Platelet and Coagulation Activation with Vascular EndothelialGrowth Factor Generation in Soft Tissue Sarcomas

Henk M. W. Verheul, Klaas Hoekman,Florea Lupu, Henk J. Broxterman,Paul van der Valk, Ajay K. Kakkar, andHerbert M. Pinedo1

Departments of Medical Oncology [H. M. W. V., K. H., H. J. B.,H. M. P.] and Pathology [P. v. d. V.], University Hospital “VrijeUniversiteit,” 1007 MB Amsterdam, the Netherlands; Weston Centrefor Experimental Research, Thrombosis Research Institute, LondonSW3 6LR, United Kingdom [F. L.]; and Department of Surgery,Imperial College School of Medicine, Hammersmith Hospital,London WI2 0NV, United Kingdom [A. K. K.]

ABSTRACTAngiogenesis and activated blood coagulation are in-

volved in tumor growth and metastasis. Although some havesuggested that activation of coagulation in tumors is notlinked to activation of platelets, no data exist to either sup-port or refute this concept. However, platelet turnover incancer patients is often increased, and platelets are carriersof angiogenic growth factors. We hypothesized that plateletsare involved in tumor-associated angiogenesis. To obtainevidence supporting this hypothesis, we have studiedwhether the angiogenic and coagulation pathways and plate-lets are concomitantly activated in cancer patients with softtissue sarcomas (STSs) using a novel method to detect acti-vated platelets in tumor specimens. Twelve patients withSTS were selected on the basis of having intratumoral ac-cumulation of fluid, which was aspirated. These accumula-tions demonstrated very high concentrations of vascularendothelial growth factor and coagulation factors (includingthrombin-antithrombin-complex). Tumor specimens showeddense vascularization with intense vascular endothelial growthfactor expression and the presence of activated platelets.Taken together, these results support the concept that an-giogenesis, blood coagulation, and platelets are concomi-tantly activated in STS and support the hypothesis thatplatelets contribute to tumor-induced angiogenesis.

INTRODUCTIONAngiogenesis is required for tumor growth and metastasis

(1). Tumors stimulate new vessel formation through the secre-

tion of angiogenic factors,e.g., basic fibroblast growth factorand VEGF2 (2). Activation of the coagulation pathway alsoenhances tumor growth and metastasis (3). Procoagulants in-volved in angiogenesis include TF and thrombin (4, 5). VEGF,the most potent proangiogenic factor, is an indirect procoagu-lant; it is capable of inducing vascular hyperpermeability and ofincreasing TF expression on endothelial cells (6, 7). Vascularhyperpermeability results in leakage of plasma proteins, includ-ing prothrombin and fibrinogen, into the extracellular matrix.Prothrombin converted into thrombin by the activated coagula-tion pathway may result in platelet activation and the productionof fibrin from fibrinogen. Fibrin is also a proangiogenic protein,and it provides an ideal matrix for the migration of endothelialcells (8). Clinical observations support the contention that co-agulation and angiogenic pathways are important in cancerbiology (9, 10). However, it is thought that activation of coag-ulation in tumors does not result in the activation of platelets(11).

Based on an increased platelet turnover in cancer patients(12), and because it has been recently shown that plateletstransport and secrete VEGF on activation (13, 14), we hypoth-esized that platelets play an important role in tumor-inducedformation of new vessels (15). To obtain more evidence for thishypothesis, we have studied whether the angiogenic and coag-ulation pathways are concomitantly activated in patients withSTSs and developed a method to detect activated platelets intumor specimens. We chose to study STSs because these tumorsare highly vascularized (16). Twelve patients with STS andintratumoral fluid accumulation were studied. Aspirated fluiddemonstrated high concentrations of VEGF and several acti-vated coagulation factors. Tumor specimens showed dense vas-cularization with intense VEGF expression. As far as we know,this is the first demonstration of the presence of activatedplatelets within the tumor vasculature, suggesting that plateletscontribute to tumor-induced angiogenesis.

PATIENTS AND METHODSPatients. We aspirated 50–600 ml of tumor fluid from

12 STS patients treated in our hospital between 1991 and 1998who had either primary or metastatic disease and had a hetero-geneous aspect on MRI suggestive of intratumoral fluid accu-mulations. These patients were diagnosed with the followingsubtypes of STS: (a) leiomyosarcoma (five patients); (b) malig-nant fibrous histiocytoma (two patients); (c) liposarcoma (twopatients); (d) gastrointestinal stromal cell tumor (one patient);(e) hemangiopericytoma (one patient); and (f) malignant schw-annoma (one patient). Tumor fluids were aspirated under ultra-

Received 7/14/99; revised 9/29/99; accepted 10/4/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 To whom requests for reprints should be addressed, at Department ofMedical Oncology, University Hospital “Vrije Universiteit,” Postbus1007 MB Amsterdam, the Netherlands. Phone: 31-20-4444300; Fax:31-20-4444355; E-mail: hm.pinedo@azvu.nl.

2 The abbreviations used are: VEGF, vascular endothelial growth factor;STS, soft tissue sarcoma; TF, tissue factor; MRI, magnetic resonanceimaging; mAb, monoclonal antibody; TAT, thrombin-antithrombin.

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sound guidance. Aspirated tumor fluids were aliquoted andimmediately frozen at220°C. Tumor tissue was obtained frompatients undergoing radical surgery.

Tissue Sampling and Immunohistochemical Staining.Tissues acquired from these patients, from different tumor areas,were histologically analyzed after H&E staining and appropriateimmunohistochemistry of formaldehyde-fixed, paraffin-embed-ded tissues (4-mm sections).

Immunohistochemical staining for VEGF was performedwith a polyclonal goat antibody against VEGF (catalogue num-ber AB-293-NA; R&D Systems, Abingdon, United Kingdom)on paraffin slides using a standard procedure. Briefly, afterdeparaffinization and rehydration, endogenous peroxidase wasblocked with 0.3% H2O2 in methanol for 30 min. Nonspecificbinding of the secondary antibody was blocked, and subsequentincubation with the primary antibody (20mg/ml), followed by abiotin-labeled secondary horse-antigoat antibody (1:150), wasperformed with washing steps in between. A complex of strepta-vidin and biotinylated horseradish peroxidase was added, andstaining was visualized with 3,39-diaminobenzidine.

Cryostat sections from seven patients (4-mm sections) werefixed in cold acetone. To get an impression of the vasculariza-tion of these STSs and discriminate between the intravascularand extravascular location of platelets, we performed a platelet-specific, double immunofluorescence labeling of thea2b3 inte-grin and an endothelial-specific lectin binding site containinga-linked fucose residues, which binds biotinylated Ulex euro-paeus agglutinin I (catalogue number FL-1061; Vector Labora-tories, Inc). Immunofluorescence labeling of platelets was per-formed using a mAb againsta11bb3 integrin (catalogue numberM7057; DAKO A/S, Glostrup, Denmark). We used streptavi-din-Texas red for the detection of the biotinylated lectin andhorse antimouse IgG-FITC for the detection of thea11bb3

integrin as secondary fluorescence-labeled antibodies.A novel method to detect activated platelets was devel-

oped, based on the codetection ofa11bb3 integrin and fibrin/fibrinogen on their surface. Thea11bb3 integrin, also known asGPIIb/IIIa, is a complex of two major platelet glycoproteins thatundergo conformational changes after platelet activation, expos-ing a high-affinity binding site for fibrin/fibrinogen and otherRGD-containing proteins (17). The principle of this doublestaining for platelet activation is based on the observation thatfibrinogen is only bound by activateda11bb3 integrin duringplatelet activation and is not bound by resting platelets. Immu-nofluorescence labeling of fibrinogen was performed using arabbit polyclonal antibody (catalogue number A 0080; DAKO)and goat antirabbit IgG-Texas red (both from Vector Laborato-ries, Inc.). The platelet marker was detected using the samesecondary antibody as mentioned above. After preincubation(1% FCS in PBS), slides were incubated with a combination ofthe primary antibodies, either mAb anti-a11bb3 and biotinylatedUlex Europaeus lectin or mAb anti-a11bb3 and polyclonal an-tibody antifibrinogen, and subsequently incubated with theabove-mentioned mixtures of the fluorescence-labeled detectionagents (antibodies or streptavidin), all in a 1:50 dilution. There-after, the slides were mounted with Vectashield antifading me-dium containing 49,6-diamidino-2-phenylindole as a nuclearcounterstaining (catalogue number H1200; Vector Laboratories,Inc.). All slides were examined within 1 week after staining

using a Bio-Rad MRC confocal laser scanning unit attached toa Nikon Diaphot inverted microscope (Bio-Rad MicroscienceLtd., Hertfordshire, United Kingdom). Thea11bb3 integrin-stained platelets appeared green, and endothelial cells or fibrin/fibrinogen appeared red, whereas areas of coincident labelingcontaining colocalized antigens appeared yellow.

ELISA Assays. VEGF concentrations were measuredwith a quantitative sandwich enzyme immunoassay (R&D Sys-tems). If necessary, samples were diluted up to 1000 times. Asindicators of coagulation and endothelial activation, we meas-ured TF, TAT-complex, and thrombomodulin concentrations,respectively, in the tumor fluids. TF and thrombomodulin con-centrations were determined by ELISA assays (DiagnosticaStago, Asnieres-sur Seine, France). Samples were diluted 1:10or, if necessary, 1:50. TAT-complex concentrations were deter-mined with an enzyme immunoassay (Enzygnost TAT micro;Behring Diagnostics GmbH, Marburg, Germany).

Statistical Analyses. Statistics were performed usingSPSS. Correlations were calculated using Spearman’s rank test.

RESULTSTumor Fluid Aspirated from STSs. MRI examination

of patients with histologically confirmed STS showed that themajority of patients with tumor diameters. 10 cm had heter-ogenous tumor masses, which represent solid elements and fluidaccumulations (Fig. 1). We succeeded in aspirating tumoralfluid under ultrasound guidance for 12 patients. H&E lightmicroscopy examination of paraffin sections of the walls ofthese tumor cavities failed to show either tumor necrosis orepithelial cell lining, but the extracellular matrix appeared veryloose with occasional extravasation of blood cells (Fig. 2).

Angiogenic Phenotype of STS. An intense vasculariza-tion in solid areas of the tumors was demonstrated by immun-ofluoresence staining. Fig. 3 shows a representative tumor. In

Fig. 1 MRI of the right upper leg of a patient with a histiocytoma(marked by anarrow) showing intratumoral fluid accumulation (twolarge cavities and one smaller cavity, marked byp).

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the tumor aspirates of these 12 patients, we found that VEGFconcentrations were substantially higher (median, 18 ng/ml;range, 0.3–345 ng/ml) than those in normal plasma (30–40pg/ml; Ref. 13), whereas basic fibroblast growth factor, anotherangiogenic factor, was only detectable at low levels (,90 pg/

ml) in a minority of the aspirates (data not shown). The medianprotein concentration of these aspirates was 52 g/liter (range,33–74 g/liter), marginally below that seen in normal plasma (60g/liter). Immunohistochemical staining reflected that VEGF wasexpressed by tumor cells and the endothelial cells (lining thevasculature) in these sarcomas (Fig. 4).

Coagulation Phenotype of STS. Markers of activatedcoagulation were measured in the tumor aspirates of these 12patients. TF concentrations were approximately two timesgreater than those seen in normal plasma, with a median valueof 723 pg/ml (range, 400-4998 pg/ml; Ref. 9). The thrombincontent, as manifested by TAT complexes, was also high, witha median value of 126mg/liter (range, 33–184mg/liter), repre-senting a 63-fold increase in thrombin generation as comparedto normal plasma (9). The marker for endothelial activation,thrombomodulin, was 181 ng/ml (range, 39–700 ng/ml) andwas only modestly elevated when compared with normal plasmalevels (Ref. 18; summarized in Table 1). Spearman rank corre-lation analyses revealed a significant correlation between in-creasing aspirate thrombomodulin concentration and that of TF(r 5 0.65;P , 0.05; Fig. 5), whereas the other markers did notshow significant correlations.

Platelet Activation within Tumor Vasculature. To ex-amine whether platelets are also activated in the tumor vascu-

Fig. 2 H&E staining. Malignant fibrous histiocytoma, a solid tumor with loose extracellular matrix containing extravasated blood cells depicted byan arrow.

Fig. 3 Immunoconfocal microscopy (310). A leiomyosarcoma, vas-culature (marker Ulex lectin) is shown inred, and platelets (markera11bb3 integrin) are shown ingreen. Dense vascularization is seen, andplatelets are confined to the vasculature.

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lature, we performed immunoconfocal microscopy to detectintratumoral activated platelets. The immunostaining confirmedthe presence of platelets in the tumor vasculature (Fig. 3). Alarge proportion of the detected platelets were associated withfibrinogen in aggregates, as demonstrated by double staining ofthea11bb3 integrin and fibrin/fibrinogen in both solid elementsand intratumoral cavities (Fig. 6,A andB). The yellow appear-ance resulting from the double staining ofa11bb3 integrin(green) and fibrin/fibrinogen (red) confirms the presence ofactivated platelets in these tumors. In addition, most of the

extracellular matrices of the tumor tissue showed abundantexpression of fibrinogen, which is indicative of leakage ofplasma proteins out of the tumor vasculature.

DISCUSSIONContrary to the current view that tumor-induced coagula-

tion does not result in platelet activation, we have shown thatplatelets do become activated within the tumor vasculature. Thisfinding suggests that platelets contribute to tumor angiogenesis

Fig. 4 Intense VEGF staining of leiomyosarcoma tumor cells and vasculature marked byan open arrowandsolid arrow, respectively.

Table 1 Concentrations of VEGF and coagulation factorsConcentration of VEGF and coagulation factors in tumor aspirates compared with normal plasma concentrations.

Normal plasmaMedian (range)

Aspirated Tumor Fluid(n 5 12)

Median (range) Fold higher

Total protein (g/liter) 60–80 52 (33–74)VEGF (ng/ml) 0.04a 18 (0.3–345) 4503Tissue factor (pg/ml) 349 (296–469)b 723 (400–4998) 23TAT complex (mg/liter) 2 (2–3.5)b 126 (33–184) 633Thrombomodulin (mg/liter) 44 (35–55)c 181 (39–700) 43

a Values are the medians (range) of normal plasma as reported in Ref. 13.b Values are the medians (range) of normal plasma as reported in Ref. 9.c Values are the medians (range) of normal plasma as reported in Ref. 18 and tumor aspirates.

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through growth factor release upon their activation. It also offersa plausible explanation for the clinically observed increasedplatelet turnover in cancer patients as compared with healthyvolunteers (12).

The involvement of the coagulation cascade in tumor-induced angiogenesis has been described previously (3). In atransgenic murine mouse model of dermal fibrosarcoma, tumorsoccurred predominantly in areas prone to wounding (19). In amurine fibrosarcoma model, it was demonstrated that the trans-fection of a TF gene is associated with up-regulation of VEGFand enhanced tumor growth (4). A strong correlation betweenTF and VEGF expression in breast carcinoma cellsin vitro hasbeen established, and a colocalization of TF and VEGF in breastcancer tissue from patients has also been reported (20). Inaddition, expression of TF was found in active angiogenic sitesin invasive human breast carcinoma (21). Through its effect onTF production and vascular permeability, VEGF is an indirectprocoagulant. The high VEGF concentration in the tumor aspi-rates and its high tumor expression lead us to hypothesize thatthe coagulation cascade may be concomitantly activated in thesesarcomas. Our data demonstrate high levels of both VEGF andTF in a human solid tumor, but without any direct correlation,suggesting that they are partly derived from different sources.Of note, VEGF and TF may both be responsible for the highconcentrations of each other in these fluids, because it has beenreported that stimulation of endothelial cells with exogenousVEGF results in up-regulation of TF (7), whereas transfection ofTF results in up-regulation of VEGF (4). The presence of highlevels of TAT complexes in sarcoma aspirates provides strongevidence that thrombin, a powerful platelet activator, is gener-ated in these tumors, probably due to TF expression. The proan-giogenic activity of thrombin is not dependentper seon fibrinformation (5). Thus, based on the observation that thrombingeneration occurs in STS, together with the finding of intratu-moral platelet activation, one may speculate that thrombin exertsits proangiogenic effect by activation of platelets. The highintratumoral concentrations of thrombomodulin, a marker ofendothelial activation (22) and a physiological anticoagulant,are consistent with previously reported observations (19). Thestrong correlation between thrombomodulin and TF may be dueto the fact that they are both markers of endothelial activation(21, 22). Taken together, these findings show that the angio-genic and coagulation pathways can be concomitantly activatedin STS.

It has been described previously that intratumoral fluid

accumulation in STS reflects necrosis (23). However, this studyprovides evidence that cystic areas in STS reflect the biologicalbehavior of these mesenchymal tumors. Our findings, includingthe dense vascularization, are in accordance with the observa-tion that STSs are highly angiogenic (16) and support the

Fig. 6 Immunoconfocal microscopy (340).A, leiomyosarcoma; fi-brinogen isred, platelets (marker mAb againsta11bb3 integrin) aregreen, and activated platelets areyellow, due to double staining. Restingand activated platelets present in small cavities of the tumor are markedby thewhite arrow, and a high expression of fibrinogen is detected (p).B, histiocytoma; fibrinogen isred, platelets (marker mAb againsta11bb3

integrin) aregreen, and activated platelets areyellow, due to doublestaining. Activated platelets are present in the tumor, and these plateletsappear to be confined to a tumor vessel marked by thewhite arrow.

Fig. 5 Correlation between the levels of TF and thrombomodulin in 12intratumoral fluids (r5 0.65;P , 0.05).

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hypothesis that tumor interstitial fluid has a biological role intumor growth (24). The exact mechanism for intratumoral fluidaccumulation remains to be clarified, but evidence for an im-portant role of VEGF is suggested from this study.

Intratumoral cavities contain proteineacious fluid with orwithout cellular elements, suggesting a spectrum from fluidextravasation secondary to endothelial hyperpermeability on theone hand, to frank bleeding into the cavities on the other. Thehigh concentrations of VEGF, which is well known for itsability to induce hyperpermeability (25), suggest that VEGFplays an important role in the formation of these intratumoralcavities. Presumably, some intracavital VEGF is derived fromthe STS cells because an intensive immunoreactivity for VEGFwas found in STS tissue, and STS cell lines produce highamounts of VEGFin vitro (data not shown). VEGF mRNAlevels are dramatically increased under hypoxic conditions (26).It may well be that hypoxia in STS contributes to the abundantVEGF production we observed. In addition, platelets transportVEGF (13), and megakaryocytes synthesize this protein (27).Taken together with the finding that platelets were activatedwithin the STS tumor vasculature and cavities, we propose thatthe extremely high VEGF levels (levels not previously reportedin biological fluids) in sarcoma aspirates are explained by acombination of both tumor- and platelet-derived VEGF.

The immunostaining we used to detect activated plateletsalso demonstrated that fibrinogen was present in abundance inthe extracellular matrix of these tumors. This finding supportsthe current belief that extravasation of plasma proteins withinthe tumor is most likely due to the hyperpermeability factor,VEGF (26).

Taken together, the very high concentrations of a numberof key coagulation factors and VEGF in these tumor fluids andthe evidence for intratumoral platelet activation reflect the im-portance of both the angiogenesis and coagulation pathways inthe biology of STS. Additional studies in a larger number ofpatients with different types of cancer are presently underway inour institute to define whether intratumoral platelet activation isa general phenomenon in solid tumors.

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2000;6:166-171. Clin Cancer Res   Henk M. W. Verheul, Klaas Hoekman, Florea Lupu, et al.   Growth Factor Generation in Soft Tissue SarcomasPlatelet and Coagulation Activation with Vascular Endothelial

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