase fusion
proteins). The C-domain bound
I-Factor IX, whereas the
P-domain, N-domain, and glutathione S-transferase control
protein displayed only background binding (data not shown). Studies
with
I-Factor IX and C-domain indicated that binding was
concentration dependent, with an apparent K
3.3 nM (Fig. 6). Similar results were obtained with
respect to the interaction of
I-Factor X and
I-prothrombin with calreticulin C-domain (apparent K
values were 4.8 and 6.0 nM,
respectively; data not shown).
Figure 6:
Interaction of I-Factor IX
with the C-domain of calreticulin. Wells were incubated with C-domain
(0.5 µg/well) for 15 h at 4 °C, excess sites were blocked with
blocking buffer, and then a binding assay was performed with the
indicated concentrations of
I-Factor IX (6.3 nM)
alone or in the presence of a 30-fold excess of the unlabeled
respective protein. Specific binding is plotted versus the
concentration of free/added tracer. Data were analyzed by the nonlinear
least-squares program, and the curve indicates the best fit line. The inset shows Scatchard analysis of the same
data.
In view of the interaction of calreticulin with multiple vitamin K-dependent coagulation factors, we investigated the effect of calreticulin on coagulant reactions involving these proteins. The following reactions were studied: tissue factor/Factor VIIa-mediated activation of Factor X (using matrices prepared from endothelial cells exposed to tumor necrosis factor as the source of tissue factor; 36), Factor IXa-VIIIa-mediated activation of Factor X (using crude cephalin as the phospholipid surface or endothelium), Factor Xa-Va-mediated activation of prothrombin (using cephalin as the phospholipid surface or endothelium), and thrombomodulin-dependent, thrombin-mediated activation of protein C (using intact bovine endothelial cells as the source of thrombomodulin; 37). No inhibition of these reactions was observed over a range of calreticulin concentrations. Furthermore, addition of calreticulin to recalcified human or bovine plasma did not delay clotting initiated with Factor IXa or Factor Xa (employing cephalin as the phospholipid source; data not shown).
Figure 7:
Infusion of recombinant rabbit
calreticulin into mice. A, clearance of infused I-calreticulin from the plasma. Mice were infused via the
tail vein with
I-calreticulin or
I-albumin,
and at the indicated times, blood was withdrawn for determination of
plasma radioactivity, with data expressed as the percent recovery (100
measured cpm in 5 µl of plasma/administered cpm). B, immunohistologic study of rabbit calreticulin infused into
mice. Mice were infused with rabbit calreticulin or rabbit serum
albumin (120 µg/animal); 5 min later the animals were sacrificed,
tissues were washed, and prepared for immunostaining with anti-rabbit
calreticulin IgG. Panel I shows lung tissue from an animal
infused with calreticulin, compared with an albumin-infused control (panel II). Magnification,
480.
, albumin;
, calreticulin.
Figure 8:
Effect of calreticulin on coronary
thrombosis in a canine model. Following instrumentation of the left
circumflex artery, current was applied to the needle electrode until a
50% increase in mean blood flow velocity occurred (this corresponds to
a 50% decrease in cross-sectional area). The current was then
turned off, a bolus of the indicated amount of calreticulin or saline
was given into the left circumflex coronary artery (in each case,
volume was 0.5 ml), and vessel patency was monitored as described in
the text.
Figure 9:
Binding of calreticulin to endothelial
cells. Dose dependence. confluent monolayers of bovine aortic
endothelial cells (passage 6; 0.32 cm/well) (endothelial
cells) were incubated with the indicated concentration of
I-calreticulin alone or in the presence of 100-fold
excess of unlabeled protein. Monolayers were washed, bound
radioactivity was eluted, and specific binding is plotted versus the free/added concentration of
I-calreticulin. Data
were analyzed by nonlinear least-squares analysis, and the curve
indicates the best fit line. Parameters of binding were K
= 7.37 ± 0.93 nM and n = 4.28 ± 0.23
fmol/well.
Figure 10: Effect of calreticulin on endothelial production of nitric oxide. Bovine endothelial cells (third passage) were plated on coverslips and placed on a warmed microscope stage (37 °C). Nitric oxide was measured using a porphyrinic microsensor as the working electrode, placed with the aid of a micromanipulator. A saturated calomel electrode served as the reference electrode and a platinum wire electrode as the counterelectrode. A, amperometric recordings were taken at a potential of 0.68 V, with an aqueous nitric oxide standard used for microsensor calibration. Recombinant rabbit calreticulin (or albumin as control) in phosphate-buffered saline was applied, and nitric oxide measurements obtained. B, nitric oxide levels at the endothelial surface as a function of applied recombinant rabbit calreticulin concentration. Measurements were obtained as described above using a porphyrinic microsensor, with total amount of NO quantified by integration of the area under the amperometric curves. Data are expressed as the mean ± S.E.
To characterize cell-associated polypeptides which interact
with Factor IX, 55-kDa polypeptide was isolated from lung extracts
based on its capacity to bind Factor IX. The
55 kDa polypeptide
co-migrated on SDS-PAGE with calreticulin and displayed virtual
sequence identity with calreticulin of five different species.
Furthermore, recombinant calreticulin bound Factor IX in a manner
analogous to the
55-kDa lung-derived polypeptide. These data
indicated that the polypeptide isolated from lung was calreticulin
(previously identified as an intracellular calcium-binding protein
present in a wide range of cell types)(46, 50) , but
it proved to be unrelated to the endothelial cell Factor IX-binding
site, as indicated by several lines of evidence: (i) the binding of
Factor IX to either the
55-kDa polypeptide or calreticulin was not
selective since the interaction with other vitamin K-dependent factors,
including Factor X, prothrombin, and protein S (for the latter, data
not shown), was very similar and the coagulation proteins
cross-competed, whereas binding to the endothelial cell surface is
selective for Factor IX(7, 8) ; (ii) addition of
excess unlabeled calreticulin to binding mixtures of
I-Factor IX-endothelial cell incubation mixtures did not
prevent specific binding; and (iii) antibody to human calreticulin did
not block the binding of
I-Factor IX to human endothelial
cells (data not shown). Thus, the interaction of Factor IX with the
endothelium does not appear to involve calreticulin.
Although the binding of calreticulin to vitamin K-dependent coagulation factors seems to be relatively non-selective with respective to the target clotting factor, it specifically involves the C-domain of calreticulin. The first evidence supporting the possibility of an interaction between calreticulin and vitamin K-dependent coagulation proteins derives from the observation that calreticulin was a persistent contaminant co-purifying with protein Z, requiring preparative electrophoresis to effect a complete separation(51) . However, the same authors found that calreticulin was present in apparently normal human plasma at low levels(51) , despite the presence of an endoplasmic retention sequence and two lysosome targeting signals (50) . These data suggest that calreticulin is likely to bind to plasma vitamin K-dependent coagulation proteins under physiologic conditions, although the functional significance of this interaction is unclear, especially in view of our inability to find any effect of calreticulin on the participation of Factor VIIa, Factors IX/IXa, X/Xa, prothrombin, protein C/activated protein C or protein S in coagulation-related reactions.
The results of our in vivo clearance studies indicated that infused calreticulin became rapidly associated with endothelium of the vessel wall. To explore a possible mechanism whereby calreticulin could limit thrombus formation at localized endothelial cell sites without the development of a systemic anticoagulant effect, modulation of endothelial cell production of NO was studied. An important role for NO produced by the endothelium is to inhibit platelet aggregation and serotonin release, even in the presence of powerful proaggregants and secretogogues, such as thrombin. Measurement of endothelial cell-derived NO using a sensitive and specific porphyrinic microsensor showed that calreticulin increased NO production and that this was dose dependent over a range of calreticulin concentrations which paralleled those resulting in occupancy of endothelial cell calreticulin-binding sites. In addition, recombinant rabbit calreticulin appears to inhibit thrombin-mediated platelet aggregation and serotonin release (data not shown), suggesting the potential clinical relevance of these results. By stimulating a potent anticoagulant mechanism localized to the vascular wall, the nonsystemic nature of calreticulin's anticoagulant effects may be understood. Although local stimulation of nitric oxide production could account for the localized antithrombotic effect of calreticulin in vivo, the relationship between occupancy of endothelial cell calreticulin-binding sites and calreticulin-stimulated NO production by endothelial cells is at present unclear.
Taken together, these data suggest the hypothesis that calreticulin, an intracellular calcium-binding protein, when infused into the intravascular space binds to endothelial cells, stimulates NO production, blocks platelet adhesion and activation, and thereby prevents thrombus formation. Although further studies will be required to define the mechanisms through which calreticulin exerts its effects in vivo and in vitro, if these findings can be extrapolated to a range of thrombosis models, calreticulin may prove to have novel antithrombotic properties of potential therapeutic value. In support of the possible utility of calreticulin in this context, our recent pilot studies have shown that calreticulin infused intravenously (i.e. not at the site of thrombosis) also has an antithrombotic effect.