The effects of chemotherapeutic agents on the regulation

of thrombin on cell surfaces

Nethnapha Paredes,1

Ling Xu,1

Leslie R. Berry1

and Anthony K. C. Chan1,2,3 1Henderson

Research Centre, 2Department of Paediatrics, McMaster University, Hamilton, and 3Department of Paediatrics,

The Hospital for Sick Children, Toronto, Ontario, Canada

Received 3 July 2002; accepted for publication 6 July 2002

Summary. Thromboembolic disorders are common in can-cer patients. Two major contributing factors are centralvenous catheters for drug delivery and the use of l-apara-ginase, which decreases the plasma antithrombin level, butthe causes of the hypercoagulable state in these patients arenot fully understood. In this study, the T24/83 cell line wasused as a model to investigate the effects of chemothera-peutic agents on cell surface thrombin regulation. Plasmathrombin generation and prothrombin consumption wasincreased in most of the treated cells, particularly vincris-tine- and adriamycin-treated cells (P < 0Æ05), comparedwith controls. However, no free thrombin generation orprothrombin consumption was observed in factor VII (FVII)-depleted plasma. No significant differences in the levels ofthrombin–a2-macroglobulin (IIa–a2M) and thrombin–anti-

thrombin (TAT) were observed between controls and any ofthe treatments, except for vincristine- and adriamycin-treated cells, which showed a significant difference in TATproduction (P < 0Æ05). Also, there was an upregulation intissue factor (TF) mRNA expression in etoposide-, metho-trexate- and vincristine-treated monolayers compared withcontrols, as well as an upregulation in TF protein produc-tion in vincristine-treated cells. The data suggests thatthrombin generation occurs via the extrinsic (TF-depend-ent) coagulation pathway on cell surfaces and that somechemotherapeutic agents are able to upregulate TF mRNAand protein expression in T24/83 cells.

Keywords: chemotherapy, tissue factor, thrombin genera-tion, hypercoagulable state, cancer.

There is considerable evidence that cancer patients have anincreased incidence of thromboembolic complications (Fran-cis et al, 1998). Blood tests for haemostatic markers revealabnormal levels of these markers in as high as 90% ofcancer patients, which could predispose them to a hyper-coagulable state, and an estimated 15% of this populationwill manifest clinically significant thromboembolic compli-cations (Gouin-Thibault & Samama, 1999). One of thepathological consequences of the impaired regulation ofthrombin is deep vein thrombosis (DVT). DVT is a significantproblem in both the adult and paediatric patient populationundergoing chemotherapy treatment for cancer. In theadult population, the most reliable information on theincidence of thromboembolism in patients receiving che-motherapy comes from patients with breast cancer (Levine,1997; Lee & Levine, 1999). It is estimated that thethrombotic risk for stage II breast cancer patients under-

going chemotherapy is 5–10%, with thrombotic eventsoccurring predominately in post-menopausal women (Weisset al, 1981; Levine et al, 1988; von Tempelhoff et al, 1996),and the risk increases up to 17Æ6% in stage IV breast cancerpatients (Goodnough et al, 1984).

Central venous catheters, a known risk factor forthrombosis (Monreal et al, 1996; Bona, 1999), are oftenrequired for the administration of chemotherapeutic agents.Monreal et al (1996) estimated the incidence of DVT incancer patients with central venous lines without prophy-laxis to be 62%. In the paediatric population, Andrew et al(1994a) reported that central venous lines were the leadingcause of DVT (33% of children with central venous lines hadDVT). Another factor that increases the thrombotic risk incancer patients is a low plasma antithrombin concentra-tion, which contributes to the pathogenesis of thrombosis inchildren receiving l-asparaginase (Steinherz et al, 1981;Priest et al, 1982; Andrew et al, 1994b; Mitchell et al,1994a). l-asparaginase depletes l-asparagine, the essentialamino acid in plasma that is required for protein synthesis(Lee & Levine, 1999). l-asparagine is required for anti-thrombin production, and therefore l-asparaginasedecreases the levels of plasma antithrombin to a variable

Correspondence: Anthony K. C. Chan, Henderson Research Centre,

711 Concession St, Hamilton, Ontario, Canada L8V 1C3. E-mail:

achan@thrombosis.hhscr.org

British Journal of Haematology, 2003, 120, 315–324

� 2003 Blackwell Publishing Ltd 315

extent (Lee & Levine, 1999) and thus gives rise to anacquired pre-thrombotic state.

Thromboembolic events in patients with cancer areprobably multifactorial and numerous studies have impli-cated tissue factor (TF) as one of the main contributingfactors (Dvorak, 1987; Kubota et al, 1991; Callander et al,1992; Kakkar et al, 1995a; Shigemori et al, 1998;Wojtukiewicz et al, 1999). TF is an integral membraneglycoprotein that binds to the zymogen, factor VII (FVII), orthe activated form, FVIIa, and plays a pivotal role in theactivation of the extrinsic coagulation pathway (Ruf &Edgington, 1994). The distribution of TF in the suben-dothelium of normal tissue represents a �haemostaticenvelope� that is primed for coagulation in the event thatthe integrity of the vessel wall is compromised (Drake et al,1989). However, studies have shown that TF is alsoexpressed in various types of tumours (Callander et al,1992; Drake et al, 1989), such as non-small-cell lungcarcinoma (Koomagi & Volm, 1998), pancreatic ductaladenocarcinomas (Kakkar et al, 1995b) and colorectalcarcinomas (Shigemori et al, 1998; Seto et al, 2000). Inaddition to tumour-associated TF expression, some cancerpatients also show elevated levels of TF in their plasmasamples (Wada et al, 1994; Asakura et al, 1995; Kakkaret al, 1995a; Takahashi et al, 1995).

Many cancer patients must undergo chemotherapytreatment and chemotherapeutic agents can perturb thehaemostatic environment by altering the balance of proco-agulant and anticoagulant proteins in the plasma (Edwardset al, 1990; Gabazza et al, 1994; Lee & Levine, 1999).Because these agents are cytotoxic, endothelial cell injurycan also occur after exposure (Bertomeu et al, 1990). Littleis known about the effects of chemotherapeutic agents onthrombin regulation on cell surfaces. We used the T24/83cell line, which constitutively overexpresses functional TFon the cell surface (Lopez-Pedrera et al, 1997), as an in vitrocell model to explore the effects of chemotherapeutic agentson the regulation of thrombin generation. In this study, wereport that some chemotherapeutic agents increased throm-bin generation through the extrinsic (TF-dependent) coagu-lation pathway on T24/83 cell surfaces and that thrombininhibitors may not be adequate to neutralize the thrombinproduced by the cells in response to exposure to anti-neoplastic agents. Some chemotherapeutic agents not onlyaffect thrombin generation on the cell surfaces but prelim-inary data show that some chemotherapeutic agents areable to upregulate TF mRNA expression and subsequent TFprotein production in these cells. Consequently, theincreased cell surface thrombin generation, in cells exposedto chemotherapeutic agents, may contribute to the throm-boembolic disorders observed in some cancer patientsundergoing chemotherapy.

MATERIALS AND METHODS

Materials. Medium 199, fetal bovine serum (FBS) andpenicillin–streptomycin were purchased from Gibco BRL(Burlington, Ontario, Canada). The chemotherapeuticagents etoposide, vincristine, adriamycin, methotrexate,

cytosine arabinoside, l-asparaginase and hydrocortisonesodium succinate, as well as standard heparin, diethylpyrocarbonate (DEPC) and alkaline phosphatase-conjugateddonkey anti-sheep IgG, were all purchased from Sigma(Mississauga, Ontario, Canada). The human coagulationfactors FVIIa and FX were both purchased from EnzymeResearch Laboratories (South Bend, IN, USA). FVII-depletedplasma was obtained from Instrumentation Laboratory(Milan, Italy), Arvin from Connaught Laboratories(Toronto, Ontario, Canada), S-2238 and S-2765 substratesfrom KabiVitum (Stockholm, Sweden), purified thrombinfrom Enzyme Research Laboratories, trypsin from Gibco BRL(Burlington, Ontario, Canada), Trypan blue stain fromGibco BRL, anti-thrombin from Bayer (Etobicoke, Ontario,Canada), purified sheep anti-human TF IgG from AffinityBiologicals (Hamilton, Ontario, Canada) and cDNA frag-ments encoding for human TF (1Æ05 kb NarI–HindIIIfragment) was purchased from American Type CultureCollection (ATCC) (Rockville, MD, USA).

Cell culture. The human transitional bladder carcinomacell line, T24/83, was purchased from ATCC (Rockville, MD,USA). T24/83 cells constitutively overexpress TF on thesurface (Lopez-Pedrera et al, 1997), making this cell line auseful in vitro model to study the effects of chemotherapeuticagents on thrombin generation. These cells were cultured inmedium 199 supplemented with 10% FBS and 1% penicil-lin–streptomycin in Falcon 24-well tissue culture plates(Becton Dickinson, Franklin Lake, NJ, USA) at 37�C in ahumidified, 5% CO2 atmosphere. Cells were grown toconfluence, followed by exposure, under culture conditions,to either supplemented media (controls) or supplementedmedia containing one of the seven chemotherapeutic agents(etoposide, vincristine, adriamycin, methotrexate, cytosinearabinoside, l-asparaginase and hydrocortisone sodiumsuccinate) at a concentration of 10 lg/ml for 20 h. In thecase of l-asparaginase, 10 U/ml concentrations were used.After cell culture with media ± chemotherapeutic agents,cells in some wells were incubated with trypsin for 10 min,and the released cells were mixed with diluted Trypan blue.The percentage of cells that excluded Trypan blue stain(viable cells) was determined by microscopic observation.

Thrombin generation. Thrombin generation studies werecarried out using either normal adult pooled plasma or FVII-depleted plasma. Normal human adult plasmas wereobtained by taking whole blood (nine volumes) into 3Æ2%Na3 citrate (one volume) and were then centrifuged for20 min at 3000 g at 4�C to obtain plasma supernatant. Theresultant plasmas from individual adults were then pooledand kept frozen at )60�C until assayed. FVII-depletedplasma was used in some experiments to determine ifthrombin generation on cell surfaces was TF dependent, asTF must bind to FVII/FVIIa to be activated, and the resultswere compared with experiments performed with normaladult pooled plasma. Five sets of cell cultures (n ¼ 5) wereperformed for each chemotherapeutic agent, except forl-asparaginase (n ¼ 4).

The method used for quantifying thrombin generationhas been reported previously (O’Brodovich et al, 1991).Briefly, the non-treated and treated confluent T24/83

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monolayers in the 24-well plates were maintained on ametal block at 37�C on a Thermolyne Dri-bath. After theremoval of media from the wells, the monolayers werewashed twice with 1Æ0 ml of acetate-barbital-saline (ABS)buffer, pH 7Æ4 (0Æ036 mmol/l Na acetate, 0Æ036 mmol/l Nabarbital and 0Æ145 mmol/l NaCl). The monolayers werethen incubated for 3 min with 100 ll of ABS buf-fer + 200 ll of defibrinated plasma (prepared by reactingplasma for 10 min at 37�C with 0Æ18 U/ml Arvin andwinding out the fibrin clot, followed by a 10-min incubationon ice and winding out any further clot formed). After3 min, 100 ll of 0Æ04 mmol/l CaCl2 in ABS buffer wasadded and the clock was started. Periodically, a 25-llaliquot was removed from the reaction mixture on thesurface of the cells and mixed with 475 ll of 0Æ005 mmol/lNa2 ethylenediamine tetra-acetic acid (EDTA) on ice.Immediately, a 25-ll aliquot of each time sample-Na2

EDTA mixture was added to 775 ll of 0Æ00016 mmol/lS-2238 substrate in ABS buffer and maintained at 37�C for10 min. Amidolysis of S-2238 was then stopped by theaddition of 200 ll of 50% acetic acid. The absorbance at405 nm was measured using a spectrophotometer. Purifiedthrombin in ABS buffer plus EDTA was reacted in a range ofconcentrations with S-2238 in the same way as describedabove and terminated in each case with 50% acetic acid.Reaction of substrate with thrombin follows a linearrelationship with respect to enzyme concentration, andcalculations from standard curves showed that an absorb-ance at 405 nm of 1Æ00 was obtained with 1Æ123 nmol/lthrombin in the final acid-neutralized mixture. This refer-ence value for activity against S-2238 was used to calculatethe concentration of total thrombin generated in theplasmas.

Inhibitor complex formation, free thrombin generation, andprothrombin consumption. As thrombin bound to a2-macro-globulin (a2M) still has activity against small substrates(Berry et al, 1991), the component of total thrombinactivity due to IIa)a2M was measured as describedpreviously (Xu et al, 1995). The IIa)a2M generated wasdetermined using the same method as that for totalthrombin except that, at each time-point, 50 ll of thereaction mixture was incubated for 1 min on ice with7 ll of 0Æ15 mmol/l NaCl containing 0Æ5 U of standardheparin and 0Æ042 U of antithrombin, followed by mixing25 ll of the incubate with 475 ll of 0Æ005 mmol/lNa2 EDTA and proceeding as described previously. Sub-traction of the concentration of IIa)a2M at each time-point from the total thrombin gave the amount of freethrombin present. This method is valid because thrombincomplexed with a2M is bound by a stable covalentlinkage and is inaccessible to large-molecular-weightsubstrates (Berry et al, 1991). Free thrombin values wereplotted against time and the area of under the curvecalculated as nmol/l · min.

Prothrombin, thrombin–antithrombin (TAT), and throm-bin–heparin cofactor II (IIa–HCII) inhibitor complexes weremeasured using commercially available enzyme-linkedimmunosorbent assay (ELISA) kits (Affinity Biologicals,Hamilton, Ontario, Canada).

Assessment of TF activity. The presence of active TF wasdetermined by measurement of FXa generation from mix-tures of FVIIa + FX incubated on the cell surface accordingto a previously published method (Nishibe et al, 2001).Briefly, cells in 24-well plate wells were washed twice with1 ml of (0Æ05 mmol/l Tris-HCl, 0Æ02 mmol/l NaCl,0Æ0027 mmol/l KCl, 3 mg bovine albumin/ml pH 7Æ4)buffer and then incubated with 300 ll of 5 nmol/l FVIIa,150 nmol/l FX, 0Æ005 mmol/l CaCl2 in buffer on a Dri-bathat 37�C. After 30 min, 250 ll of supernatant in the wellwas mixed with 25 ll of 0Æ2 lmol/l S-2765 substrate. Afterincubation of supernatant with substrate for 3 min, thereaction was stopped by addition of 20 ll of 50% acetic acidand the absorbance at 405 nm was determined. FXaactivity was calculated as DA405/min.

Northern blot analysis. Total RNA was isolated fromconfluent T24/83 cell monolayers, cultured either undercontrol conditions or exposed to one of the seven chemo-therapeutic agents, using the RNeasy Mini kit (Qiagen,Mississauga, Ontario, Canada). The quantity and purity ofthe isolated RNA was assessed by A260 and A280 absor-bances. Samples with A260/A280 ratios above 1Æ6 werefrozen at )70�C until assayed.

RNA levels in the samples were analysed by Northernblotting. Briefly, 10 lg of total RNA per lane was fraction-ated by electrophoresis on a 2Æ2 mmol/l formaldehyde/1Æ2%agarose gel and transferred overnight onto a Zeta-Probe GTblotting membrane (Bio-Rad, Hercules, CA, USA) in 10·saline sodium citrate (SSC). An ultraviolet cross-linker(Stratagene, La Jolla, CA, USA) was used to cross-link theRNA to the membrane. cDNA fragments encoding forhuman TF (1Æ05 kb NarI–HindIII fragment) were radiola-belled with a-32P-dCTP (NEN Life Sciences Products, Boston,MA, USA) using the Random Primed DNA Labeling Kit(Boehringer Mannheim, Laval, Quebec, Canada). Themembranes were then hybridized with the radiolabelledprobes in a solution containing final concentrations of 40%formamide, 5% sodium dodecyl sulphate (SDS), 0Æ12 mmol/lNa2HPO4, 0Æ025 mmol/l NaCl, and 0Æ001 mmol/l EDTA inDEPC-treated water overnight at 42�C. After washing offthe unbound probes, the membranes were exposed to KodakX-Omat AR film (Mandel Scientific, Guelph, Ontario,Canada) and subject to autoradiography. A Storm 860PhosphorImager and imagequant image analysis software(Molecular Dynamics, Sunnyvale, CA, USA) was used toquantify the differences in gene expression. Human glycer-aldehyde 3-phosphate dehydrogenase (GAPDH) was used tonormalize the total RNA in each lane (loading control).

TF protein expression. T24/83 confluent cell monolayerswere exposed to either culture media (controls) or mediacontaining 10 lg/ml of vincristine for 20 h. Vincristine waschosen because it elicited one of the greatest thrombingenerations in these cells. Cell-associated TF was measuredby Western blot according to previously established proce-dures (Towbin & Gordon, 1984). Briefly, the monolayerswere lysed in 0Æ1% SDS to extract the protein and thensolubilized in SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The samples were separated on a 7Æ5%SDS-polyacrylamide gel under reducing conditions and

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transferred onto an Immobilon membrane (Bio-Rad,Hercules, CA, USA). The membrane was then probed forTF using purified sheep anti-human TF IgG followed byalkaline phosphatase-conjugated donkey anti-sheep IgG.The membranes were developed using the Renaissancechemiluminescence reagent kit (Mandel Scientific, Guelph,Ontario, Canada).

Statistical analysis. Differences between controls andtreated groups were determined by analysis of variance(anova) followed by Tukey’s pairwise comparison. A valueof P < 0Æ05 was considered significant. All values areexpressed as mean ± SEM.

RESULTS

Cell culture of T24/83 cells was carried out under physio-logical conditions in nutrient medium with or without thepresence of chemotherapeutic agents. Determination of cellviability (as measured by Trypan blue exclusion) inchemotherapeutic drug-treated cultures gave values of91%, 87%, 99%, 97%, 97%, 97% and 98% for etoposide,vincristine, adriamycin, methotrexate, cytosine arabinoside,l-asparaginase and hydrocortisone sodium succinaterespectively. These results compared with 97% viability foruntreated cells.

As thrombin plays a major role in coagulation, weexamined the capacity of T24/83 cell surfaces to generatethrombin in response to exposure to chemotherapeuticagents. Free thrombin generation, over the time-course of30 min, was significantly increased for all T24/83 mono-layers treated for 20 h with the various chemotherapeuticagents compared with controls (P < 0Æ05), except foretoposide- and methotrexate-treated cells (Fig 1). Therewere no significant differences in the starting concentrationof free thrombin between controls and all treated groups attimes < 1 min after plasma recalcification (P > 0Æ05).However, at the peak of free thrombin production, cellstreated with the chemotherapeutic agents showed elevatedlevels compared with control cells, but the concentrationsultimately decreased to similar levels at the end of 30 min.Areas under the thrombin generation time-course curveswere calculated and values confirmed the differences forpeak-free thrombin found between chemotherapeutic agent-treated cells and non-treated controls (Table I).

Prothrombin consumption and production of thrombin-inhibitor complexes (IIa–a2M, TAT, and IIa–HCII) duringthrombin generation were investigated to determine if theincrease in free thrombin generation was due to an increasein thrombin production or a decrease in thrombin inhibi-tion. Prothrombin consumption was greatest in the first10 min, which corresponded to the highest amount of freethrombin generated within that time (thrombin generationpeaking at 4 min) for both the control and treated groups.However, over the initial 4 min, the amount of prothrombinconsumed was not significantly increased for cells exposedto the chemotherapeutic agents, except for vincristine- andadriamycin-treated cells (P < 0Æ05), compared with controls(Fig 2). The significant increase in prothrombin consump-tion for vincristine- and adriamycin-treated groups correla-

ted with their significantly increased free thrombingeneration.

A dose–response study was carried out by measuring theeffect on plasma thrombin generation of pretreating cells

Fig 1. Free thrombin generation on the surface of T24/83 cells.

Confluent T24/83 monolayers were exposed to various chemo-

therapeutic agents for 20 h and thrombin generation assays were

performed as described in Materials and methods. Levels of free

thrombin (IIa) generation were calculated by subtracting the con-

centration of IIa–a2M at each time-point from the amount of total

thrombin. Values are expressed as mean ± SEM in five independent

experiments performed for control, and for each chemotherapeutic

agent-treated group, except for l-asparaginase-treated group

(n ¼ 4). *P < 0Æ05, significantly different from control cultures.

Table I. Area under the curve values for thrombin generation

on T24/83 cells.*

Chemotherapeutic treatment

Area under the curve

(nmol/l · min)

Controls 1114 ± 238

Etoposide 2346 ± 438

Vincristine 3453 ± 547

Adriamycin 3962 ± 409

Methotrexate 2580 ± 563

Cytosine arabinoside 4807 ± 1523

l-asparaginase 3955 ± 575

Hydrocortisone sodium succinate 3727 ± 607

*Area under the curve values are for plasma thrombin

generation time-courses on T24/83 cell surfaces exposed for

20 h to media containing varying concentrations of chemo-

therapeutic agents (results for control incubations with media

alone (no chemotherapeutic agent) are shown for compar-

ison). Values are mean ± SEM (n ‡ 5).

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with varying concentrations of vincristine (one of the morepotent agents). Peak thrombin generation and total throm-bin potential (area under thrombin generation time-coursecurve) were reduced on cells that had been treated withreduced concentrations of vincristine (Table II). Thus, thelinkage of the presence of drug with procoagulant activitywas verified. Further tests showed a trend for slightlydecreased cell viability when cells were incubated withincreased concentrations of vincristine (Table II), however,the differences were not statistically significant.

To further elucidate the pathway of thrombin generationin T24/83 cells exposed to chemotherapeutic agents, FVII-depleted plasma was used in thrombin generation andprothrombin consumption experiments, instead of normal

adult pooled plasma. No thrombin generation or prothrom-bin consumption was observed with controls and chemo-therapeutic agent-treated cells when normal adult pooledplasma was replaced by FVII-depleted plasma (data notshown). It was further confirmed that the increasedthrombin generation with chemotherapeutic agent pre-treatment was due to increased TF activity by measuringthe generation of FXa from incubations of FVIIa + FX onT24/83 surfaces. Measurements showed that chromo-genic substrate hydrolysis due to generated FXa was0Æ128 ± 0Æ002 DA405/min/well for vincristine-treated cells(10 lg/ml) compared with 0Æ049 ± 0Æ003 DA405/min/wellfor non-treated controls.

Formation of IIa–a2M, TAT and IIa–HCII complexes wereinvestigated to determine if there were significant differencesin thrombin inactivation between untreated and treatedcells. There was a trend toward increased IIa–a2M produc-tion, but there were no significant differences in the totalamount of IIa–a2M produced between treated and non-treated groups during thrombin generation on the cellsurfaces (P > 0Æ05, Table III). Although TAT productionfollowed a similar trend to IIa–a2M production, significantincreases in TAT formation was found only in vincristine-and adriamycin-treated cells compared with controls(P < 0Æ05, Table III), which is consistent with the signifi-cant increases observed in thrombin generation andprothrombin consumption experiments where monolayerswere exposed to either vincristine or adriamycin. Themajority of the thrombin-inhibitor complexes formed duringthrombin generation consist mostly of TAT and IIa–a2M,but a small proportion (�3%) of this total is IIa–HCII(Table III).

To assess the effects of chemotherapeutic agents beyondcell surface thrombin generation and inhibition, we lookedat TF mRNA expression. Cells treated for 20 h withetoposide, methotrexate and vincristine showed a 7Æ3-,4Æ5- and 3Æ4-fold increase in TF mRNA expression,respectively, compared with controls. The increase in TFmRNA expression correlated with the increase in thrombingeneration for vincristine-treated cells suggesting that, atleast for vincristine, the effects of thrombin regulation is notonly on the cell surface but also at the molecular levelwhere it is able to upregulate TF mRNA.

Fig 2. Prothrombin consumption on the surface of T24/83 cells

exposed for 20 h to the various chemotherapeutic agents during

thrombin generation. Subsamples were taken during the thrombin

generation assay and prothrombin levels were determined by

enzyme-linked immunosorbence assay (ELISA). Results are

expressed as mean ± SEM (n ¼ 5, except for l-asparaginase where

n ¼ 4). *P < 0Æ05, significantly different from control cultures.

Table II. Effect of vincristine concentration on thrombin generation.*

Vincristine concentration

(lg/ml)

Peak-free thrombin

concentration (nmol/l)

Area under the curve

(nmol/l · min)

Cell viability

(% of cells, excluding

Trypan blue)

10Æ0 352 ± 15 3636 ± 206 86 ± 8

3Æ3 311 ± 2 2906 ± 78 95 ± 2

1Æ0 239 ± 12 2503 ± 282 91 ± 4

0Æ0 78 ± 3 799 ± 108 98 ± 1

*Peak-free thrombin concentration and area under the curve values are for thrombin generation on T24/83

cell surfaces exposed for 20 h to media containing varying concentrations of vincristine (results for incubations

with media alone (no chemotherapeutic agent) are shown for comparison). Values are mean ± SEM (n ‡ 3).

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Vincristine-treated cells showed one of the most signifi-cant differences in free thrombin generation and prothrom-bin consumption compared with untreated cells. There wasalso an upregulation of TF mRNA expression in thistreatment group. Thus, we wanted to determine if therewas a corresponding upregulation in TF protein production,by Western blot analysis of total cellular TF proteinexpression, in cells treated with vincristine. Results indica-ted that TF protein expression in vincristine-treated cellswas increased compared with that of non-treated cells(Fig 3). TF protein expression in other treated cells was alsoupregulated compared with controls, but only cells treatedwith vincristine showed a multifold increase compared withthe untreated group.

DISCUSSION

Cancer patients experience a higher incidence of thrombo-embolic complications, commonly know as Trousseausyndrome (Trousseau, 1865). Although the association iswell documented, determining the aetiology of increasedthrombotic risk in these patients is difficult. The pathogen-esis of cancer-associated thrombosis is complicated and mayinvolve more than one mechanism, such as the activation ofthe coagulation and fibrinolytic systems, vascular endothe-lium perturbation, and the activation of platelets andmonocytes (Bertomeu et al, 1990; Gabazza et al, 1994;Monreal et al, 1996). Other confounding factors includeinfection, type and stage of cancer, surgery, prolonged bedrest, age of the patient, use of central venous catheters, andthe type of chemotherapeutic regimen used to treat thecancer (Lee & Levine, 1999).

Thromboembolic events in cancer patients are probablymultifactorial and complicated by the use of anti-neoplasticdrugs in cancer treatment. Anti-cancer drugs can beharmful to endothelial cells by exposing TF in thesubendothelial matrix (Nicholson & Custead, 1985) orinducing the expression of adhesion molecules on endothe-

lial cell surfaces increasing the reactivity of these cells toplatelets (Bertomeu et al, 1990), potentially increasing therisk of coagulation. However, the effects of chemotherapeu-tic agents on cell surface thrombin regulation are not wellknown. In this study, we demonstrated that when confluent

Table III. Thrombin-inhibitor complexes*.

Treatment TAT (nmol/l) IIa–a2M (nmol/l) IIa–HCII (nmol/l)

Controls 378Æ8 ± 39Æ7 477Æ0 ± 23Æ9 13Æ8 ± 0Æ1Etoposide 422Æ6 ± 52Æ9 465Æ3 ± 14Æ7 14Æ1 ± 0Æ2Vincristine 461Æ3 ± 74Æ8 467Æ2 ± 24Æ5 13Æ7 ± 0Æ2Adriamycin 459Æ0 ± 61Æ1 453Æ1 ± 19Æ3 13Æ7 ± 0Æ2Methotrexate 422Æ9 ± 61Æ1 472Æ1 ± 25Æ1 13Æ2 ± 0Æ3Cytosine arabinoside 407Æ7 ± 52Æ9 461Æ6 ± 18Æ5 13Æ0 ± 0Æ2l-asparaginase 376Æ8 ± 51Æ2 499Æ1 ± 64Æ4 12Æ5 ± 0Æ5Hydrocortisone sodium succinate 380Æ3 ± 41Æ6 441Æ4 ± 47Æ2 12Æ8 ± 0Æ2

*Thrombin–inhibitor complexes formed during thrombin generation on T24/83 cell surfaces exposed

for 20 h to media or media containing one of the seven chemotherapeutic agents. Data shown represent

the final end-point concentration in the sample after equilibrium was reached. Thrombin–anti-thrombin

(TAT) and thrombin–heparin cofactor II (IIa–HCII) levels were measured by enzyme-linked immuno-

sorbence assay (ELISA), and IIa–a2M levels were determined by chromogenic substrate, after neutral-

ization of free thrombin. Results are expressed as mean ± SEM (n ¼ 5, except l-aparaginase where

n ¼ 4).

1 2 3

Fig 3. Western blot analysis of total tissue factor (TF) protein

expression in T24/83 cells. The confluent cell monolayers were

exposed to culture media or media containing 10 lg/ml of vincr-

istine for 20 h. Extracts of total cellular proteins were separated by

SDS-PAGE, transferred onto a membrane and then incubated with

purified sheep anti-human TF IgG, followed by an incubation with

alkaline phosphatase-conjugated donkey anti-sheep IgG and

developed by a chemiluminescent as described in Materials and

methods. Lane 1, molecular weight markers, from top to bottom: 98,

64, 50, 36, 30 kDa; lane 2, cells treated with culture media; lane 3,

cells treated with culture media containing 10 lg/ml of vincristine.

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T24/83 cells were exposed to chemotherapeutic agents,thrombin generation was significantly increased in cellstreated with the anti-cancer drugs compared with controls,except for etoposide- and methotrexate-treated cells. Cyto-sine arabinoside, adriamycin and vincristine had thegreatest effect in eliciting thrombin generation. Otherstudies have shown that coagulation can be directlyactivated by neoplastic cells, which could result in increasedthrombin generation (Fischer et al, 1995; Rao & Pendurthi,1998) or it can be indirectly activated by microvesicle-shedding tumour cells, which can then stimulate mono-nuclear cells to produce various procoagulants such as TFor factor X activators (Bastida et al, 1984; Silberberg et al,1989; Kubota et al, 1991; Rao, 1992; Piccioli et al, 1996).Therefore, perturbing tumour-associated cells (which mayalready express TF or TF-like procoagulant activity) withchemotherapeutic agents may induce these cells to enhancetheir procoagulant state. General tests for cell viabilityshowed no significant relationship to either drug treatmentor increase in thrombin-generating activity.

The choice of concentrations for study of chemothera-peutic agents arose from the high plasma levels that wouldbe expected for these agents, given the dosages administeredduring clinical treatment. Plasma concentrations over timedepend on the method of administration (i.e. intravenousbolus, intravenous infusion, etc.) and dosage. Significantranges of plasma concentrations have been reported forapplication of the different drugs. Typical long-term plasmaconcentrations that have been observed were: 0Æ35 lg/ml(Krishna et al, 2001) and 0Æ9 lg/ml (Mendelsohn et al,1998) estimated for vincristine, 11–24 lg/ml for methat-rexate (Dukic et al, 2000), 2Æ4–4Æ9 lg/ml for cytosinearabinoside (Avramis et al, 1998), 50–100 lg/ml estimatedpeak value for bolus injection of etoposide (Mendelsohnet al, 1998), and > 100 lg/ml estimated peak value forbolus adriamycin (Kim et al, 2000). Thus, the 10 lg/mlconcentrations used in our incubations are well within therange of concentrations reported in vivo. All the agents wereused at similar concentrations (except for asparaginase thathad a significantly different molecular weight) to enable anequal comparison for potency.

We have shown that T24/83 cells increase thrombingeneration on their cell surfaces in response to certainchemotherapeutic agents. This parallels the increase inhaemostatic activation markers found in the plasmas ofsome cancer patients undergoing chemotherapy. Routineblood tests on cancer patients reveal that up to 90% haveabnormal levels of haemostatic markers (clotting factors V,VII, IX and XI) and approximately 15% of these patients willexhibit clinically significant symptoms such as venousthromboembolic episodes, post-operative venous thrombosisand disseminated intravascular coagulation (Gouin-Thiba-ult & Samama, 1999). Constantini et al (1998) reportedelevated plasma levels of FVIIa, as well as increasedthrombin generation, in patients with various types ofcancer compared with healthy controls. Gabazza et al(1994) also reported higher levels of haemostatic activationmarkers and a lower fibrinolytic activity in the plasmas of25 lung cancer patients during multidrug combination

chemotherapy compared with pretreatment values. Giventhe increased thrombin generation in the presence of someof the chemotherapeutic agents, we also looked at pro-thrombin consumption and thrombin-inhibitor complexformation. The increase in thrombin generation seen inmost of the chemotherapeutic agent-treated cells corres-ponded to the amount of prothrombin consumed. Therewas a trend towards increased prothrombin consumption,but only vincristine- and adriamycin-treated cells showedsignificant differences compared with non-treated cells.Although there was a trend towards increased thrombin-inhibitor complex formation (mainly IIa–a2M and TAT),there were no significant differences between treated andnon-treated cells except for TAT production in the cellstreated with either vincristine or adriamycin, which showeda significant increase over controls. As antithrombin is themost efficient inhibitor of thrombin (Downing et al, 1978),the higher amounts of free thrombin generated in thevincristine- and adriamycin-treated cells were probablyresponsible for the increase in TAT production. There mayalso be some glycosaminoglycans present on these cellsurfaces that could facilitate the inactivation of thrombin byantithrombin. This suggests that elevated levels of freethrombin generated on the surface of cells treated with thechemotherapeutic drugs, are most likely to be due to theconversion of prothrombin to thrombin and not as aconsequence of decreased thrombin inhibition, as the levelsof thrombin-inhibitor complex formation in the groupstreated with the anti-cancer drugs were relatively similar tothe control groups, with the exception of vincristine- andadriamycin-treated monolayers, which showed a significantincrease compared with controls.

To determine the pathway of thrombin generation on thesurfaces of T24/83 cells, both thrombin generation andprothrombin consumption assays were performed on sam-ples from similar experiments carried out in the presence ofFVII-depleted plasma instead of normal adult pooled plasma.No free thrombin generation or prothrombin consumptionwas observed in any of the treated cells when FVII-depletedplasma was used in the experiments. These results indicatedthat TF is involved in thrombin generation as TF requiresFVII or FVIIa to be catalytically active (Ruf & Edgington,1994) and that the regulation of thrombin occurs via theextrinsic coagulation pathway. This is in agreement withthe most currently accepted view that cancer-associatedcoagulation is initiated through the extrinsic coagulationpathway, where TF plays a major role in the coagulationcascade. Furthermore, TF activity (measured as FXageneration from cell surface incubations of FVIIa + FX)was increased on cells treated with vincristine comparedwith that on non-treated control cells. Studies have shownthat TF is expressed by tumour cells (Callander et al, 1992;Sturm et al, 1992; Contrino et al, 1996), as well as tumour-associated macrophage and monocytes (Edwards et al,1981; Lorenzet et al, 1983). Plasmas of cancer patientshave also been shown to have higher haemostatic indicesof extrinsic coagulation factors than healthy individuals.Kakkar et al (1995a) found that, not only was there anexcess of thrombin generation in the plasmas of cancer

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patients, but the levels of TF and FVIIa were 67% and 46%higher, respectively, compared with normals.

To further determine the effects of chemotherapeuticagents on these cells, we investigated TF mRNA regulationand protein expression. Analysis of TF mRNA expression inT24/83 cells showed an increase in TF mRNA upregulationin cells treated with certain chemotherapeutic agents, mainlyetoposide, vincristine and methotrexate. Furthermore, TFprotein production was increased in vincristine-treated cells.

Taken together, this data demonstrated that increasedthrombin generation is the result of the conversion ofprothrombin to thrombin, and not a decrease in thrombininactivation. Also, thrombin regulation occurs via theextrinsic (TF-dependent) coagulation pathway on the cellsurface when these cells are exposed to chemotherapeuticagents in vitro and that certain chemotherapeutic agents arecapable of inducing TF mRNA upregulation and subsequentTF protein expression.

In conclusion, exposure of T24/83 cells to some chemo-therapeutic agents can lead to increases in thrombingeneration and TF mRNA and protein expression. Wespeculate that chemotherapeutic agents may have similareffects on the endothelium. Although administration ofanticoagulant prophylaxis, such as warfarin, heparin andlow-molecular-weight heparin (Edwards et al, 1990; Mon-real et al, 1996; Levine, 1997; Kakkar & Williamson, 1999;Levine & Lee, 1999), or treatments such as antithrombinsubstitution (Nowak-Gottl et al, 1996), in conjunction withchemotherapy, have been shown to decrease the incidenceof thrombosis, more studies are required to investigate howthese anti-neoplastic drugs affect thrombin regulation, notonly on the surfaces of tumour-related cells, but onendothelial cells as well.

ACKNOWLEDGMENTS

This work was supported by a grant-in-aid (#NA4020) fromthe Heart and Stroke Foundation of Ontario. Dr AnthonyChan holds a Research Scholarship award from the Heartand Stroke Foundation of Canada.

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