|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From The Center for Blood Research and the Department
of Pathology, Harvard Medical School, Boston, MA, and Wyeth/Genetics
Institute, Cambridge, MA.
Interleukin (IL)-11 is a cytokine with thrombopoietic activity that
has been shown to increase plasma von Willebrand factor (vWf) in
preliminary clinical studies. This led to further evaluation of
the effect of recombinant human (rh)IL-11 on vWf and factor VIII
(FVIII) secretion. In vitro, rhIL-11 did not increase vWf production by
cultured endothelial cells, which suggests an indirect mechanism. Also,
in vivo, plasma vWf was not elevated in mice shortly after a single
intravenous (IV) bolus injection of 250 or 1000 µg/kg rhIL-11. The
effect of continuous exposure to rhIL-11 was accessed by treating wild
type mice for 7 consecutive days with subcutaneous 250 µg/kg/d
rhIL-11. Platelet counts increased by 25% and 40% after 4 and 7 days,
respectively. Plasma vWf and FVIII levels increased 2-fold after 4 and
7 days. Surprisingly, no effect of rhIL-11 on vWf or FVIII messenger
RNA was observed, which suggests that the regulation by rhIL-11
occurs after transcription. No increase in soluble P-selectin
was observed after rhIL-11 treatment, indicating that platelet
activation is not the source of elevated vWf. Similarly to wild type
mice, vWf heterozygous mice responded to rhIL-11 treatment by a
significant increase in platelet counts and vWf and FVIII levels.
Importantly, in vWf-deficient mice, rhIL-11 also induced a significant
increase in FVIII independent of vWf and was able to reduce skin
bleeding time. These results suggest that a clinical evaluation of the
effects of rhIL-11-induced vWf/FVIII elevation in maintaining
hemostasis in mild hemophilia A or von Willebrand disease would be worthwhile.
(Blood. 2001;97:465-472) von Willebrand factor (vWf) facilitates
platelet adhesion to the subendothelium, especially at high shear
rates, and is the plasma carrier for factor VIII
(FVIII).1-4 A quantitative or functional deficiency in vWf
causes von Willebrand disease (vWd), which is the most common
hereditary hemorrhagic blood disease.5-8 Symptoms of vWd
include bruising, nosebleeds, bleeding gums, bleeding mucous membranes,
and gastrointestinal blood loss.9 In severe vWd, joint
tissue bleeds may resemble those seen in hemophilia.9 In
affected females, heavy menstrual bleeding is the primary
symptom.10 The gene for vWf is large (178 kilobases) and
complex (52 exons), and a wide range of mutations has been described in
patients with vWd.11,12 The vWf deficiencies resulting
from these mutations are categorized as either qualitative or
quantitative and are further classified by disease
type.9,11,12 Type 1 and type 3 disease encompass mild and
severe quantitative deficiency.9,11,12 Type 2 deficiency
refers to qualitative abnormalities in which there is a normal level of
vWf that is functionally deficient.9,11,12 The majority of
patients with vWd have mild disease and are treated with desmopressin
(1-deamino-8-D-arginine vasopression
[DDAVP]).13,14 The benefit of DDAVP is its ability to
stimulate tissue storage sites to release FVIII and vWf, resulting in
increased circulating factors. Continued exposure to DDAVP results in a
diminished response to the product.13,14 Patients with
more severe forms of vWd and those nonresponsive to DDAVP therapy are
treated with plasma concentrates containing FVIII and
vWf.13,15
Recombinant human interleukin 11 (rhIL-11, Neumega; Wyeth/Genetics
Institute, Cambridge, MA) is a pleiotropic cytokine with both
thrombopoietic and anti-inflammatory activity that is approved for
platelet restoration following high-dose
chemotherapy.16-18 During preliminary clinical trials, in
addition to increases in platelet count, elevations in vWf and
fibrinogen were also observed.19 Because this increase in
vWf might suggest potential benefit to the vWd patient, we were
interested in further evaluating this response in appropriate cell
culture systems and animal models. The results presented here
demonstrate that both the wild type mouse and the vWf+/ Mice
Placebo and rhIL-11 treatment regimens
Blood counts Mice were anesthetized with Metofane (Schering-Plough Animal Health, Union, NJ), and blood was collected by retro-orbital venous plexus sampling in polypropylene eppendorf tubes containing ethyelendiamine tetraacetic acid (EDTA). Complete blood counts were determined using an automatic cell counter (Coulter Electronics, Hialeah, FL).vWf antigen determination Mice were anesthetized, and blood was obtained by retro-orbital venous plexus sampling in polypropylene eppendorf tubes containing 129 mM trisodium citrate. Plasma was prepared by centrifugation of the blood at 2500g for 15 minutes at room temperature. We measured vWf antigen level using an enzyme-linked immunosorbent assay (ELISA) technique as described.20 The number of units in each sample was determined based on the absorbance value of a normal pooled plasma obtained from 10 wild type mice. We defined normal pooled plasma as containing one unit vWf antigen per mL plasma. For the in vitro studies with human umbilical vein endothelial cells (HUVECs), vWf antigen level was determined in the culture media, and human plasma was used as the reference plasma (Sigma Chemical, St Louis, MO).FVIII activity determination FVIII concentration was determined using a one-stage clotting assay. Plasma was harvested as described for the vWf assay and kept frozen until the assay. Plasma was diluted 1:40 with Owren's buffer (Sigma), and normal reference plasma (Sigma) was used as a standard. FVIII-deficient plasma (Helena Laboratories, Beaumont, TX) and diluted mouse plasma were mixed in a glass tube with activated partial thromboplastin time reagent (Instrumentation Laboratory, Lexington, MA). The tube was then transferred to a 37°C water bath, and after 5 minutes, clotting was initiated by the addition of 25 mM calcium chloride. Clotting was monitored visually during constant mixing in the 37°C water bath, and the time necessary for the clotting to occur was recorded. FVIII activity in the samples was determined using the standard curve obtained with the reference plasma.Quantitative reverse transcriptase-polymerase chain reaction Heart, lungs, liver, and spleen were collected after 1 or 4 days from wild type mice injected subcutaneously with 250 µg/kg/d rhIL-11. Total RNA was extracted from liquid nitrogen snap-frozen tissues using the Tri-Reagent (Molecular Research Center, Cincinnati, OH) according to manufacturer's specification. In brief, 100 mg tissue was homogenized in 1 mL Tri-Reagent and stored at room temperature for 5 minutes. One-tenth volume of 1-bromo-3-chloropropane (BCP) was added, shaken vigorously for 15 seconds, and kept at room temperature for 10 minutes prior to centrifugation at 12 000g for 15 minutes at 4°C. The aqueous phase was removed and placed into a new tube, where RNA was precipitated for 10 minutes at 20°C using 0.5 mL
isopropanol. RNA was collected by centrifugation at 12 000g
for 10 minutes at 4°C. The supernatant was removed, and RNA was
washed with 1 mL 75% ethanol and then followed by subsequent
centrifugation at 12 000g for 10 minutes. Ethanol was removed, and RNA was allowed to dry for 5 minutes. RNA was resuspended in diethylpyrocarbonate-treated (DEPC) sterile water. Total RNA was
treated with RQ1 DNase I (Promega, Madison, WI) and RNase inhibitor (Five Prime Three Prime, Boulder, CO) for one hour at 37°C. RNA clean was performed using Qiagen Rneasy Minicolumns (Qiagen, Valencia, CA) according to manufacturer's specification. Recombinant thermostable DNA polymerase was used to reverse
transcribe and amplify 125 ng total RNA using the Perkin Elmer Taqman
EZ RT-PCR (reverse transcriptase-polymerase chain reaction) kit
(Perkin Elmer Applied Biosystems, Foster City, CA) with gene-specific forward and reverse primers and a fluorescently labeled probe at the 5'
end with 6-carboxy-flourescein (6-FAM).23-25 Primer and probe sequences were generated using Primer Express software (Perkin Elmer).
Murine FVIII forward primer 5'-TGTTTGTAGAGTACAAGGACCAGCTT-3', reverse primer 5'-GAACCTCAGTCCAAATGGTAGGA-3', and Taqman probe 5'-TGCCAAGCCCAGGCCACCCT-3' were used. Murine vWf forward primer 5'-ATCCGCGTGGCAGTGGTA-3', reverse primer 5'-CGCTTCCGGGCCTTG-3', and Taqman probe 5'-CATGATGGCTCCCGTGCCTACCTTG-3' were used. Duplicate samples were reverse transcribed for 30 minutes at 60°C, followed by 40 cycles of amplification for 15 seconds at 95°C and one minute at 60°C using the ABI Prism 7700 sequence detection system (Perkin Elmer) as described by the manufacturer. Gene-specific amplification was detected as a fluorescent signal during the amplification cycle. Gene-specific message quantification was evaluated by fluorescence intensity levels of unknown samples compared to fluorescence intensity of known messenger RNA (mRNA) levels. Amplification of a housekeeping gene, murine glyceraldehyde 3 phosphate dehydrogenase (GAPDH), was performed on all samples to account for RNA-level variations. All genes were normalized to GAPDH mRNA levels, and levels of gene-specific messages were depicted as normalized Taqman units as determined by standard curve. P-selectin ELISA Blood was collected as described in the vWf ELISA, except that blood samples were placed in polypropylene tubes containing 0.1 volume of 38 mM citric acid, 75 mM trisodium citrate, and 100 mM dextrose. The P-selectin ELISA was completed as described by Hartwell et al.26Endothelial cell cultures HUVECs (second or third passages) were grown as described.27 To visually assess the release of storage granules by rhIL-11, cells were grown on glass coverslips and incubated for 30 minutes, 3 hours, or 24 hours at 37°C with 1, 10, 50, and 1000 ng/mL rhIL-11 added to the media. Control cells were treated with rhIL-11 diluting solution (PBS). For vWf ELISA, the HUVECs were grown in 35-mm dishes (Falcon), and after confluency, cells were rinsed twice with fresh media and treated with 100 or 1000 ng/mL rhIL-11. After the designated incubation time, media were collected, centrifuged, and stored at20°C prior to vWf assay.
Immunofluorescence staining Cells were grown on coverslips, fixed in 3.7% (vol/vol) formaldehyde, and permeabilized with 0.5% Triton X-100 in PBS. The cells were stained for vWf using rabbit polyclonal antibody to vWf (American Bioproducts, Parsippany, NJ) at 1:100 dilution followed by fluorescein-conjugated sheep antibody to rabbit immunoglobulin G (IgG) (ICN Pharmaceuticals, Costa Mesa, CA) at 1:1000 dilution. All incubations with antibodies were performed for 30 minutes at 37°C.Multimer analysis Plasma samples diluted 1:20 in 10 mM Tris-HCl (tris[hydroxymethyl aminomethane-hydrochloride) and 1mM EDTA (pH 8) containing 2% sodium dodecyl sulfate (SDS) were layered on 1% agarose gels (agarose IEF, Amersham Pharmacia Biotech, Piscataway, NJ) poured on Gelbond (FMC, Rockland, ME). Gel electrophoresis was run horizontally at a constant amperage of 10 mA. The gel was fixed in 25% isopropyl alcohol and 10% acetic acid for 30 minutes and rinsed. Multimers were revealed by incubating the dried Gelbond with a polyclonal antibody to human vWf labeled with iodine 125 (125I) and visualized by autoradiography.Skin bleeding time We injected 8-week-old female vWf / mice
subcutaneously with a placebo or 250 µg/kg/d rhIL-11 for 7 days. The
mice were anesthetized, and hair from the internal side of the hind leg
was removed with electric clippers. A standard incision (5-mm long by
1-mm deep) was made with an automatic device (Surgicutt; ITC, Edison,
NJ). Every 15 seconds a filter paper was applied to the wound for 5 seconds until the blood no longer stained the paper. Bleeding time was
determined to the nearest 15 seconds.
Clearance of rhvWf in vWf / mice were injected subcutaneously with a
placebo or 250 µg/kg/d rhIL-11 for 5 days. On the fifth day following
rhIL-11 injection, the mice were injected intravenously with 40 U/kg
rhvWf (Wyeth/Genetics Institute). Blood was collected from 4 mice injected with rhIL-11 and from 4 mice injected with placebo
immediately after the rhvWf injection and at various time points
following the injection: 30 minutes and after 3, 6, 14, and 24 hours.
Each mouse was bled only once except for the 14- and 24-hour time
point, where the same set of mice was bled twice. Plasma vWf antigen levels were determined by ELISA.
Statistical analysis Data are presented as the mean ± SEM. Statistical significance was calculated using Student t test for all analyses. Unpaired or paired tests were performed as indicated in the figure legends.
Platelet count and vWf and FVIII levels were increased in mice by daily subcutaneous rhIL-11 injections C57BL6J mice were injected subcutaneously with a placebo or 250 µg/kg/d rhIL-11 for a total of 7 days. A group of mice was bled and killed at day 4 and another on day 7. The remaining animals were killed on day 14, 7 days after the last injection. As an internal control for rhIL-11 activity, platelet counts were measured in the peripheral blood in all mice. Similar to the preliminary report on humans,19 the number of platelets was significantly increased in the rhIL-11-treated mice at day 4 and day 7 (Figure 1A). Platelet numbers declined after treatment was stopped and returned to preinjection levels by day 14. The white blood cell count and hemoglobin level did not change in any group during the evaluation period. Plasma vWf was increased 2.0- and 1.8-fold at day 4 and 7, respectively, in the rhIL-11-treated mice compared to the vehicle-injected mice (P = .005 and P = .01, respectively) (Figure 1B). Similar to the platelet counts, the vWf levels returned to pre-injection levels by day 14. FVIII levels were increased the most by rhIL-11 administration (Figure 1C). The rhIL-11 injection enhanced FVIII level 2.6-fold at day 4 and 2.9-fold at day 7 compared to day 0. These increases were highly significant (P = .0002) when compared to preinjection levels or to placebo-injected mice (P < .0001). Interestingly, FVIII level at day 14 was still significantly higher in the rhIL-11-treated mice than in the controls (P < .04). We also tested whether the effect of rhIL-11 could be long-lasting. For this purpose we injected the mice with rhIL-11 daily for up to 21 days, and we found that the elevation in platelet numbers and vWf and FVIII levels was sustained for as long as the treatment lasted (not shown).
Multimer analysis of plasma vWf The multimeric composition of vWf from wild type mice injected with rhIL-11 for 7 days was similar to that of mice injected with placebo (Figure 2). We could not observe an increase of the higher molecular weight multimers characteristic of vWf stored in the endothelial cell storage granules (Weibel-Palade bodies)25 after rhIL-11 treatment.
Dose-dependence of rhIL-11 activity To determine if lower doses of rhIL-11 were active, we compared the effect of 3 doses of rhIL-11 (30, 100, and 300 µg/kg) in wild type mice. The 2 highest concentrations, 100 and 300 µg/kg, produced significant elevations in platelet numbers and in vWf and FVIII levels (Figure 3A-C), with the 300 µg/kg dose being the most effective. At 30 µg/kg, rhIL-11 did not reliably induce an increase in platelet numbers and vWf and FVIII levels. Thus, a dose of 100 µg/kg is necessary to achieve biological activity in mice.
Evaluation of vWf release from HUVECs To investigate whether rhIL-11 treatment directly induces release of Weibel-Palade bodies containing vWf in endothelial cells, we tested the effect of rhIL-11 on vWf release by HUVECs in vitro. HUVECs were grown to confluency and treated with placebo or rhIL-11 at concentrations of 0-1000 ng/mL for different incubation times (30 minutes to 72 hours). We evaluated the release of Weibel-Palade bodies in 2 different ways. First, we stained treated HUVECs by immunofluorescence with an antibody to vWf to visualize Weibel-Palade bodies. The rhIL-11 treatment of HUVECs did not result in a loss of Weibel-Palade bodies, and we could not detect the typical release patches28 of cell-surface-associated vWf (data not shown). Second, we collected HUVEC culture medium after treatment with or without rhIL-11 and measured vWf levels by ELISA (Table 1). Again, we did not observe any rhIL-11-dependent vWf secretion.
Effect of a single IV injection of rhIL-11 in mice To determine whether rhIL-11 could stimulate Weibel-Palade-body secretion in vivo, we injected 250 or 1000 µg/kg rhIL-11 via the tail vein in wild type mice and measured vWf and FVIII levels 3 hours after injection. Three hours was estimated to be sufficient time for the secretion of the granules to occur when endothelial cells were treated by secretagogues, but it was not long enough for significant de novo synthesis of the vWf protein.27 Platelet counts and vWf and FVIII concentrations in the plasma were not changed by rhIL-11 treatment (Table 2). White blood cell count and hemoglobin level were also unchanged. Thus rhIL-11 does not appear to induce vWf or FVIII release from storage compartments.
Effect of rhIL-11 on soluble P-selectin concentration in the plasma P-selectin, like vWf, is located both in the Weibel-Palade bodies of endothelial cells and the granules of platelets, and therefore
P-selectin is expressed on the surface of these cells by the same
secretagogues or agonists that stimulate vWf release. Soluble
P-selectin in the plasma is generated by shedding of the membrane form
of P-selectin and usually reflects platelet activation.29 We tested the effect of daily subcutaneous injections of 250 µg/kg rhIL-11 on the level of soluble P-selectin in wild type mice. The
conditions chosen were the ones found to be optimal for vWf increase.
There was no significant increase in P-selectin levels observed after
rhIL-11 (Table 3). This result indicates
that excessive platelet activation by rhIL-11 did not contribute
significantly to the vWf increase.
Effect of rhIL-11 on vWf and FVIII mRNA To investigate whether an rhIL-11 effect on vWf and FVIII levels occurred through a stimulation of their synthesis, we used a quantitative method to determine the amount of vWf and FVIII mRNA after injection of wild type C57BL/6J mice with a placebo or 250 µg/mL rhIL-11 for 4 days. We confirmed the significant increase in platelet counts and in vWf and FVIII levels in rhIL-11-injected mice and measured mRNA for vWf in lung, kidney, and spleen and for FVIII in liver, kidney, and spleen. There were no differences detected in vWf and FVIII message between rhIL-11-treated mice and controls (Figure 4). The same result was obtained when we looked at an earlier time point (24 hours after the beginning of the rhIL-11 injections) (data not shown). Thus, rhIL-11 does not appear to lead to increased synthesis or stabilization of vWf and FVIII mRNA.
Effect of rhIL-11 on vWf heterozygous mice To determine whether rhIL-11 would be able to produce an increase of vWf and FVIII in a model of vWd type 1, we used vWf-heterozygous mice, which have only 50% vWf antigen level and approximately 60% FVIII compared to wild type mice.20 The vWf-heterozygous mice injected for 7 days with 250 µg/kg rhIL-11 responded in a similar fashion to wild type mice (Figure 5). Platelet numbers and vWf and FVIII levels were significantly increased in the rhIL-11-treated group compared to the placebo-treated group. A slight increase was seen in the placebo-treated mice, likely due to the stress of daily injections. After rhIL-11 treatment of vWf+/ mice, vWf levels
increased very close to the baseline level observed in wild type mice
(Figure 5). Strikingly, FVIII level after rhIL-11 treatment of the
heterozygous mice was higher than the baseline level seen in wild type
mice (P = .0004). Using these
vWf+/ mice, we also performed a
long-term rhIL-11 injection study and observed that the high levels of
vWf and FVIII could be sustained at their maximum for as long as the
treatment was continued, ie, 21 days of daily subcutaneous
injections of 100 µg/kg rhIL-11 (data not shown).
Effect of rhIL-11 on FVIII levels in vWf-deficient mice In the vWf-deficient mice, the level of FVIII is about 20% that of wild type mice.20 We then questioned whether rhIL-11, in the complete absence of vWf, would still be able to increase FVIII levels. After injecting the vWf/ mice with 250 µg/kg
rhIL-11 subcutaneously for 7 consecutive days, platelet numbers
increased in a fashion similar to those in wild type mice.
Surprisingly, as in vWf+/+ and vWf+/ mice, we
noticed a significant increase in FVIII levels, up to the level of
untreated vWf+/ mice, after rhIL-11 treatment of
vWf/ mice (Figure 6).
Therefore, it appears that the FVIII increase observed after rhIL-11
treatment is not simply a consequence of the increase in vWf. In terms
of absolute values, the FVIII level in vWf/ mice after
rhIL-11 treatment was lower than the FVIII level in vWf+/+
and vWf+/ mice. This suggests that without the
concomitant increase in vWf, FVIII level cannot be optimally elevated,
probably due to an increased rate of clearance. However, the FVIII
level was increased to 0.8 U/mL, which is similar to the baseline level
observed in vWf+/ mice. To test whether rhIL-11
injections could improve the hemostatic status of vWf/
mice, we measured bleeding time in these mice using a skin model. We
found that rhIL-11 injections were able to significantly reduce the
skin bleeding time of vWf/ mice compared to placebo
injections (115 ± 12 seconds vs 265 ± 37 seconds, respectively)
(Figure 7).
Effect of rhIL-11 on vWf clearance in vWf-deficient mice To investigate whether rhIL-11 induces increase in vWf levels through a prolongation of its half-life, we measured clearance of rhvWf injected in vWf/ mice treated with placebo or rhIL-11.
We did not detect any differences in the clearance rate of rhvWf
between and placebo- and rhIL-11-treated mice, with a half-life of
about 2 hours in each group (Figure 8).
Thrombocytopenia is associated with a number of pathological situations or can arise as a complication of medical treatments such as chemotherapy. As a consequence, numerous studies have focused on isolating growth factors that are able to stimulate megakaryocyte development and platelet production. Several of these factors have been identified including IL-6,30 IL-11,32 and thrombopoietin.31 During the preliminary trials evaluating the potential of IL-11 in reducing chemotherapy-induced thrombocytopenia, it was observed that rhIL-11 increased vWf and fibrinogen levels.19 Because of the potential therapeutic interest of this effect in preventing bleeding complications, we decided to confirm this observation in mice and investigate the mechanisms involved. We found that rhIL-11 treatment of wild type mice for 7 days resulted not only in elevation of vWf levels but also in FVIII levels. The vWf increase was rapidly reversible on discontinuation of rhIL-11 treatment. However, we noted that the FVIII level remained elevated even after the vWf level had returned to baseline. Similar observations about the kinetics of FVIII increase have been reported following infusions of plasma or cryoprecipitates in vWd patients.33,34 IL-11 is a member of the IL-6 family of cytokines that includes ciliary
neurotrophic factor, leukemia inhibitory factor, oncostatin M, and
cardiotrophin.35-37 While each of these cytokines uses the gp130 chain as part of their receptor complex, the high-affinity binding is determined by the association of this common chain with a
cytokine-specific The effect of IL-11 on the expression of acute phase proteins was first
reported in the liver. Indeed, exposure of hepatocytes to rhIL-11
induces expression of fibrinogen, The origin of the increase in plasma vWf was next investigated. Plasma
vWf usually comes almost exclusively from the endothelial cells,42 but we could not exclude the possibility that the
stimulated bone marrow megakaryocytes and/or the increased circulating
platelets could also be a source of vWf. However, we could not detect
any increase of soluble P-selectin after rhIL-11 treatment, which would
indicate Taking advantage of our mouse model of vWd, we tested the effect of rhIL-11 in this animal model. Studies performed with the vWf heterozygous mice provided evidence that rhIL-11 can bring vWf and FVIII levels up to normal levels in a model of type 1 vWd. Similar to the wild type mice, the elevation of FVIII in the vWf heterozygous mice could be explained by the availability of more carrier sites provided by the elevated vWf levels. Our results obtained with the vWf-deficient mice were surprising because in these animals, rhIL-11 treatment resulted in a significant elevation in plasma FVIII in the absence of vWf, which indicates that to some extent, FVIII can be regulated independently of vWf. We then investigated whether vWf or FVIII mRNA was increased after rhIL-11, which would indicate either a stimulation of the synthesis or an increased stability of the mRNA. Under the same conditions that resulted in protein level increase, we could not detect a change in mRNA levels. In an attempt to identify the mechanism(s) underlying rhIL-11-mediated increase of vWf and FVIII protein expression, we measured the clearance rate of rhvWf injected in vWf-deficient mice after rhIL-11 treatment. Proteolytic degradation is likely to contribute to vWf clearance, and a vWf degrading protease enzyme was partially purified and characterized from human plasma.44 The rhIL-11-mediated vWf increase could therefore possibly be mediated through a reduced activity of this protease. However, we did not detect any prolongation of vWf half-life following rhIL-11 treatment. This result does not preclude a possible effect of rhIL-11 on FVIII half-life. In the vWf-deficient mice, an in vivo prolongation of FVIII half-life can be achieved by the receptor-associated protein (RAP), which inhibits the clearance mediated by the low-density lipoprotein receptor-related protein.45 A stimulatory effect of rhIL-11 on the RAP protein is possible. The most plausible hypothesis based on our results is that rhIL-11 plays a role at the translational level. Indeed there is growing evidence demonstrating the importance of RNA-protein interactions in the regulation of mRNA translation, which results in different amounts of protein produced.46,47 There are a few examples of interleukins acting at the translational and/or post-translational level. IL-6 can inhibit the expression of the syndecan-1 proteoglycan without any effect on the mRNA levels.48 Similarly, IL-10 can modulate intercellular adhesion molecule (ICAM)-1 expression by glial cells by inhibiting the increase in protein expression mediated by a number of other cytokines, but it has no effect on the accompanying increase in mRNA.49 Our results demonstrate that rhIL-11, in addition to its effect on platelet production, can significantly increase FVIII levels in a mouse model of severe vWd and can improve hemostasis in these mice. A significant increase of vWf and FVIII levels can also be obtained in wild type mice and in heterozygote mice representing a model of type 1 vWd. Importantly, this increase can be sustained for long periods of time, in contrast to the effect observed with DDAVP, where patients treated repeatedly may become less responsive, perhaps due to the depletion of the storage granules.50 These results suggest that rhIL-11 might be helpful in alleviating the bleeding symptoms in vWd. Treatment with rhIL-11 might prove to be especially useful in those clinical situations in which vWf and/or FVIII levels must be maintained above baseline for a prolonged period of time. For example, it is possible to imagine a preventive use of rhIL-11 treatment before surgical procedures in order to achieve an optimal hemostatic balance in patients suffering from mild hemophilia A or vWd.
We wish to thank Charlene DeClercq for technical help, Sangeetha Subbarao and Jessie Papalia for mouse husbandry, and Lesley Cowan for assistance with preparation of the manuscript. We also would like to thank Drs Richard Hynes and Bruce Ewenstein for reading the manuscript and for helpful discussions.
Submitted March 31, 2000; accepted September 25, 2000.
Supported by grants R37 HL41002 and PO1 HL56949 (both to D.D.W.) from the National Institutes of Health, Bethesda, MD.
C.V.D. and K.K. contributed equally to this work. Presented in part at the 40th annual meeting of the American Society of Hematology, Miami Beach, FL, December 1998.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Denisa D. Wagner, Center for Blood Research, Harvard Medical School, 800 Huntington Ave, Boston, MA 02115-6399.
1. Sakariassen KS, Bolhuis PA, Sixma JJ. Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-von Willebrand factor bound to the subendothelium. Nature. 1979;279:636-638[CrossRef][Medline] [Order article via Infotrieve].
2.
Turitto VT, Weiss HJ, Zimmerman TS, Sussmann II.
Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion.
Blood.
1985;65:823-831 3. Weiss JH, Sussman II, Hoyer LW. Stabilization of factor VIII in plasma by the von Willebrand factor: studies on post-transfusion and dissociated factor VIII and in patients with von Willebrand's diseases. J Clin Invest. 1977;60:390-404. 4. Ruggeri ZM. von Willebrand factor. J Clin Invest. 1997;99:559-564[Medline] [Order article via Infotrieve]. 5. Scott JP, Montgomery RR. Therapy of von Willebrand disease. Semin Thromb and Hemostasis. 1993;19:37-47[Medline] [Order article via Infotrieve]. 6. Holmberg L, Nilsson IM. Von Willebrand disease. Clin Hematol. 1985;14:461-488.
7.
Rodeghiero F, Castaman G, Dini E.
Epidemiological investigation of the prevalence of von Willebrand's disease.
Blood.
1987;69:454-459 8. Miller CH, Lenzi R, Breen C. Prevalence of von Willebrand's disease among US adults. Blood. 1987;70:377-383. 9. Federici AB. Diagnosis of von Willebrand disease. Haemophilia. 1998;4:654-660[CrossRef][Medline] [Order article via Infotrieve]. 10. Kouides PA. Females with von Willebrand disease: 72 years as the silent majority. Haemophilia. 1998;4:665-676[CrossRef][Medline] [Order article via Infotrieve]. 11. Ginsburg D, Sadler JE. Von Willebrand disease: a database of point mutations, insertions and deletions. Thromb Haemost. 1993;69:177-194[Medline] [Order article via Infotrieve]. 12. Sadler JE. A revised classification of von Willebrand disease. Thromb Haemost. 1994;71:520-525[Medline] [Order article via Infotrieve].
13.
Mannucci PM.
Desmopressin (DDAVP) in the treatment of bleeding disorders: the first 20 years.
Blood.
1997;90:2515-2521 14. Mannucci PM. Treatment of von Willebrand disease. Haemophilia. 1998;4:661-664[CrossRef][Medline] [Order article via Infotrieve]. 15. Menache D, Aronson DL. New treatments of von Willebrand disease: plasma derived von Willebrand factor concentrates. Thromb Haemost. 1997;78:566-570[Medline] [Order article via Infotrieve].
16.
Tepler I, Elias L, Smith JW II, et al.
A randomized placebo-controlled trial of recombinant human interleukin-11 in cancer patients with severe thrombocytopenia due to chemotherapy.
Blood.
1996;87:3607-3614
17.
Gordon MS, McCaskill-Stevens WJ, Battiato LA, et al.
A phase I trial of recombinant interleukin-11 (Neumega rhIL-11 growth factor) in women with breast cancer receiving chemotherapy.
Blood.
1996;87:3615-3624
18.
Issacs C, Robert NJ, Bailey FA, et al.
Randomized placebo controlled study of recombinant human interleukin-11 to prevent chemotherapy-induced thrombocytopenia in patients with breast cancer receiving dose-intensive cyclophosphamide and doxorubicin.
J Clin Oncol.
1997;15:3368-3377 19. Kaye J, Loewy J, Blume J, et al. Recombinant human interleukin eleven (Neumega rhIL-11 growth factor) increases plasma von Willebrand factor and fibrinogen concentrations in normal human subjects [abstract]. Blood. 1994;84:1088a
20.
Denis C, Methia N, Frenette PS, et al.
A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis.
Proc Natl Acad Sci U S A.
1998;95:9524-9529 21. Schwertschlag US, Trepicchio WL, Dykstra KH, Keith JC, Turner KJ, Dorner AJ. Hematopoietic, immunomodulatory and epithelial effects of interleukin-11. Leukemia. 1999;13:1307-1315[CrossRef][Medline] [Order article via Infotrieve]. 22. Goldman SJ. Preclinical biology of interleukin-11: a multifunctional hematopoietic cytokine with potent thrombopoietic activity. Stem Cells. 1995;13:462-471[Abstract]. 23. Kruse N, Pette M, Tokya K, Rieckman P. Quantification of cytokine mRNA expressio |
Top
Abstract
Introduction
)
or 250 µg/kg rhIL-11 (
). At the indicated times, blood was
collected, and platelet counts were performed with a Coulter counter.
We measured vWf concentration using ELISA, and FVIII activity was
determined with a clotting assay. (A) Platelet counts, (B) vWf
concentration, and (C) factor VIII level in placebo- and
rhIL-11-treated mice (at each time point, n 
4).
*P < .05 compared with placebo-treated
animals.
). Organs were then collected, and mRNA was
prepared. The quantity of message for (A) vWf or (B) FVIII was measured
for 125 ng total RNA by the Taqman method. Levels were normalized to
murine GAPDH mRNA levels. No significant differences were found between
placebo or rhIL-11-injected animals (n = 5).
) or 250 µg/kg rhIL-11 (
) for 7 days. An incision (5-mm long by 1-mm deep)
was made on the hind leg with an automatic device, and filter paper was
applied to the wound for 5 seconds every 15 seconds until cessation of
bleeding. The rhIL-11 treatment significantly reduced the skin bleeding
time of the vWf