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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:2110.)
© 2003 American Heart Association, Inc.

Thrombosis

Heparin Inhibition of Endothelial Cell Proliferation and Organization Is Dependent on Molecular Weight

Alok A. Khorana; Abha Sahni; Owen D. Altland; Charles W. Francis

From the James P. Wilmot Cancer Center and Hematology/Oncology Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY.

Correspondence to Charles W. Francis, MD, Hematology/Oncology Unit, Box 610, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642. E-mail charles_francis{at}urmc.rochester.edu

	Abstract

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Objective— Studies have shown improved survival in cancer patients treated with low molecular weight heparins (LMWHs). Tumors depend on an expanding vasculature, and heparins may affect vessel growth and function. We investigated the effect of heparins differing in M_r on selected endothelial cell properties.

Methods and Results— Human umbilical vein endothelial cells were cultured with fibroblast growth factor-2 and heparins differing in M_r. Cell proliferation was assessed by [³H]thymidine incorporation, and vascular organization was assessed by in vitro assays. Maximum inhibition of 94±2% was observed with 6-kDa LMWH, greater than the inhibition seen with unfractionated heparin (58±8%) or 3-kDa LMWH (60±9%, P=0.02 for both). No inhibition of proliferation was observed with heparin tetrasaccharide, octasaccharide, or pentasaccharide (fondaparinux). Three- and 6-kDa fractions decreased endothelial tube formation in Matrigel to 58±15% and 67±9% (P<0.05), respectively, of that with fibroblast growth factor-2, whereas no inhibition was observed with unfractionated heparin, tetrasaccharide, pentasaccharide, or octasaccharide. LMWH (6 kDa) also inhibited vessel formation in a placental explant.

Conclusions— Heparin inhibition of endothelial cell proliferation and organization requires a chain length of >8 saccharide units, with maximal inhibition at M_r of 6 kDa. This M_r dependence differs from that required for anticoagulant activity.

Key Words: low molecular weight heparins • cancer • angiogenesis • endothelium • thrombosis

	Introduction

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Unfractionated heparin is a heterogeneous mixture of polysaccharide molecules with a mean M_r between 12 and 15 kDa containing glycosaminoglycan chains from 200 to 300 saccharide units.¹ Low molecular weight heparins (LMWHs) are fragments of unfractionated heparin produced by controlled enzymatic or chemical depolymerization with lower mean M_r between 3 and 6 kDa and chain lengths of 12 to 18 saccharide units.² These structural differences result in important functional and pharmacokinetic differences, inasmuch as LMWHs have an increased anti-Xa/anti-IIa activity ratio, reduced plasma protein binding, decreased interaction with platelets and platelet factor 4, a prolonged half-life, and increased bioavailability after subcutaneous administration.^2,3 These changes give LMWHs an improved therapeutic profile compared with unfractionated heparin and have led to their increasing use in the treatment of venous and arterial thrombosis.

See page 1954

Individual studies^4,5 and meta-analyses^6,7 have found significant improvements in 3- and 6-month mortality in patients with cancer who were treated with LMWHs compared with unfractionated heparin. This survival advantage was found in subgroups differing in age, sex, and primary site of malignancy and could not be attributed to differences in thrombosis or bleeding complications.⁸ The mechanism of this effect remains unknown but appears to be independent of the anticoagulant action of LMWH and may be related to effects on the vasculature. Prior studies have shown the effects of heparin on angiogenesis in rat mesentery⁹ and cornea¹⁰ models and have suggested a differential effect of fractions varying in M_r without defining a relationship between M_r and specific vascular cell functions.

Tumor growth is dependent on an expanding vascular supply, and the development of metastasis involves the transvascular passage of malignant cells and new blood vessel development at the site of new tumor formation. These are complex processes requiring endothelial and progenitor cell recruitment, proliferation, migration, and organization into new vessels.¹¹ Glycosaminoglycans play a role in endothelial cell function, inasmuch as heparin and heparan sulfate can both modulate the activities of angiogenic growth factors, including fibroblast growth factor (FGF)-2,¹² by facilitating the interaction of FGF-2 with its receptor and promoting receptor activation.^13,14 Of note, specific structural variations in heparin and heparan sulfate can result in differential effects on FGF-2 signaling and downstream effects.^15,16

We hypothesized that the effect of heparins on endothelial cells is dependent on M_r and have evaluated this by using in vitro models of endothelial cell function, including proliferation and organization, with the use of unfractionated heparin, heparin fractions varying widely in M_r, and commercial LMWHs. The results demonstrate a difference between the M_r dependence of the effects of heparin on endothelial cells and on coagulation. Maximum inhibition of endothelial cell proliferation and organization occurred at 6-kDa M_r, with less inhibition observed at both higher and lower M_r fractions.

	Methods

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Cell Culture
Primary endothelial cells were obtained as described previously,¹⁷ seeded on 0.2% (wt/vol) gelatin-coated 25-cm² tissue culture flasks, and cultured in McCoy’s 5A medium (Flow Laboratories) containing 20% FBS and 50 µg/mL endothelial cell growth supplement (ECGS) (Collaborative Research, Inc) until they reached confluence, within 2 or 3 days. The cells were passaged up to 2 times before use and then placed in suspension by trypsinization of monolayers. Cells were suspended by rinsing in Hanks’ balanced salt solution, followed by brief incubation with trypsin-EDTA (GIBCO Life Technologies, Inc). The cells were pelleted by centrifugation for 10 minutes at 2000 rpm and resuspended in McCoy’s 5A medium in the absence of serum. This wash procedure was repeated twice before use in experimental protocols.

[³H]Thymidine Incorporation
Approximately 2x10⁴ endothelial cells suspended in McCoy’s 5A medium supplemented with 20% FBS and 50 µg/mL ECGS were plated in gelatin-coated 12-well, non–tissue culture-treated plates (Becton Dickinson & Co) and allowed to adhere for 6 hours. The medium was then removed, and the cells were washed twice with serum-free McCoy’s 5A medium. Serum-free medium containing 1% Nutridoma (Boehringer-Mannheim Corp), 25 ng/mL human recombinant FGF-2 (R&D Systems, Inc), or 25 ng/mL vascular endothelial growth factor (VEGF¹⁶⁵, R&D Systems, Inc) and 1 µCi/mL [³H]thymidine (New England Nuclear) was then added. To this medium was added 10 µg/mL of various heparins. Tetrasaccharide at 1.2 kDa and octasaccharide at 2.4 kDa (Neoparin Inc) were prepared from unfractionated heparin by controlled deaminative cleavage and subsequent purification. LMWH at 3 kDa, LMWH at 6 kDa, and unfractionated heparin (Sigma) were manufactured from porcine intestinal mucosa. Tinzaparin (DuPont Pharma), enoxaparin (Aventis Pharmaceuticals), dalteparin (Pharmacia and Upjohn Co), and fondaparinux (Sanofi-Synthelabo) were obtained as pharmaceutical products. After incubation at 37°C for 24, 48, or 72 hours, nonadherent cells were removed by washing twice with ice-cold PBS. To each well, 500 µL of 10% ice-cold trichloroacetic acid was added, and precipitates were collected on a filter by using a filtration manifold. Filters were washed twice with ice-cold 5% trichloroacetic acid, followed by 95% ethanol. They were allowed to air-dry and were then suspended in scintillation fluid. Acid-precipitable counts were quantified by using a scintillation counter.

In Vitro Models of Vascular Development
Matrigel Model
Matrigel (BD Biosciences) was thawed on ice at 4°C, and 24-well plates were coated with 300 µL Matrigel per well for 15 minutes at 25°C. Confluent human umbilical vein endothelial cells (HUVECs) were detached with trypsin-EDTA as described above and suspended in McCoy’s 5A medium containing 20% FBS, and 192 µL of the suspension was plated into each well. Heparins were added at a total volume of 8 µL, followed by incubation in 5% CO₂/95% air. After 16 hours, medium was aspirated from each well, and 200 µL Diff-Quik fixative (Dade Behring) was added and incubated for 30 seconds. The fixative was then aspirated, and the cells were stained for 2 minutes with Diff-Quik solution II (Dade Behring) diluted 1:1 with water. Tube structures were observed by using an inverted microscope, photographed, and quantified as (1) the average total length of tube structures per low-power field (LPF) and (2) the number of tube structures per LPF with the use of Image-Pro Plus 3.0 software (Media Cybernetics).

Placental Explant Model
A placental explant model was prepared as previously described.¹⁸ Briefly, superficial vessels were excised from the apical surface of human placentas within 24 hours of elective cesarean births. The vessels were cut into 1- to 2-mm fragments with the use of fine dissecting forceps and placed into Hanks’ balanced salt solution containing 2.5 µg/mL fungizone (GIBCO Life Technologies). Fifteen microliters of thrombin was added to each well of a 48-well culture plate, followed by 0.5 mL of 3 mg/mL fibrinogen per well (Enzyme Research) in medium 199 (GIBCO Life Technologies). One vessel fragment was placed quickly in the center of the well before clot formation. After gel formation, 0.5 mL medium 199 per well supplemented with 20% FBS (Gemini Bioproducts), L-glutamine, gentamicin (GIBCO Life Technologies), and fungizone was added. Vessels were cultured at 37°C in an incubator for 18 days, and the medium was changed every 2 days. New vessel growth was observed microscopically and was quantified by using Image-Pro Plus 3.0 software every second day.

Anticoagulant Activity
Anticoagulant activity of various heparins was determined by using a heparin anti-Xa chromogenic assay. The anticoagulant activity of heparins declined progressively with size from 176 anti-Xa units per milligram for unfractionated heparin, to 85 anti-Xa units per milligram for 6-kDa LMWH, 29 anti-Xa units per milligram for octasaccharide, and 2 anti-Xa units per milligram for tetrasaccharide.

Statistical Analysis
Results are expressed as mean±SE. Statistical significance of differences in means between groups was determined by using a 2-tailed Student t test or ANOVA. A value of P0.05 was considered significant for our analysis. All experiments were repeated at least twice, in duplicate or triplicate.

	Results

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The effect of heparins on endothelial cell proliferation was evaluated as [³H]thymidine incorporation at 48 hours, selected on the basis of previous experience indicating that this was an optimal time to observe inhibitory effects. Compared with medium alone, FGF-2 increased cell proliferation by 60±14% (P=0.02), as expected (Figure 1). The addition of unfractionated heparin, 3-kDa or 6-kDa fractions, inhibited FGF-2–dependent proliferation as observed microscopically, although the cells appeared morphologically normal otherwise. As quantified by [³H]thymidine incorporation, the 6-kDa fraction inhibited proliferation by 94±2%, significantly greater than that with unfractionated heparin (58±8%) or with the 3-kDa fraction (60±9%, P=0.02 for both). The addition of heparin fractions of 1.2 kDa (tetrasaccharide), 2.4 kDa (octasaccharide), and 1.7 kDa (pentasaccharide [fondaparinux]) appeared to have no inhibitory effects microscopically. This was confirmed by using [³H]thymidine uptake, which increased by 61±10% (P=0.04) with octasaccharide, 8±15% (P=0.7) with pentasaccharide, and 38±9% (P=0.14) with tetrasaccharide, compared with culture with FGF-2 alone. Similar results were observed when HUVECs were stimulated with the use of a different angiogenic growth factor, VEGF¹⁶⁵. LMWH at 6 kDa inhibited VEGF¹⁶⁵-stimulated cell proliferation by 61±10% (P<0.01).

View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of various heparins on endothelial cell proliferation in the presence of FGF-2. Endothelial cells were plated on gelatin-coated wells in McCoy’s 5A medium supplemented with 20% FBS and 50 µg/mL ECGS and allowed to adhere for 6 hours. The cells were then washed twice with McCoy’s medium and incubated for 48 hours in serum-free medium containing 1% Nutridoma, 1 µCi/mL of [3H]thymidine with 25 ng/mL FGF-2, and 10 µg/mL of the following heparins: unfractionated heparin (UFH), 6-kDa fraction, 3-kDa fraction, 2.4-kDa octasaccharide, 1.7-kDa pentasaccharide (fondaparinux), and 1.2-kDa tetrasaccharide. Isotope incorporated into DNA was precipitated with trichloroacetic acid, collected by vacuum filtration, and measured by scintillation counting. For each condition, data are shown normalized to cells incubated with FGF-2 with no heparin.

We further characterized the time dependence of inhibition with the 3-kDa and 6-kDa fractions, which showed the greatest activity (please see online Figure I, available at http://atvb.ahajournals.org). The addition of FGF-2 increased growth over medium alone at each time point from 8774±935 cpm at 24 hours to 27 280±5874 cpm at 72 hours. FGF-2–stimulated proliferation was inhibited by the 3-kDa and 6-kDa heparin fractions at each time point, with inhibition by the 6-kDa fraction of 84±4%, 90±3%, and 93±3% at 24, 48, and 72 hours, respectively (P<0.05 for each). The cells remained viable at 72 hours, with a normal microscopic appearance in the presence of the heparin fractions. Inhibition with the 3-kDa fraction was less but significant, with reduction of [³H]thymidine incorporation by 75±2%, 81±1%, and 78±3% compared with FGF-2 at 24, 48, and 72 hours, respectively (P<0.05 for each).

The concentration dependence of FGF-2–stimulated HUVEC proliferation was characterized at concentrations of various heparin fractions from 0.1 to 50 µg/mL (Figure 2). For 6-kDa LMWH, progressive inhibitory activity was seen at higher concentrations, up to 5 µg/mL (P<0.05 for each). Maximum inhibition of proliferation was achieved with 6-kDa LMWH concentrations at 5 µg/mL without an increase in inhibition at higher concentrations. Inhibition was also observed with 3-kDa LWMH and unfractionated heparin, although the degree of inhibition was less than that observed with 6-kDa LMWH. Heparin fractions of 1.2 kDa (tetrasaccharide), 2.4 kDa (octasaccharide), and 1.7 kDa (pentasaccharide) were not inhibitory at any of the concentrations tested (please see online Figure II, available at http://atvb.ahajournals.org).

View larger version (15K): [in this window] [in a new window]   Figure 2. Concentration dependence of heparin inhibition of endothelial cell proliferation. Endothelial cells were plated on gelatin-coated wells in McCoy’s 5A medium supplemented with 20% FBS and 50 µg/mL ECGS and allowed to adhere for 6 hours. The cells were then washed twice with McCoy’s medium and incubated in serum-free medium containing 1% Nutridoma, 1 µCi/mL of [3H]thymidine with 25 ng/mL FGF-2 and with concentrations of 3-kDa (solid diamond), 6-kDa (solid square), and unfractionated (solid triangle) heparins, ranging from 0.1 to 50 µg/mL. After 48 hours of incubation, nonadherent cells were removed, and isotope incorporated into DNA was precipitated with trichloroacetic acid, collected by vacuum filtration, and measured by scintillation counting as cpm per well. For each condition, data are shown normalized to cells incubated with FGF-2 with no heparin.

The effect of heparins on endothelial cell function was also evaluated by culturing the cells in Matrigel, leading to the formation of capillary-like tube structures (Figure 3A through 3F). In the presence of medium alone, there was little cell organization, whereas a complex network developed in the presence of FGF-2. The inclusion of unfractionated heparin in the medium appeared to have no effect on tube length. In contrast, only minimal tube formation was observed visually in the presence of LMWH fractions, and the complex networks observed in the presence of FGF-2 or unfractionated heparin were not observed in any area (Figure 3A through 3F). When analyzed quantitatively, unfractionated heparin had no significant effect on tube formation (Figure 3G). However, compared with FGF-2 alone, both the 6-kDa and 3-kDa LMWH fractions had similar inhibitory activity, decreasing tube length by 67±9% and 58±15%, respectively (P<0.05 for each). Inhibition was also observed with all 3 commercial LMWHs: enoxaparin, 46±3%; tinzaparin, 58±22%; and dalteparin, 68±13% (P<0.05 versus FGF-2 for each). Tetrasaccharides, pentasaccharides, and octasaccharides had no significant inhibitory effects, as observed with cell proliferation. Similar results were obtained when organization was quantified as the average number of endothelial tubes observed per LPF (please see online Figure III, available at http://atvb.ahajournals.org). In the presence of 6-kDa LMWH, 65±11% inhibition was observed, and in the presence of 3-kDa LMWH, 64±10% inhibition was observed (P<0.05 versus FGF-2 for each). Similar amounts of inhibition were seen with the 3 commercial LMWHs: enoxaparin, 52±10%; dalteparin, 61±8%; and tinzaparin, 50±21% (P<0.05 for each). In the presence of the tetrasaccharide, pentasaccharide, octasaccharide, and unfractionated heparin, no significant inhibitory effects were seen. Similar results were observed when the cells were stimulated by using a different angiogenic growth factor, VEGF¹⁶⁵. LMWH at 6 kDa inhibited VEGF¹⁶⁵-stimulated tube formation by 60±9% (P=0.04), whereas no inhibition was observed with unfractionated heparin.

View larger version (62K): [in this window] [in a new window]   Figure 3. Effect of heparins on endothelial tube formation. Twenty-four–well plates were coated with 300 µL Matrigel per well and rested at 25° for 15 minutes. Confluent HUVECs were detached with trypsin-EDTA, suspended in McCoy’s 5A medium supplemented with 20% FBS, glutamine, and penicillin/streptomycin and with 25 ng/mL FGF-2, and plated into each well. Various heparins (10 µg/mL) were added as follows: A, FGF-2 alone; B, unfractionated heparin; C, 6-kDa fraction; D, 3-kDa fraction; E, octasaccharide; and F, tetrasaccharide. The dishes were incubated in a 5% CO2/95% air incubator. After 16 hours, medium was aspirated, the wells were fixed and stained, and tube structures were observed and photographed (A through F). Tube formation was quantified by using image analysis software as total average length of tubelike structures per LPF (G).

The inhibitory effect of 6-kDa LMWH was confirmed by culturing vessel fragments obtained from human placentas in a fibrin clot over 18 days (Figure 4). In the absence of 6-kDa LMWH, initiation of new vessel growth was observed as early as day 2. New vessel formation continued to occur thereafter, reaching a peak on day 8 and persisting up to day 18. In wells containing 6-kDa LMWH, new vessel growth initiation was observed at day 6. Although this initial growth was not significantly different from that observed with medium alone, growth subsequent to day 6 was considerably inhibited. After day 14, nearly 100% inhibition was seen in the presence of 6-kDa LMWH, whereas new vessels continued to be observed in wells without 6-kDa LMWH.

View larger version (15K): [in this window] [in a new window]   Figure 4. Effect of 6-kDa LMWH on new vessel formation in a placental explant model. The rate of growth of microvessels from human placental vessel fragments cultured in a fibrin gel with medium 199 and 20% FBS, for 18 days, in the presence (solid square) or absence (solid diamond) of 6-kDa LMWH is shown. New vessel growth was quantified by image analysis software.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The vascular adaptations required for tumor growth and metastasis are complex. We have chosen to evaluate the specific endothelial cell properties of proliferation and organization into tubular structures. The findings presented demonstrate that heparins have significant effects on these activities that are dependent on M_r. Unfractionated heparin inhibited endothelial cell proliferation but had no effect on endothelial tube formation, whereas heparin fractions with a mean M_r of 3 and 6 kDa had greater inhibitory effects on proliferation and also reduced tube formation, with maximum inhibition observed in the presence of the 6-kDa fraction. No inhibitory effects on either proliferation or tube formation were observed with fractions <3 kDa. The anticoagulant activity of heparin was also related to size, but the M_r dependence of the endothelial and anticoagulant activities differed (Figure 5).

View larger version (20K): [in this window] [in a new window]   Figure 5. Comparison of anti-Xa and antiangiogenic activities of heparins as a function of molecular weight (MW). Anti-Xa activity (dashed line) increases as a function of increasing MW of heparin, with maximal activity seen with 14-kDa unfractionated heparin. In contrast, inhibition of vascular formation (straight line) is greatest between 3- and 6-kDa heparins and less at lower and higher MWs.

Heparin and LMWHs are the most widely used parenteral anticoagulants, and they accelerate the rate of Xa and thrombin inhibition through binding to antithrombin.^2,19 The anticoagulant properties of heparins are related to size and chain length, as reflected by the increased anti-Xa/anti-IIa activity of LMWHs, resulting from loss during depolymerization of the larger molecules, which are needed for anti-IIa activity.² Other functional properties that also depend on chain length include half-life, bioavailability after subcutaneous administration, protein binding, release of tissue factor pathway inhibitor, and interactions with platelet factor 4.^20,21 The mean M_r value of LMWHs in clinical use ranges from 4 to 5.5 kDa, close to that of the 6-kDa fraction, which we found to exert maximum endothelial inhibitory effects.

Previous reports have evaluated the effects of heparins in models of angiogenesis but have not defined specific antiproliferative or antiangiogenic effects on M_r or chain length. Folkman et al²² demonstrated angiogenesis inhibition and tumor regression with unfractionated heparin or a hexasaccharide heparin fragment, but they observed this only in the presence of cortisone. Norrby and Ostergaard⁹ showed that FGF-2–mediated angiogenesis in a rat mesentery model was more effectively suppressed by a 2.6-kDa fraction compared with 4 higher M_r fractions varying in charge density and anticoagulant activity.⁹ More recently, Collen et al²³ evaluated the effect of unfractionated heparin and LMWHs on vessel development in vitro in a fibrin matrix. They demonstrated similar inhibition of endothelial cell proliferation by both unfractionated heparin and LMWH and observed that the formation of tubular structures was inhibited by LMWH but stimulated by unfractionated heparin. They related these effects to alterations in the fibrin matrix structure caused by heparins. However, such effects on fibrin cannot explain the differential effects observed in our experiments in the absence of fibrin.

FGF-2 interacts with both specific tyrosine kinase receptors and with heparan sulfate proteoglycans (HSPGs) as small as disaccharides and trisaccharides,²⁴ which function as coreceptors, promoting receptor binding and activation.¹³ This is consistent with our findings and explains the potentiation of FGF-2–mediated proliferation and endothelial cell organization observed with heparin tetrasaccharides and octasaccharides. Interestingly, Ornitz et al²⁴ observed that small and large heparin fractions could both induce FGF receptor dimerization; however, at higher concentrations, the amount of dimerization remained elevated with the disaccharide or trisaccharide but approached basal levels with hexadecasaccharide. The authors suggested that the larger hexadecasaccharide sterically inhibits receptor dimerization at high ratios of heparins to FGF. This is consistent with the differential effects of tetrasaccharide and octasaccharide compared with the 3- and 6-kDa fractions observed in the present study.

In addition to chain length, specific structural modifications also play an important role in HSPG function. The formation of a ternary complex of FGF-2, heparin, and FGF receptor 1 can be prevented by certain structural changes, such as selective 6-O-desulfation.^16,25 Similarly, specific saccharide sequences within HSPGs play critical roles, influencing which FGF receptor isoform is activated and also acting as positive or negative regulators of FGF signaling.^15,26 Liu et al²⁷ have demonstrated that tumor cell surface heparan sulfate contains both cryptic promoters and inhibitors of growth and metastasis that can be differentially released by specific heparanases, and these influence the activity of FGF-2. Similarly, Zhang et al²⁸ have recently shown that membrane-associated HSPGs were effective promoters of FGF-2–initiated FGF receptor 1 phosphorylation, whereas equal amounts of soluble trypsinized HSPG were not. However, the soluble HSPG could be converted into a potent activator of FGF receptor signaling on further treatment with heparitinase.²⁸ The relation of specific structural modifications in the processing of heparin fractions to inhibitory activity against endothelial cell properties is not known.

The antiproliferative effect on endothelial cells may be relevant in the biology of malignancy and also important therapeutically. Two recent meta-analyses evaluating randomized controlled trials of LMWHs versus unfractionated heparin have found a significant risk reduction in mortality rates in the LMWH arm.^6,7 Because the cytotoxic effects of LMWHs have not been reported, the effects on endothelial cells and vascularity may explain slower tumor growth. Prospective randomized trials are currently under way to evaluate effects on survival. Our results demonstrate critical and variable effects of the heparin chain length on coagulation and endothelial cell proliferation that must be considered in interpreting the results, inasmuch as a variety of antithrombotic agents are now available for use.^29,30

	Acknowledgments

 
Acknowledgments

This work was supported by a grant (HL-30616) from the National Heart, Lung, and Blood Institute, Bethesda, Md. Dr Khorana was supported by the James P. Wilmot Cancer Research Fellowship.

Received July 16, 2003; accepted July 31, 2003.

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