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Tumor Biology |
Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York 14263 [L. O.]; and Department of Oncology, Montefiore Hospital and Albert Einstein Cancer Center, Bronx, New York [N. B., M. D-L.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15% and 
88%, respectively (n = 20; P < 0.0001). Finally, with an in vitro model system, tumor-induced platelet aggregation was enhanced by 127% (P < 0.001) in study patient platelet-rich plasma (PRP) compared to control PRP and could be completely inhibited (P < 0.0009) when both tumor cells and their PRP substrates were incubated with monoclonal antibodies directed against the vWf binding epitope of GPIb
and against the GPIb binding epitope of plasma vWf, respectively. Unusually large vWf multimers observed in patients with disseminated tumors probably result from deficient vWf-cleaving protease activity and may represent a novel mechanism regulating primary platelet-tumor adhesive interactions involved in the metastatic process. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mounting evidence supports the notion that a platelet-related GPIb3
receptor, expressed by both cultured tumor cell lines (2)
and fresh human carcinoma specimens (3
, 4)
, participates in primary adhesive events required for the initiation of the metastatic process (3
, 5)
. Additionally, recent reports demonstrate that the functional binding properties of these two homologous receptors also vary (5)
. In contrast to platelet GPIb, which requires the nonphysiological cofactor, ristocetin, for in vitro binding under static conditions to its ligand, vWf, tumor GPIb functions as a receptor for vWf in the absence of ristocetin (5)
. Hence, extrapolating to static flow conditions in the capillary or venule network, it is likely that spontaneous tumor GPIb binding to plasma vWf could initiate the process of tumor-induced platelet aggregation, thrombus formation, and the evolution of a metastatic colony.
Endothelial or megakaryocyte-derived vWf exists in the plasma as a series of multimers with molecular weights ranging up to 15 x 106 (6) . The distinctive property of the vWf molecule resides in its ability to regulate adhesive interactions with platelets depending upon its molecular size. Highly polymeric forms of vWf are the most effective in promoting platelet adhesion and aggregation (7) . Although vWf is secreted from endothelial cells as an extremely large polymer (8) , these molecules are converted in the plasma to a series of multimers (9) by a plasma vWf-cleaving protease (10 , 11) , which serves to limit their adhesive activity.
In view of the aberrant, metastasis-facilitating properties of tumor GPIb, we wondered whether plasma vWf also manifested comparable structural anomalies in patients with metastatic carcinoma. Several hemostatic abnormalities result from perturbations in the multimeric organization of plasma vWf, including TTP (12)
, a microangiopathy characterized by disseminated intravascular platelet aggregation. This syndrome is associated with an increased predominance of high molecular weight vWf multimers resulting from either a deficiency of vWf-cleaving protease activity (13)
or an IgG plasma inhibitor directed toward the enzyme (14)
. Although many of the pathoetiological features of TTP bear striking similarities to those involved in the metastatic cascade, vWf anomalies in cancer patients have yet to be reported, with the exception of elevated FVIIIR:Ag levels in patients with metastatic versus localized prostatic carcinoma (15)
.
On the basis of these observations, we hypothesized that similar pathological events with respect to adhesive interactions between tumor GPIb and plasma vWf might generate potent stimuli that are conducive to the initiation of the metastatic process. As such, in this study, we examined the quality, quantity, and functional activity of plasma vWf in patients with disseminated neoplasms. Additionally, we used an in vitro model system to investigate the participation of plasma vWf and its adhesive ligand, GPIb, in tumor-platelet aggregation and thrombus formation, an early event required for the generation of a viable tumor metastasis (16)
. Prior studies assessing the predictive value of in vitro tumor-induced platelet aggregation have demonstrated its correlation with a tumor’s in vivo metastatic (1
, 17 , 18)
and thrombogenic (19)
potential.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Study patients were eligible only if they had widely metastatic solid tumors requiring a hematogenous route, as opposed to local spread. Control patients were required to have localized nonmetastatic disease at one site or at contiguous local sites. All eligible patients, based on protocol inclusion and exclusion criteria, were sequentially accrued. Patients were originally evaluated in a hospital inpatient setting or an outpatient clinic setting. Patients with a known history of coagulopathies, sepsis, or platelet disorders were excluded from accrual. For control patients, blood specimens were obtained prior to their tumors’ surgical resection, and for study patients, blood specimens were obtained prior to any treatment. This protocol was approved by the Institutional Review Board, and all patients signed an informed consent prior to study entry. A second set of 10 control specimens consisted of blood samples from healthy, consenting hospital personnel. Citrated blood specimens for vWf assays were immediately centrifuged at 800 x g for 20 min at 25°C, after which aliquots were frozen at -70°C.
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MCF-7 Tumor Cells, PRP, and Tumor-induced Platelet Aggregation.
MCF-7 cells were grown routinely as a monolayer in T80 flasks using Eagle’s MEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies, Inc.). The aggregation experiments were carried out with cells grown at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were harvested with 5 mM EGTA, 5 µM leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride in HBSS (Life Technologies, Inc.), enumerated in a Nageotte counting chamber and adjusted with HBSS to a concentration of 
2.5 x 106 cells/ml for aggregation experiments, as described previously (2)
. Viability, as determined by trypan blue exclusion, was >80%.
Blood obtained from study and control patients was drawn through a 19-gauge needle, into a plastic syringe containing 3.8% trisodium citrate, was centrifuged at room temperature for 10 min at 160 x g, and the PRP was collected. The number of platelets was determined with a cell counter (Sysmex-K-100 Cell Analyzer; Baxter Diagnostics Inc., McGraw Park, IL) and then diluted to a final concentration of 300,000 cells/µl using the autologous patient’s PPP (or control PPP for mixing experiments), which was prepared by centrifuging the remaining aliquot of PRP at 800 x g for 20 min. Tumor-induced platelet aggregation experiments were performed using optical aggregometry, as outlined in prior reports (2 , 3 , 5) , using a dual-channel optical aggregometer (Chrono-Log model 560Ca).
For aggregation experiments with MoAbs, MCF-7 cells or PRP was preincubated for 30 min at 37°C with the following MoAbs: SZ2 (anti-GPIb, mouse IgG1, directed against the vWf binding epitope of the subunit of GPIb, Immunotech, Westbrook, ME), P2 (anti-IIbß3, mouse IgG1
, reacts with IIbß3 complex, precluding its activated state, and preventing ligand binding; Immunotech), AMF-7 (anti-vitronectin receptor, mouse IgG1, recognizes the Mr 120,000 chain of the vitronectin receptor, precluding ligand binding; Immunotech), and/or RFF VIII R/1 (anti-vWf, mouse IgG1, specific for the GPIb binding epitope of human vWf; Harlan Sera Laboratory, Sussex, United Kingdom). SZ2, P2, and AMF-7 were used at a final concentration of 80 µg/ml, whereas the final concentration of RFF VIII R/1 was 24 µg/ml, as described previously (2
, 3
, 5)
. For assays requiring an isotype-matched nonspecific MoAb, mouse IgG1 (MOPC-21; Sigma Chemical Co., St. Louis, MO) served as a control. These tumor preparations were washed with HBSS, pelleted, resuspended in HBSS, and then added at a final concentration of 2.5 x 106 tumor cells/ml to PRP that had previously equilibrated at 37°C in the aggregometer cuvette. The subsequent change in absorbance was registered on the chart recorder. For each specimen, the maximum aggregation were calculated by a computer interfaced with a Chrono-Log integrator.
vWf Multimer Analysis.
vWf Multimer analysis was performed in SDS-0.8% agarose gels [SeaKem HGT(P); FMC, Rockland, ME] according to Ruggeri and Zimmerman (20)
. Plasma samples containing 0.0025 units of vWf:Ag were electrophoretically separated. These preparations were subsequently subjected to Western blot analysis using an anti-vWf MoAb (A0082; Dako, Carpinteria, CA) and an alkaline phosphatase labeled 
-chain specific for antihuman IgG (D0478; Dako). Analysis of subunit composition and proteolytic fragments of plasma vWf was performed by electrophoresis on SDS-7% polyacrylamide gels. To quantify the vWf multimers, we scanned the blots with a densitometer (EC Densitometer; EC Apparatus, St Petersburg, FL) attached to a Hewlett Packard Integrator (HP 3396A, Purchase, NY). The proportion of large multimers was determined by the fraction consisting of the intensity of all but the five smallest bands, divided by that of all of the bands, as described previously (21)
.
Assays Measuring vWf-cleaving Protease Activity.
The activity of vWf-cleaving protease in patients’ plasma samples was assayed as described previously (10)
. Briefly, 100 µl of an incubation mixture consisting of dilutions of control or patient’s plasma containing the protease inhibitor, Prefabloc SC (Boehringer, Mannheim, Germany), at a final concentration of 10 mmol/liter were added to 50 µl of the purified vWf substrate (American Bioproducts Co., Parsippany, NJ). The resulting solution was transferred onto a hydrophilic filter membrane (VSWP, 25-mm diameter; Millipore, Bedford, MA) floating on the surface of 50 ml of dialysis buffer [1.5 mol/liter urea-5 mmol/liter Tris-HCl (pH 8.0)] in a screw-cap plastic tube and incubated for 24 h in a dry oven at 37°C. The multimeric size distribution of vWf and its proteolytic fragments were assayed by 1.4% SeaKem HGT(P) agarose and SDS-7% polyacrylamide gels, respectively, as described above. vWf multimers were identified with peroxidase-conjugated antibodies against human vWf (P226; Dako). A standard curve was constructed using data from dilutions of normal control plasma subjected to the vWf cleaving assay. The percentage of cleaving activity (with normal human plasma defined as 100% activity and TBS as 0%) was plotted as a function of the mean size of multimers (defined as the distance between the peak of the tracing and the lowest molecular weight band) from scans of the autoradiographs.
Screening for Inhibitors.
Protease activity in patient plasma specimens was assayed by measuring vWf cleaving in 1:1 (v/v) mixtures of control plasma from hospital personnel and patients’ test plasmas. Additionally, dot blot analysis was performed to explore the possibility of an autoantibody directed against a putative vWf proteolytic cleaving site. Five µl of a 0.5 mg/ml vWf solution were applied onto nitrocellulose and overlaid for 4 h at room temperature with plasma dilutions of 1:500 or 1:50, and bound antibodies were detected with alkaline phosphate-labeled goat antihuman IgG.
Statistical Analysis.
The Student’s t test (P) was used for comparisons of vWf parameters and aggregation measurements in study versus control patient samples. The Mann-Whitney and Fisher’s exact tests were used to determine the significance of the association between the clinical presence of metastases and the semi-quantitative D-dimer values (P) and between metastases and the level of vWf cleaving activity (P), respectively. All values are expressed as mean ± SD, unless otherwise indicated.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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lists clinical characteristics of 20 patients with widely disseminated solid tumors and 15 corresponding control patients with localized malignancies. Although about one-quarter of these patients manifested mild thrombocytopenia (platelet count, <150,000 cells/µl) as well as a modest prolongation in their partial thromboplastin or prothrombin times, these abnormalities were not statistically significant. A significant increase, however, was observed in their mean D-dimer levels (P < 0.023) in comparison to control patients. The study patients demonstrated no abnormalities in their fibrinogen levels relative to controls. Fig. 1
displays the study patients’ mean levels of FVIII:C activity, vWf FVIII-related antigen activity (vWf:Ag), and ristocetin cofactor activity (vWf:RCof), which were elevated by 68% (P < 0.013), 91% (P < 0.0009), and 207% (P < 0.0009), compared to those from control patients. Values from both sets of control samples, i.e., those from patients with localized disease versus samples from hospital personnel, showed no significant differences.
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. Plasma samples from six representative study patients bearing disseminated renal cell carcinoma (Lane D), metastatic prostatic carcinoma (Lane E), metastatic colorectal carcinomas (Lanes F and G), and metastatic breast carcinomas (Lanes I and J) are illustrated, along with samples from four corresponding controls. To quantify the size distribution of vWf multimers, these gels were subjected to densitometry scanning, with the percentage of large multimers illustrated in Fig. 3
. The fraction of the highly polymeric vWf forms was increased by 165% (P < 0.001) in the study patient plasma specimens compared to the controls. These data are consistent with either a deficiency or functional impairment of the vWf-cleaving protease, or alternately (but not mutually exclusively), enhanced vWf secretion/synthesis due to endothelial perturbations from metastatic tumor.
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. Incubation mixtures from study patients with metastatic renal cell, testicular, prostate, and colorectal carcinomas (Fig. 4B)
or a TBS (non-plasma-containing) buffer sample (Fig. 4C)
, showed no degradation of the vWf substrate, whereas control patient specimens demonstrated significant degradation (Fig. 4B)
. An analysis of 10 pairs of plasma specimens demonstrated a 96% (P < 0.009) reduction in vWf-cleaving activity in the study patient plasmas, with mean values of 95.0 ± 4.6% for the control patients and 4.0 ± 4.9% for the study patients. The clinical presence and absence of tumor metastases were associated with vWf-cleaving enzyme activities of 15% and 88%, respectively (n = 20; P < 0.0001). Furthermore, when study patients’ reaction mixtures were subject to SDS-PAGE under reduced conditions, minor amounts of the proteolytic fragments at Mr 170,000 and 140,000 were observed, whereas the vWf subunit was extensively degraded after incubation with control plasmas (Fig. 5)
. These results confirm that the disappearance of the large multimers results from proteolytic degradation of the vWf substrate rather than cleavage of the disulfide bridges linking the protomeric subunits of vWf.
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illustrates, the pattern of digested vWf in this mixture is comparable to that observed with diluted control plasma and is consistent with deficient activity of the vWf-cleaving enzyme opposed to a plasma inhibitor. A dot blot analysis also aimed at detecting the presence of possible autoantibodies, demonstrated no binding of any such antibodies to immobilized vWf (data not shown).
To investigate the adhesive interactions between tumor cells and plasma vWf, we carried out tumor-induced platelet aggregation assays. When study patients’ PRP was used as substrate, maximum aggregation was increased by 127% (P < 0.001), compared to PRP from control patients (Fig. 6)
. Maximum aggregation could be abrogated by 72% (P < 0.005) or 54% (P < 0.014) when MCF-7 cells were pretreated with MoAbs directed against the vWf epitope of GPIb or with MoAbs inhibiting the functional binding domain of IIbß3, respectively. When study patient PRP was incubated with a MoAb directed against the GPIb binding domain of vWf, tumor-induced platelet aggregation was inhibited by 77% (P < 0.001). In assays in which both tumor cells and PRP substrates were incubated with anti-GPIb and anti-vWf, respectively, tumor-induced platelet aggregation was nearly abolished (P < 0.0009). Additionally, when tumor cells were incubated with a MoAb directed against the functional domain of the vitronectin receptor, MCF-7-induced platelet aggregation was not significantly perturbed. Finally, preincubation of study patients’ PRP with MoAbs directed against the vWf binding domains of GPIb with MoAbs directed against the functional binding domain of IIbß3, resulted in an 84 ± 5.3% and 68 ± 7.1% inhibition in MCF-7-induced platelet aggregation, respectively (n = 4 for both experiments). These observations, however, represent inhibition resulting from MoAbs bound to both tumor and platelet adhesive receptors because excess unbound MoAbs were present in the PRP.
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. These findings are consistent with the ristocetin cofactor data and support a mechanism involving an excess of a plasma aggregation-enhancing factor present in study patients’ plasmas.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IIbß3 receptors. A recent study establishing platelet GPIb and IIbß3 as receptors for these unusually large vWf multimers (22)
lends support to the observations reported herein. Also supportive are the findings by Grossi et al. (23
, 24)
, who have identified immunorelated GPIb and IIbß3 receptors on a variety of cultured tumor cell lines. When these tumor receptors were simultaneously blocked with MoAbs, tumor-induced platelet aggregation was completely inhibited.
Although the vitronectin receptor did not appear to participate in MCF-7 tumor-induced platelet aggregation, it is possible that a heretofore undescribed vWf receptor expressed by tumor cells may additionally augment primary GPIb-mediated vWf adhesive attachments. Hence, extrapolating from our in vitro model, hemostatically active, highly polymeric vWf molecules, under static conditions, may function as a potent nidus for adhesive interactions with both platelets and circulating tumor cells, resulting in tumor-induced platelet aggregation, thrombus formation, and the presumptive development of a metastatic colony (16, 17, 18, 19)
. Also, constitutive expression of both GPIb and IIbß3 by circulating tumor cells could result in their interaction with other plasma proteins, triggering platelet aggregation and tumor-induced thrombus formation. Therefore, the abnormal function of these receptors could also facilitate the hemostatic spread of tumors.
vWf is secreted from endothelial cells by either a constitutive or a regulated pathway (25) . The most biologically potent molecules are released from endothelial Weibel-Palade bodies in response to thrombin, vasoactive amines, purine nucleotides, and a variety of cytokines (26) . Because enhanced quantities of platelet-tumor aggregates were generated using study patient PRP in our in vitro assays, these thrombi could conceivably constitute potent sources of vWf secretagogues (27) , sequentially catalyzing the release of high molecular weight vWf multimers from stimulated endothelium. Notwithstanding, our findings reveal deficient activity of the vWf-cleaving enzyme, which regulates the size and, hence, the adhesive activity of plasma vWf. The extent to which tumor-platelet aggregates themselves might contribute to augmented secretion of highly polymeric vWf is unclear and will require further study using different approaches. It is clear, however, that increased quantities of highly polymeric vWf were associated with enhanced in vitro generation of tumor-platelet aggregates, as well as being highly correlated with the clinical presence of metastatic disease in patients bearing malignancies.
In a subset of patients, disseminated carcinoma is believed to function as the chief stimulus for TTP, generating a "carcinoma-associated microangiopathy" that is characterized by widespread endothelial-adherent platelet-tumor emboli occluding the microcirculation (28) . Although most of these patients display mucinous adenocarcinomas, recent studies have also linked almost all other common tumors with this TTP-related process (29 , 30) , with unusually large vWf multimers detected in the few cancer patients examined (30) . Curiously, a similar array of hemostatic abnormalities are observed in both patients with disseminated metastases and the carcinoma-associated microangiopathies. Previously recognized as a hypercoaguable state and attributed to an acute phase reaction in patients with disseminated metastases, these hemostatic abnormalities characteristically consist of elevated fibrin degradation products (Ref. 31 and this study), platelet activation (32) , coagulopathies (33) , and increased thrombotic tendencies (34) . Hence, it seems reasonable to speculate that similar pathoetiological stimuli may contribute to a common mechanistic process both in the TTP subgroup of carcinoma-associated microangiopathies and in the clinical setting of widely disseminated tumor.
Although the mechanism whereby the reduced functional activity of the vWf-cleaving enzyme developed in these cancer patients is speculative, several recent observations may provide clues to its etiopathogenesis. A frequent event accompanying cellular neoplastic transformation involves altered expression of extracellular proteinases, which could directly result in perturbations in vWf-cleaving activity. Various oncogenes have been shown to regulate the expression of proteins such as the matrix-degrading metalloproteinases and plasminogen activators, consistent with the transforming and metastatic response induced by oncogenes (35) . Alternately, protein phosphorylation, modulated by a network of oncogene-mediated kinases, could directly or indirectly impact on the functional properties of the vWf-cleaving enzyme (36) . Conceivably, the activity of the enzyme itself or associated regulatory molecules, cofactors, or docking-type receptors could be altered by phosphorylation, either positively or negatively (37) . Further studies will be required to identify the process through which the vWf-cleaving enzyme is reduced or impaired in patients with metastatic tumors.
In conclusion, high concentrations of vWf antigen, organized in unusually large multimers, were observed in plasma samples from patients with disseminated neoplasms. The functional activity of this aberrant vWf was significantly enhanced, as demonstrated by ristocetin cofactor and tumor-induced platelet aggregation assays. Tumor-induced platelet aggregation and subsequent thrombus formation could be completely inhibited by simultaneously blocking the vWf epitope of tumor GPIb and the GPIb epitope of plasma vWf. These unusually large vWf multimers appear to result from either a deficiency or a functional aberration of the vWf-cleaving protease, similar to the pathogenesis of TTP and other microangiopathies. Not only do these laboratory findings have important implications with regard to the formation of tumor metastases, but they also have significance with regard to their prognostic utility. Elevated levels of highly polymeric vWf may constitute a novel biomarker indicative of the presence of or the risk of metastatic disease.
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FOOTNOTES |
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1 Supported in part by a grant from the Bruce Cuvelier Endowed Research Foundation and by Cancer Center Core Grants P30CA1330-21 and CA16056-24 from the National Cancer Institute. 
2 To whom requests for reprints should be addressed, at Department of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-7614; Fax: (716) 845-8008; E-mail: oleksowicz{at}sc3101.med.buffalo.edu
3 The abbreviations used are: GPIb, glycoprotein Ib; vWf, von Willebrand’s factor; TTP, thrombotic thrombocytopenic purpura; PRP, platelet-rich plasma; PPP, platelet-poor plasma; MoAb, monoclonal antibody.
Received 10/27/98. Accepted 3/ 3/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-related protein is expressed by fresh human breast carcinoma tissue and is regulated by a PKC-sensitive mechanism. Exp. Cell Res., 237: 110-117, 1997.[Medline]
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, maximum and minimum values; 
, median values. The mean fractions of the largest multimers for study and control patients were 0.82 ± 0.06 and 0.31 ± 0.09, respectively. The fraction of the highly polymeric vWf forms was increased by 165% (P < 0.001) in the study patients’ plasmas (n = 20) compared to control patient specimens (n = 15). These data are consistent with either a deficiency of the vWf-cleaving protease or an enzyme with impaired activity.
