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Am J Physiol Heart Circ Physiol 287: H1207-H1213, 2004. First published May 6, 2004; doi:10.1152/ajpheart.00020.2004
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Intracellular signaling pathways involved in Gas6-Axl-mediated survival of endothelial cells

Ines Hasanbasic,1 Jessica Cuerquis,1 Brian Varnum,2 and Mark D. Blostein1

1Lady Davis Institute for Medical Research and Division of Hematology, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, Quebec, Canada H3T 1A4; and 2Amgen Corporation, Thousand Oaks, California 91320

Submitted 9 January 2004 ; accepted in final form 3 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Gas6 is a {gamma}-carboxylated ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions can rescue endothelial cells from apoptosis, and this study examined the intracellular signaling mechanisms responsible for this phenomenon. Using flow cytometry, we first confirmed that Gas6 can abrogate apoptosis induced by serum starvation of primary cultures of human umbilical vein endothelial cells (HUVECs). This effect is mediated through phosphorylation of the serine-threonine kinase Akt, with maximal phosphorylation observed after 4 h of treatment with 100 ng/ml Gas6. Inhibition of Akt phosphorylation and abrogation of gas6-mediated survival of HUVECs by wortmannin implicated phosphatidylinositol 3-kinase as the mediator of Akt phosphorylation. Dominant negative Akt constructs largely abrogated the protective effect of Gas6 on HUVECs, underscoring the importance of Akt activation in Gas6-mediated survival. Several downstream regulators of this survival pathway were identified in HUVECs, namely, NF-{kappa}B as well as the antiapoptotic and proapoptotic proteins Bcl-2 and caspase 3, respectively. We showed that NF-{kappa}B is phosphorylated early after Gas6 treatment as evidenced by doublet formation on Western blotting. As well, the level of Bcl-2 protein increased, supporting the notion that the Bcl-2 antiapoptotic pathway is stimulated. The levels of expression of the caspase 3 activation products p12 and p20 decreased with Gas6 treatment, consistent with a reduction in proapoptotic caspase 3 activation. Taken together, these experiments provide new information about the mechanism underlying Gas6 protection from apoptosis in primary endothelial cell cultures.

apoptosis

GAS6 IS A LIGAND for the receptor tyrosine kinase Axl (41). It contains four protein domains that comprise, from the NH2 to COOH terminus, a {gamma}-carboxyglutamic acid-containing domain, two epidermal growth factor-like domains, and a COOH-terminal steroid hormone binding globulin-like domain. This structure is homologous to that of protein S, a vitamin K-dependent blood coagulation protein and, like the vitamin K-dependent proteins, contains several {gamma}-carboxyglutamic acid residues at its NH2 terminus. Gas6 is a ligand for the receptor Axl (18, 40), which is a member of a family of receptor tyrosine kinases that include c-mer and rse. Axl was first isolated from a T-cell leukemia cell line in 1991 (35) and is a type I transmembrane protein whose extracellular portion is composed of two immunoglobulin-like domains and two fibronectin type III domains (18, 40).

Gas6-Axl interactions have been examined in a variety of different cell systems. The pleotropic effects of Gas6-Axl interactions in different tissues may be summarized as follows. In vascular smooth muscle, Gas6 was initially purified as a mediator of vascular smooth muscle proliferation (34). Later studies showed Axl upregulation at sites of vascular injury, suggesting a role for this receptor in vascular remodeling (29). In the kidney, Gas6-Axl interactions are important for mesangial cell proliferation (43) and play a role in nephrotoxic glomerular injury (45) as well as diabetic nephropathy (32). Interestingly, one intracellular signaling pathway that mediates mesangial cell proliferation occurs through signal transducer and activator of tranduction 3 (STAT3) activation (44). Other tissues where Gas6 may play a functional role include bone, where Gas6-Axl interactions upregulate osteoclast function (27, 33), the central nervous system, where Gas6 protects neurons from amyloid-induced apoptosis (42), and the eye, where Gas6 mediates outer retinal pigment epithelial function (22, 23). Finally, homozygous null Gas6 mice have a platelet dysfunction that protect mice against lethal intravascular thrombosis (1). The role of Gas6-Axl interactions in endothelial physiology is discussed below.

The sequence and nature of the mitogenic and antiapoptotic events resulting from Gas6-Axl interactions have been most extensively studied in murine NIH-3T3 fibroblasts (19–21). In this system, Gas6-Axl interactions activate Akt through phosphorylation. Akt itself is a serine-threonine kinase that has been shown to be a key intracellular regulator of cellular survival. Its activation by phosphorylation is carried out by phosphatidylinositol 3-kinase (PI3K), a kinase that can be activated by upstream events such as ligand-receptor interactions or through the recruitment of adaptor proteins to activated cell surface receptors. Activation of Akt leads to downstream signaling events, including those associated with the mitochondrial regulation of apoptosis (7).

Recent studies of endothelial cell physiology have demonstrated that the endothelium undergoes apoptosis. Information about mechanisms underlying the regulation of endothelial cell survival is fundamentally important for understanding angiogenesis and vascular remodeling, crucial processes in a wide variety of disease states ranging from atherosclerosis to tumor metastasis. Known inducers of endothelial apoptosis include TNF-{alpha}, oxidized LDL, and reactive oxygen species. On the other hand, VEGF, angiopoietin 1, basic FGF, and insulin have all been shown to have antiapoptotic/survival effects on endothelium (4). Both VEGF- and angiopoietin-mediated protection from apoptosis in endothelial cells have been particularly well studied (15–17, 38). These mediators utilize classical survival pathways such as Akt phosphorylation and NF-{kappa}B activation, suggesting that these pathways are functional in endothelium (16, 38).

The present study concerns the nature of intracellular signaling pathways responsible for Gas6-Axl-mediated protection from apoptosis. Although mouse NIH-3T3 fibroblasts have been used as a model system for studying the antiapoptotic effects of Gas6-Axl interactions, the present experiments address cell signaling events using freshly isolated primary human endothelial cells maintained for limited periods in culture. The results underscore the important antiapoptotic role of Gas6 and demonstrate that this antiapoptotic effect involves "classical" survival pathways including Akt phosphorylation, NF-{kappa}B activation, Bcl-2 stimulation, and, ultimately, caspase 3 inhibition.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Recombinant human Gas6 was produced as previously described (41).

Cells. Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords by collagenase digestion (26) and cultured in complete medium comprising endothelial cell basal medium (EBM-2) supplemented with an endothelial cell Bullet Kit (Cambrex) containing 2% FCS, human endothelial growth factor-2 (EGF-2), human fibroblast growth factor-2, human vascular endothelial growth factor, R3-insulin-like growth factor I, ascorbic acid, hydrocortisone, heparin, gentamicin, and amphotericin B [endothelial cell growth medium (EGM-2)]. Cultures were passaged in Corning tissue culture dishes coated with 0.1% gelatin (Sigma). Cells were grown at 37°C in a humidified atmosphere with 5% CO2. HUVECs used for the experiments described herein were passaged between three and six times. For serum starvation, EGM-2 was replaced by EBM-2 without supplements.

Detection and quantification of apoptosis. For analysis of apoptosis by flow cytometry, 1 x 106 cells were plated in 100-mm tissue culture plates. Upon reaching 70% confluency, cells were placed in serum-free media in the presence or absence of 100 ng/ml recombinant human gas6 and incubated for 24, 48, and 72 h. The cells were then harvested by trypsinization and washed twice with PBS (GIBCO Invitrogen). Apoptosis was quantified by flow cytometry either by costaining harvested HUVECs with FITC-conjugated annexin V and propidium iodide (BD Biosciences) or by staining with propidium iodide only and enumerating the hypodiploid apoptotic cells in the sub-G1 fraction. Cells analyzed by flow cytometry (Becton Dickinson) were quantified using Cell Quest software. For experiments performed with wortmannin, 1 µM wortmannin (Sigma) was present in the medium for 30 min before gas6 treatment and/or serum starvation. For experiments performed with dominant negative Akt constructs, HUVECs were transfected with adenoviral constructs containing dominant negative Akt that were obtained as a gift from Dr. S. Richard at the Lady Davis Institute for Medical Research.

Western blot analysis. HUVECs were grown in 100-mm dishes. After reaching 70% confluency (5 x 106 cells), they were serum starved for 0.5, 1, 4, 24, and 48 h in the presence or absence of 100 ng/ml recombinant human Gas6. HUVECs were trypsinized, pelleted at 300 g, and then lysed by the addition of 150 µl lysis buffer [50 mM NaF, 50 mM Tris·HCl (pH 7.5), 1% Igepal, 0.1 mM EDTA (pH 8.0), 150 mM NaCl, 10 mM NaPO4, 10% glycerol, 1 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride plus Complete Protease Inhibitor Cocktail (Roche)]. Samples comprising equal amounts of total protein were analyzed by 7.5% and/or 12% SDS-PAGE and then transferred to a nitrocellulose membrane (Bio-Rad). Western blot analysis was carried out using the following polyclonal antibodies and dilutions: anti-NF-{kappa}B p65 (1:500), anti-Bcl-2 (1:1,000), and anti-caspase 3 (1:1,000) (Santa Cruz Biotechnology) as well as anti-Akt (1:1,000) and anti-phospho-Akt (1:1,000) (Cell Signaling Technology) rabbit polyclonal antibodies followed by the addition of a goat anti-rabbit peroxidase conjugated secondary antibody (Santa Cruz Biotechnolgy). The blots were developed with an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech) according to the manufacturer's protocol.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The present work examines the intracellular signaling pathways in endothelial cells that are important for Gas6-Axl-mediated protection from apoptosis. Axl is present in freshly isolated HUVECs (cells passaged 3–6 times) as confirmed by Western blot analysis (data not shown). These results confirm previous findings that demonstrate Axl expression by Western blot analysis in HUVECs (36) and by Northern blot analysis as well as Western blot analysis in human pulmonary endothelial cells (24).

Effect of Gas6 on apoptosis induced by serum starvation. When HUVECs were cultured in serum-free medium for 72 h, ~55% of cells underwent apoptosis as detected by annexin V staining. This is apparent from the comparison of the flow cytometric analysis shown in Fig. 1A (complete medium) to that in Fig. 1B (serum-free medium). However, if Gas6 (100 ng/ml) was included in the serum-free medium, only 11.5% of cells underwent apoptosis (Fig. 1C). The time course of apoptosis is shown in Fig. 1D (mean of 4 experiments). These results indicate that serum starvation induces apoptosis in primary HUVECs in culture and that this can be abrogated with the addition of Gas6.



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  		Fig. 1. Gas6 is a survival factor for human umbilical vein endothelial cells (HUVECs). HUVECs were grown in culture as described in MATERIALS AND METHODS. To measure apoptosis, the cells were stained with FITC-conjugated annexin V and propidium iodide, and the percentage of apoptotic cells (annexin V positive; propidium iodide negative) were determined on a Becton Dickinson FACS Analyzer. A: HUVECs in 2% FCS containing supplemental growth factor medium as outlined in MATERIALS AND METHODS. B: HUVECs were serum starved without supplemental growth medium for 72 h before being analyzed by flow cytometry. C: serum-starved HUVECs as described in B but with the addition of 100 ng/ml recombinant human Gas6. The percentage of apoptotic cells is indicated in the bottom right quadrant. The results shown are representative of 4 independent experiments. D: HUVECs were treated for 24, 48, and 72 h in serum-free media (open bars) or serum-free media supplemented with 100 ng/ml recombinant human Gas6 (solid bars). The percentage of apoptotic cells was then analyzed by flow cytometry as described above. Data shown are means ± SE of 4 independent experiments.

 
Role of Akt in Gas6-mediated protection from apoptosis. We then examined the intracellular signaling pathways responsible for this Gas6-mediated protection from apoptosis. Because VEGF and angiopoietin mediate protection of endothelial cells via survival pathways involving Akt activation, (16, 38), the participation of Akt in the signaling pathways activated by Gas6 was examined.

Akt is activated through phosphorylation at Ser473 and Thr308. As shown in Fig. 2A, Akt remained phosphorylated as cells survived in culture but became dephosphorylated as cells underwent apoptosis under serum deprivation. However, in the presence of 100 ng/ml Gas6, Akt remained phosphorylated for up to 72 h, as depicted in Fig. 2B. Gas6 phosphorylation of Akt was dose dependent, with maximal phosphorylation at 100–200 ng/ml Gas6 (Fig. 3). Furthermore, the experiment shown in Fig. 4 indicates that a dominant negative Akt construct resulted in a marked reduction in the protective effect of Gas6 on apoptosis in HUVECs, thus providing clear evidence for a critical role of the PI3K/Akt pathway for Gas6-mediated survival in HUVECs.



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  		Fig. 2. Gas6 phosphorylates Akt in HUVECs during serum starvation. HUVECs were serum starved for the indicated time points in the absence (A) or presence (B) of 100 ng/ml Gas6, after which the whole cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against Akt and phospho-Akt (p-Akt) as described in MATERIALS AND METHODS. The experiment shown is representative of 1 of 3 performed.

 


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  		Fig. 3. Concentration dependence of Gas6-mediated phosphorylation of Akt. HUVECs were serum starved for 4 h in the presence of varying concentrations of Gas6 as indicated. Their lysates were subject to SDS-PAGE followed by Western blotting as described in Fig. 2. The experiment shown is representative of 1 of 3 performed.

 


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  		Fig. 4. Akt mediates the survival effect of Gas6 on HUVECs. HUVECs were serum starved for 48 h in the presence of 100 ng/ml Gas6 and/or transfected with dominant negative Akt (dnAkt). The percentage of cells undergoing apoptosis by sub-G1 analysis was measured by flow cytometry as outlined in MATERIALS AND METHODS. Data shown are means ± SE of 3 independent experiments each normalized to the controls without Gas6.

 
Akt has been shown to be phosphorylated by PI3K (3, 13). To demonstrate whether PI3K phosphorylates and activates Akt in HUVECs, we examined the effect of wortmannin, a specific PI3K inhibitor, on Akt phosphorylation. Figure 5 shows the time course of Akt phosphorylation in the presence and absence of 100 ng/ml Gas6 with (B) and without wortmannin (A). As shown, 1 µM wortmannin largely abrogated Akt phosphorylation induced by Gas6, indicating that Gas6 activates Akt through PI3K. To confirm that the survival effect of Gas6 is dependent on PI3K, apoptosis was measured in the presence of Gas6 alone and in the presence of Gas6 plus wortmannin. As shown in Fig. 5C, a significant Gas6-mediated protection of HUVECs from serum starvation-induced apoptosis was not detected in the presence of 1 µM wortmannin.



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  		Fig. 5. Gas6 stimulates Akt phosphorylation through phosphatidylinositol 3-kinase (PI3K). A: HUVECs were serum starved for the indicated times in the absence and presence of 100 ng/ml Gas6. Cell lysates were then subjected to SDS-PAGE followed by Western blotting as described in Fig. 2. B: HUVECs were incubated as in A but pretreated with wortmannin as described in MATERIALS AND METHODS. The experiment shown is representative of 1 of 3 performed. C: HUVECs were serum starved in the absence (control) or presence of 100 ng/ml Gas6 with or without 1 µM wortmannin for 72 h, and the percentage of cells undergoing apoptosis by sub-G1 analysis was measured by flow cytometry as outlined in MATERIALS AND METHODS. Data shown are means ± SE of 3 independent experiments each normalized to the controls without Gas6.

 
Downstream mediators of Gas6-Axl-mediated survival in endothelial cells. Downstream mediators of cell survival in HUVECs cultured in the presence of Gas6 were then analyzed. The experiments shown in Fig. 6 were designed to examine the participation of NF-{kappa}B, a well-known effector of cell survival that is active when phosphorylated. As shown in Fig. 6A, Gas6 addition to serum-starved cells resulted in the phosphorylation of the p65 subunit of NF-{kappa}B. Phosphorylation is evident by the doublet seen with Gas6 treatment. In the absence of Gas6, NF-{kappa}B remained a singlet (Fig. 6B). This phosphorylation of NF-{kappa}B occurs early and is maintained for up to 48 h. This finding provides evidence for the involvement of NF-{kappa}B in Gas6-mediated endothelial cell survival. To establish more downstream effectors of cell survival induced by Gas6-Axl interactions, two well-described proteins important in apoptosis were examined, namely, Bcl-2 and caspase 3. Bcl-2 is an antiapoptotic protein whose mechanism of action is to prevent cytochrome c release from mitochondria, thereby preventing apoptosis. As shown in Fig. 7A, Bcl-2 protein expression was reduced during apoptosis induced by serum starvation, and, after treatment with Gas6, Bcl-2 protein expression was increased.



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  		Fig. 6. Gas6 activates NF-{kappa}B during endothelial cell survival. HUVECs were serum starved for the indicated time points in the presence (A) and absence (B) of 100 ng/ml Gas6. Lysates were then subjected to SDS-PAGE, followed by Western blotting with antibodies to the p65 subunit of NF-{kappa}B as described in MATERIALS AND METHODS. The experiment shown is representative of 1 of 3 performed.

 


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  		Fig. 7. Gas6 increases Bcl-2 protein levels and reduces caspase 3 activation during protection of endothelial cells from apoptosis. HUVECs were serum starved for 24 h in the presence and absence of 100 ng/ml recombinant human gas6. Lysates were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with antibodies to Bcl-2 (A) or caspase 3 (B) as described in MATERIALS AND METHODS. Also shown are lysates from nonapoptotic, non-serum-starved HUVECs as well as protein loading controls detected with anti-actin antibodies (Santa Cruz Biotechnology). The experiment shown is representative of 1 of 3 performed.

 
In contrast to these findings, caspase 3 activation, a known effector of cell death, was reduced by Gas6 treatment (Fig. 7B). Pro-caspase 3, the zymogen, is activated to caspase 3, which consists of two subunits, p12 and p20. Both of these activation products are increased during apoptosis (2). However, in the presence of 100 ng/ml Gas6, these activation products were significantly reduced. The decrease in levels of p12 and p20 indicates a reduction in caspase 3 activation consistent with Gas6's antiapoptotic effect.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Gas6 is a novel vitamin K-dependent protein that is a ligand for the receptor tyrosine kinase Axl (18, 40, 41). One property of Gas6 is to mediate endothelial cell survival induced by serum starvation or TNF-{alpha} treatment (24, 36). To further our understanding of the role of endothelial cell survival in the etiology of atherosclerosis and in vascular development (4, 14), the experiments described in this paper were aimed to address the nature of the endothelial cell intracellular signaling pathways that are activated by Gas6-Axl interactions. Intracellular signaling pathways mediated by Gas6-Axl interactions have previously been studied in NIH-3T3 fibroblasts (19–21), immortalized vascular smooth muscle cells (31), and mesangial cells (44). The first two cell systems are immortalized cell lines that are unlikely to resemble closely their primary cell counterparts. Mesangial cells, albeit primary cells, are derived from the murine kidney and involve the STAT3 activation pathway (44). The cells used in the present study are primary cells derived from freshly isolated human endothelium and were used within a very short period thereafter, i.e., three to six passages. Accordingly, these cells (HUVECs) represent a cellular phenotype that is more physiologically relevant. In the present study in which apoptosis was induced by serum starvation, we have shown both endothelial cell apoptosis induced by serum starvation and the rescue from apoptosis imparted by Gas6-Axl interactions. The latter is based on the following observations: 1) exogenously added Gas6 abrogated endothelial cell apoptosis; 2) endogenous Axl is expressed in HUVECs; and 3) Gas6-mediated survival of endothelial cells has been shown to be mediated through Axl (24, 36).

The present study shows that Gas6 rescues HUVECs from apoptosis through PI3K activation and subsequent Akt phosphorylation. Involvement of Akt in the signaling pathways of other mediators of endothelial cell survival has been reported. Thus VEGF protects endothelial cells from apoptosis via Akt/PI3K intracellular signaling events (16). The same holds true of insulin, another mediator of endothelial cell survival that prevents TNF-{alpha}-induced apoptosis (25). In the present study, wortmannin inhibited Akt phosphorylation and subsequent Gas6-mediated protection of HUVECs from apoptosis (see Fig. 5), underscoring an important role of PI3K in Akt phosphorylation and Gas6-mediated HUVEC survival (3, 13). This is consistent with other studies examining Akt phosphorylation in endothelial cells. For example, wortmannin largely abrogated Akt phosphorylation in endothelial cells induced by shear stress (10). Furthermore, experiments with dominant negative Akt constructs (see Fig. 4) demonstrate that Akt activation is necessary for mediating the survival effect of Gas6.

Our experiments have identified several downstream signaling proteins involved in Gas6-mediated cell survival, presumably via Akt phosphorylation. Thus, in HUVECs, Gas6-mediated protection from apoptosis resulted in increased levels of Bcl-2 protein expression. The increase in Bcl-2 protein is similar to the protection from apoptosis mediated by VEGF (16). Another potential downstream target of Akt is NF-{kappa}B (37, 39). NF-{kappa}B is known as a ubiquitous regulator of gene expression in both inflammation and cell survival in many different cellular systems (30). It has also been shown to be active in vascular biology (8). For example, NF-{kappa}B activation has been linked to antiapoptosis in endothelial cells, although some mediators, such as TNF-{alpha}, activate antiapoptotic pathways in endothelial cells via Akt that are independent of NF-{kappa}B (28). In the present study, NF-{kappa}B phosphorylation occurs early in Gas6-mediated rescue of HUVECs from apoptosis, consistent with the rapid upregulation of NF-{kappa}B seen in NIH-3T3 fibroblasts (9).

During apoptosis, one of the final biochemical events leading to programmed cell death is the activation of the caspase cascade and generation of caspases from zymogens or procaspases. The resultant caspases are cysteine proteases whose peptide bond cleavage site precedes aspartic acid residues. These proteins are responsible for effecting such biochemical processes as DNA fragmentation, nuclear membrane breakdown, and mitochondrial damage (2). Caspase 3 is one of the final caspases that is activated leading to apoptosis. As shown in Fig. 7B, Gas6 treatment of HUVECs undergoing apoptosis resulted in a reduction in the formation of its active p12 and p20 components, consistent with a reduction in caspase 3 activation. The results are consistent with Gas6's antiapoptotic effect and are consistent with a key role of this protease described in several other cell systems. For example, shear stress promotes endothelial cell survival and does so by reducing caspase 3 activity (11).

The importance of Gas6-Axl interactions in endothelial cell physiology is evidenced in Gas6-mediated protection of HUVECs and human pulmonary artery endothelial cells from both serum starvation and TNF-{alpha}-induced apoptosis (24, 36). The question of whether Gas6-Axl is involved in the protection of endothelial cells from other apoptotic stimuli that are more physiologically relevant is a timely issue. It is notable that protection from serum-starved apoptosis in endothelial cells is effected by acidification (5), and D'Arcangelo et al. (12) have shown that this protection is associated with Gas6-Axl interactions. Studies in our laboratory (preliminary experiments, not shown) suggest that Gas6 protects HUVECs from reactive oxygen species-induced apoptosis. Ongoing studies are underway to determine whether the signaling pathways described in the present study underlie Gas6-mediated protection of HUVECs from apoptotic stimuli other than serum starvation.

In conclusion, this study shows that Gas6-Axl interactions promote endothelial cell survival through Akt phosphorylation, NF-{kappa}B activation, increased Bcl-2 protein expression, and a reduction in caspase 3 activation. A most intriguing feature of Gas6 as a mediator of cell survival is its unusual posttranslational modification, {gamma}-carboxylation. The question of the role this modification plays in Gas6 modulation of cell survival, particularly in endothelial cells, is currently under investigation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a grant-in-aid from The Heart and Stroke Foundation of Canada as well as by an operating grant from the Canadian Institutes of Health Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. D. Blostein, Div. of Hematology, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, Quebec, Canada H3T 1A4 (E-mail: mark.blostein{at}mcgill.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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