- Email: [email protected]
Monocyte-derived microparticles may be a sign of vascular complication in patients with lung cancer
Lung Cancer 39 (2003) 145 /149 www.elsevier.com/locate/lungcan
Monocyte-derived microparticles may be a sign of vascular complication in patients with lung cancer Shigenori Kanazawa, Shosaku Nomura *, Midori Kuwana, Mikiko Muramatsu, Kazuyuki Yamaguchi, Shirou Fukuhara The First Department of Internal Medicine, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan Received 13 May 2002; received in revised form 23 September 2002; accepted 26 September 2002
Abstract We measured and compared the levels of plasma monocyte-derived microparticles (MDMP) and platelet activation markers [plasma platelet-derived microparticles (PDMP), CD62P binding to platelets; plt-CD62P, CD63 binding to platelets; plt-CD63], to develop a better understanding of their potential contribution to vascular complications of lung cancer. The concentrations of MDMP and PDMP in lung cancer patients were significantly higher (P B/0.01) than those in normal subjects. Levels of plt-CD62P and plt-CD63 were significantly higher (P B/0.001 for each) in lung cancer patients than in controls. Levels of sE-selectin were also higher in lung cancer patients than in control subjects. MDMP correlated positively with plt-CD62P, plt-CD63, and PDMP with its relation to PDMP being particularly significant. The number of MDMPs and PDMPs are patients who are non-small cell lung cancer were significantly higher than that in small cell lung cancer patients. In addition, levels of sE-selectin were higher in non-small cell lung cancer than in small cell lung cancer patients. These findings suggest that elevated MDMPs may be a sign of vascular complication in lung cancer patients, particularly those who suffer from non-small cell lung cancer. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Non-small cell lung cancer; Monocyte-derived microparticle; Platelet activation markers; sE-selectin
1. Introduction In malignant disease, there is an imbalance in activity of the coagulation and fibrinolytic systems that results in abnormal activation of coagulation, and this results in disseminated intravascular coagulation and deep vein thrombosis [1]. Abnormal hemostasis is a fundamental characteristic of malignant disease, and hemorrhagic and thromboembolic disorders are an important cause of morbidity and mortality in patients with cancer [2]. Tissue factor (TF) is the normal cellular activator of the coagulation system and is also an important activator of coagulation in malignant disease [3,4]. Particularly rich sources of TF include the lung, brain, and placenta, but TF is also found in epithelial cells, mononuclear cells, and many other cells and tissues [5,6]. Recently, it has
* Corresponding author. Tel.: /81-66-992-1001; fax: /81-728-333990 E-mail address: [email protected] (S. Nomura).
been suggested that TF also regulates tumor growth and angiogenesis, and Koomagi et al. reported the expression of TF and angiogenesis in non-small cell lung carcinomas (NSCLC)[7]. Sawada et al. also reported the significance of TF in NSCLC [8]. Many tumors have macrophage infiltrates that are activated by tumor cells. These macrophages have long been implicated in the coagulation abnormalities associated with solid tumors. There is increased TF activity in these tumor-associated macrophages [6,9]. Exposure of monocytes to tumor cells induces monocyte procoagulant activity [10]. Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules following stimulation by lipopolysaccharide [11]. This ability of endotoxin-stimulated monocytes to release heterogeneous monocyte-derived microparticles (MDMPs) may be an important mechanism regulating vascular cell effector functions [12]. The TF-exposing MDMPs promote prothrombinase complex assembly, thereby facilitating intravascular generation of thrombin and
0169-5002/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 9 - 5 0 0 2 ( 0 2 ) 0 0 4 4 1 - 5
S. Kanazawa et al. / Lung Cancer 39 (2003) 145 /149
146
enhanced procoagulant activity [13]. Procoagulant activity is also found in markers of platelet activation, such as platelet-bound CD62P (plt-CD62P) and PDMPs [14,15]. Thus, monocytes, platelets, and E-selectin could be very important in progression of vascular complications in lung cancer patients. To our knowledge, no other investigators have measured MDMP levels in lung cancer patients. We, therefore, measured and compared the levels of plasma MDMP and platelet activation markers [plasma PDMP, CD62P binding to platelets; plt-CD62P, CD63 binding to platelets; plt-CD63], to develop a better understanding of their potential contribution to vascular complications of lung cancer.
2. Patients and methods 2.1. Patients The study group included 30 healthy controls and 64 unrelated Japanese patients (45 men and 19 women) with lung cancer. All gave informed consent according to the Declaration of Helsinki. Table 1 shows the characteristics of patients and control subjects. The subjects ranged in age from 33 to 79 years. Twenty patients had small cell carcinoma, 16 had squamous cell carcinoma, and 28 had adenocarcinoma. Clinical staging of carcinoma was performed using the TMN classification. 2.2. Flow cytometry of activated platelets and microparticles Blood samples were collected into tubes containing 3.8% sodium citrate (9:1 vol/vol). Platelet-rich plasma (PRP) was prepared by centrifugation at 200 g for 10 min at room temperature. Washed platelets were prepared by centrifuging PRP at 1400 g for 10 min at room temperature. The resultant pellet was washed twice with washing buffer (9 mM/l Na2EDTA, 140 mM/l NaCl, and 26 mM/l Na2HPO4, pH 7.2) and resuspended in
HEPES-Tyrode’s buffer (129 mM/l NaCl, 8.9 mM/l NaHCO3, 0.8 mM/l KH2PO4, 0.8 mM/l MgCl2, 5.6 mM/ l glucose, and 10 mM/l HEPES, pH 7.4). An equal volume of 2% paraformaldehyde was added to the washed platelets, and theywere incubated for 15 min at room temperature. Platelets were then washed twice, resuspended in stock solution (9 mM/l Na2EDTA, 26.4 mM/l Na2HPO4 2H2O, 140 mM/l NaCl, 0.1% NaN3, and 2% fetal bovine serum, pH 7.2), and stored at 4 8C until analysis. PDMPs were detected with a modified version of a previously-reported method [16]. Ten microliters of platelets (3 /108/ml) added to 100 ml of HEPESTyrode’s buffer containing 5 nM/l EGTA. Both intact and aggregated platelets were removed by centrifugation at 1000 /g for 15 min to yield a supernatant containing microparticles only. Ten microliters of washed intact platelets (3 /108/ml) was then added to the supernatant, and the mixture was incubated with KMP-9 [17] (a FITC-labeled monoclonal antibody against platelet GPIX) for 30 min in the dark at room temperature. After incubation, samples were diluted 1:10 with HEPES-Tyrode’s buffer containing 5 nM/l EGTA and analyzed with an Ortho Cytoron Absolute Analyzer (Ortho Diagnostic Systems, Tokyo, Japan). Only cells and particles positive for GPIX were gated to distinguish platelets and PDMPs from electronic noise. To differentiate between platelets and PDMPs, the lower limit of the platelet gate was set at the left limit of the forward-scatter profile of resting platelets. Ten thousand FITC-positive particles in the PDMP gate were then counted to determine the number of PDMPs released per 10 000 platelets. As an index of platelet activation, CD62P expression was quantified by immunostaining with anti-CD62P monoclonal antibody (CLB-thromb/6, Immunotech, Marseille, France). Platelet expression of GPIIb/IIIa and of GPIb was analyzed with anti-GPIIb/ IIIa (NNKY1-32) [16] and anti-GPIb (NNKY5-5)[18] monoclonal antibodies, respectively.
2.3. Assessment of MDMP Table 1 Clinical characteristics of patients and healthy controls
Men/Women (no.) Age (years) WBC (/ml) PLT ( 104/ml) SCLC (no.) NSCLC (no.)
Control (n 30)
Lung cancer (n 64)
19/11 5195 46309502 22.494.9 0 0
48/16 5698 56509688 31.396.2 20 44
Control, non-lung cancer. Data are shown as mean9S.E.M. WBC, white blood cell count; PLT, platelet count; SCLC, small cell lung carcinoma; NSCLC, non-small cell lung carcinoma (squamous cell carcinoma 16; adenocarcinoma 28).
MDMP was detected by a previously reported method with some modifications [19]. Ten microliters of washed intact platelets (3 /108/ml) was added to the plasma, and the mixture was incubated with FITClabeled Annexin V (FITC-Ann V) and phycoerythrin (PE)-labeled CD14 (PE-CD14) for 30 min in the dark at room temperature. Samples were diluted 1:10 with HEPES-Tyrode’s buffer containing 5 nM/l EGTA and analyzed with an Ortho Cytoron Absolute Analyzer (Ortho Diagnostics). Flow cytometry was set to detect only particles bound to FITC-Ann V and PE-CD14 (Fig. 1).
S. Kanazawa et al. / Lung Cancer 39 (2003) 145 /149
147
Table 2 Microparticles, platelet activation markers, and sE-selectin in control subjects and lung cancer patients
4
MDMP (/10 plt) PDMP (/104plt) plt-gpIIb/IIIa (%) plt-gpIb (%) plt-CD62P (%) plt-CD63 (%) sE-selectin (ng/ml)
Control (n 30)
Lung cancer (n 64)
4697 245911 95.993.4 93.393.9 9.290.9 8.990.6 42.5916.1
79917* 543949* 95.793.0 94.194.1 24.391.9** 25.191.6** 84.7913.2*
MDMP, monocyte-derived microparticle; PDMP, platelet-derived microparticle; Other abbreviation, see Table 1. Values are shown as mean9S.E. *P B 0.01; **P B 0.001.
Fig. 1. Assessment of MDMP. Washed intact platelets was added to the plasma, and the mixture was incubated with FITC-labeled Annexin V and PE-labeled CD14 for 30 min in the dark at room temperature. Samples were diluted 1:10 with HEPES-Tyrode’s and analyzed with Flow cytometry. Only cells and particles positive for Annexin V were gated to distinguish platelets and microparticles from electronic noise (G1 gate). To differentiate between platelets and MPs, the lower limit of the platelet gate was set at the left limit of the forward-scatter profile of resting platelets. FITC-positive particles in the G2 gate were then counted to determine the number of MDMPs. Only particles bound to FITC-labeled Annexin V and PE-labeled CD14 were detected as MDMP.
2.4. Measurement of sE-selectin Blood samples from patients and healthy controls were collected into tubes with sodium citrate or tubes without anticoagulant. Blood was allowed to clot at room temperature for a minimum of 1 h serum or citrated plasma was isolated by centrifugation for 20 min at 1000 /g at 4 8C and then stored at /30 8C until analysis. As positive controls, in each assay we used the recombinant products and standard solutions provided with the commercial kits. Levels of serum sEselectin were measured with a monoclonal antibodybased ELISA kit from R & D Systems according to the manufacturer’s instructions. 2.5. Statistics Statistical analysis was performed with the paired or unpaired t-test, and P -values less than 0.05 were considered statistically significant.
than that in normal subjects. When levels of platelet activation markers were compared between the control and lung cancer groups, no between-group differences were detected in binding of anti-GPIIb/IIIa and antiGPIb monoclonal antibodies (Table 2). Levels of pltCD62P and plt-CD63, however, were significantly higher (P B/0.001 for each) in lung cancer patients than in controls (Table 2). Levels of sE-selectin were higher in lung cancer patients than in control subjects (Table 2). MDMP correlated positively with plt-CD62P, pltCD63, and PDMP with its relation to PDMP being particularly significant (Table 3). The number of MDMPs and PDMPs are patients who are NSCLC were significantly higher than that in SCLC (Table 4). In addition, levels of sE-selectin were higher in NSCLC than in SCLC patients (Table 4). The sE-selectin levels correlated with MDMP, PDMP, plt-CD62P, and plt-CD63 in NSCLC (Table 5).
4. Discussion The present study shows that levels of plt-CD62P, pltCD63, PDMP, and MDMP are elevated in patients with lung cancer, and that these elevations are the highest in NSCLC. Hyperactive platelets may lead to capillary microembolization because of the formation of microaggreTable 3 The correlation between MDMP and platelet activation markers (pltCD62P, plt-CD63, and PDMP) MDMP
3. Results We compared the results of MDMP and PDMP in normal subjects with those in patients with lung cancer (Table 2). The concentration of MDMP and PDMP in lung cancer patients was significantly higher (P B/0.001)
plt-CD62P plt-CD63 PDMP
r
P value
0.429 0.418 0.588
B 0.05 B 0.05 B 0.001
MDMP correlated positively with the platelet activation markers.
S. Kanazawa et al. / Lung Cancer 39 (2003) 145 /149
148
Table 4 Microparticles and sE-selectin in SCLC and NSCLC patients
MDMP (/104plt) PDMP (/104plt) sE-selectin (ng/ml)
SCLC (n 20)
NSCLC (n 44)
58913 422939 69.5910.2
99911* 601953* 101.4914.6*
Abbreviation, see Table 1. Values are shown as mean9S.E. *P B 0.01. Table 5 Relation between microparticles, platelet activation markers, and sEselectin in NSCLC sE selection
MDMP PDMP plt-CD62P plt-CD63
r
P value
0.423 0.413 0.491 0.447
B 0.05 B 0.05 B 0.01 B 0.01
n 44 patients.
gates [2]. In the present study, we measured PDMP and MDMP levels to assess coagulation in lung cancer. Levels of plasma PDMP and MDMP were both significantly higher in lung cancer patients than in control subjects. PDMPs play an important role in coagulation. Sims et al. [20] found that C5b-9 and thrombin increase the number of binding sites for factor V on PDMPs. They also showed that PDMPs play a major role in production of thrombin by the factor VaXa complex and that PDMPs are responsible for the procoagulant activity of platelets. Thus, increased levels of PDMPs may cause hypercoagulability. Increased PDMP levels are also observed in uremia and cerebral infarction [21]. In addition, increased PDMP levels occur in disease states where activated platelets have been detected in vivo [21]. Recently, White et al. [22] reported that NSCLC induce monocytes to increase expression of angiogenic activity. In the present study, we found that the levels of MDMPs in lung cancer patients are higher than those in controls. Mesri et al. [12] reported that co-culturing leukocytes with endothelium induces endothelial activation and inflammatory gene induction and that this pathway is mediated by membrane microparticles released from the activated leukocytes. Our current findings suggest that increased PDMPs and MDMPs in lung cancer patients may contribute to the development of angiogenesis. In the present study, no significant differences were detected in binding of anti-GPIIb/IIIa and anti-GPIb monoclonal antibodies between the control and lung cancer groups. Levels of CD62P- and CD63-positive platelets were higher in lung cancer patients than in control subjects. CD62P is glycoprotein located in the a -
granule membrane, and CD63 is located in the lysosomal integral membrane. When platelets are stimulated, CD62P and CD63 are rapidly redistributed to the platelet surface. This change in CD62P and CD63 localization is considered a marker of platelet activation [14]. Flow cytometric analysis of platelet CD62P and CD63 levels has been standardized [14,23]. With this technique, it has been shown that circulating levels of CD62P- and CD63-positive platelets are elevated in patients with thrombotic disease [23]. Our findings are consistent with those of these previous reports. Our present results on PDMP, MDMP, CD62P, and CD63 levels also suggest that enhanced platelet activity and related procoagulant activity in lung cancer may induce hypercoagulation. In the present study, both PDMP and MDMP levels were more increased in NSCLC patients than in those with SCLC, despite the fact that SCLC is the most metastasizing type of lung cancer. Levels of sE-selectin were also significantly increased in NSCLC patients. Glycochains as ligands of lung cancer include sialyl Lea and sialyl Lex. It has been proven that sialyl Lex is the ligand of selectin, and sialyl Lex level is correlated with matastatic ability in lung cancer [24]. Sialyl Lex is utilized as sialyl Lewisx-I antigen (SLX), and its specificity for adenocarcinoma is high. The importance of cell adhesion molecules and E-selectin in NSCSC has also been reported previously. Staal-van den Brekel et al. [25] described the immunohistochemical staining patterns of E-selectin, intercellular adhesion molecules (ICAM)-1 and VCAM-1 on endothelial cells of the vessels in tumor stroma and other types of cells in NSCLC associated with inflammatory cells. Tsumatori et al. [26] suggested relationship between serum Eselectin level and prognosis in patients with NSCLCs. The increased expression of adhesion molecules was also associated with activated monocytes. These activated monocytes could bind to vascular endothelial cells, and this binding could contribute to the development of angiogenesis in lung cancer patients. PDMPs and activated platelets may stimulate the activation of monocytes and promote the production of MDMPs. In our study, both MDMPs and PDMPs showed a positive correlation with activated platelets. Because MDMP concentration was especially high in the our NSCLC, we suggest that MDMP concentration in lung cancer patients may be a useful indicator of NSCLC progression. In conclusion, we measured and compared the levels of plasma MDMP and platelet activation markers to develop a better understanding of their potential contribution to vascular complications of lung cancer. Levels of CD62P- and CD63-positive platelets, and of plasma PDMP, MDMP, and sE-selectin were significantly higher in lung cancer patients than in controls. The number of MDMPs and PDMPs are patients who
S. Kanazawa et al. / Lung Cancer 39 (2003) 145 /149
are NSCLC were significantly higher than that in SCLC. In addition, levels of sE-selectin were higher in NSCLC than in SCLC patients. These findings suggest that elevated MDMPs may be a sign of vascular complication in lung cancer patients, particularly those who suffer from NSCLC.
Acknowledgements This study was partly supported by a grant from the Japan Foundation of Neuropsychiatry and Hematology Research, A Research Grant for Advanced Medical Care from the Ministry of Health and Welfare of Japan, and a Grant (13670760 to S.N.) from the Ministry of Education, Science and Culture of Japan.
References [1] Gordon SG. Cancer cell procoagulants and their role in malignant disease. Semin Thromb Hemost 1992;18:424 /33. [2] Honn KV, Tang DG, Chen YQ. Platelet and cancer metastasis: more than an epiphenomenon. Semin Thromb Hemost 1992;18:392 /415. [3] Dvorak HF. Thrombosis and cancer. Hum Pathol 1987;18:275 / 84. [4] El-Baruni K, Taylor I, Roath S, Francis JL. Factor X-activating procoagulant in normal and malignant breast tissue. Hematol Oncol 1990;8:323 /32. [5] Prydz H, Pettersen KS. Synthesis of thromboplastin (tissue factor) by endothelial cells. Haemostasis 1988;18:215 /23. [6] Adany R, Kappelmayer J, Berenyi E, Szegedi A, Fabian E, Muszbek L. Factors of the extrinsic pathway of blood coagulation in tumour associated macrophages. Thromb Haemost 1989;62:850 /5. [7] Koomagi R, Volm M. Tissue-factor expression in human nonsmall-cell lung carcinoma measured by immunohistochemistry: correlation between tissue factor and angiogenesis. Int J Cancer 1998;79:19 /22. [8] Sawada M, Miyake S, Ohdama S, Matsubara S, Masuda S, Yakumaru K, Yoshizawa Y. Expression of tissue factor in nonsmall-cell lung cancers and its relationship to metastasis. Br J Cancer 1999;79:472 /7. [9] Semeraro N, Montemurro P, Conese M, et al. Procoagulant activity of mononuclear phagocytes from different anatomical sites in patients with gynecological malignancies. Int J Cancer 1990;45:251 /4. [10] Rambaldi A, Alessio G, Casali B, Passerini CG, Donati MB, Mantovani A, Semeraro N. Induction of monocyte-macrophage procoagulant activity by transformed cell lines. J Immunol 1986;136:3848 /55.
149
[11] Satta N, Toti F, Feugeas O, et al. Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol 1994;153:3245 /55. [12] Mesri M, Altieri DC. Endothelial cell activation by leukocyte microparticles. J Immunol 1998;161:4382 /7. [13] Robinson RA, Worfolk L, Tracy PB. Endotoxin enhances the expression of monocyte prothrombinase activity. Blood 1992;79:406 /16. [14] Murakami T, Komiyama Y, Masuda M, et al. Flow cytometric analysis of platelet activation markers CD62P and CD63 in patients with coronary artery disease. Eur J Clin Invest 1996;26:996 /1003. [15] Miyazaki Y, Nomura S, Miyake T, et al. High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles. Blood 1996;88:3456 /64. [16] Nomura S, Suzuki M, Kido H, et al. Differences between platelet and microparticle glycoprotein IIb/IIIa. Cytometry 1992;13:621 / 9. [17] Miyake T, Nomura S, Komiyama Y, et al. Effect of a new monoclonal anti-glycoprotein IX antibody, KMP-9, on high shear-induced platelet aggregation. Thromb Haemost 1997;78:902 /9. [18] Yanabu M, Ozaki Y, Nomura S, et al. Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody. Blood 1997;89:1590 /8. [19] Xie GL, Nomura S, Fukuhara S. Annexin V expression and membrane vesiculation during activation of leukemic cell lines. Haemostasis 1997;27:259 /68. [20] Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and expres prothrombinase activity. J Biol Chem 1988;263:18205 /12. [21] Nomura S. Function and clinical significance of platelet-derived microparticles. Int J Hematol 2001;74:397 /404. [22] White ES, Strom SR, Wys NL, Arenberg DA. Non-small cell lung cancer cells induce monocytes to increase expression of angiogenic activity. J Immunol 2001;166:7549 /55. [23] Tschope D, Spangenberg P, Esser J, Schwippert B, Kehrel B, Rosen P, Gries FA. Flow cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actins status of activated platelets. Cytometry 1990;11:652 /6. [24] Inufusa H, Kojima N, Yasutomi M, Hakomori S. Human lung adenocarcinoma cell lines with different lung colonization potential (LCP), and a correlation between expression of sialosyl dimetric Le(x)(defined by Mab FH6) and LCP. Clin Exp Metastasis 1991;9:245 /57. [25] Staal-van den Brekel AJ, Thunnissen FB, Buurman WA, Wouters EF. Expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in non-small-cell lung carcinoma. Virchows Arch 1996;428:21 /7. [26] Tsumatori G, Ozeki Y, Takagi K, Ogata T, Tanaka S. Relation between the serum E-selectin level and the survival rate of patients with resected non-small cell lung cancers. Jpn J Cancer Res 1999;90:301 /7.
Monocyte-derived microparticles may be a sign of vascular complication in patients with lung cancer Shigenori Kanazawa, Shosaku Nomura *, Midori Kuwana, Mikiko Muramatsu, Kazuyuki Yamaguchi, Shirou Fukuhara The First Department of Internal Medicine, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8507, Japan Received 13 May 2002; received in revised form 23 September 2002; accepted 26 September 2002
Abstract We measured and compared the levels of plasma monocyte-derived microparticles (MDMP) and platelet activation markers [plasma platelet-derived microparticles (PDMP), CD62P binding to platelets; plt-CD62P, CD63 binding to platelets; plt-CD63], to develop a better understanding of their potential contribution to vascular complications of lung cancer. The concentrations of MDMP and PDMP in lung cancer patients were significantly higher (P B/0.01) than those in normal subjects. Levels of plt-CD62P and plt-CD63 were significantly higher (P B/0.001 for each) in lung cancer patients than in controls. Levels of sE-selectin were also higher in lung cancer patients than in control subjects. MDMP correlated positively with plt-CD62P, plt-CD63, and PDMP with its relation to PDMP being particularly significant. The number of MDMPs and PDMPs are patients who are non-small cell lung cancer were significantly higher than that in small cell lung cancer patients. In addition, levels of sE-selectin were higher in non-small cell lung cancer than in small cell lung cancer patients. These findings suggest that elevated MDMPs may be a sign of vascular complication in lung cancer patients, particularly those who suffer from non-small cell lung cancer. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Non-small cell lung cancer; Monocyte-derived microparticle; Platelet activation markers; sE-selectin
1. Introduction In malignant disease, there is an imbalance in activity of the coagulation and fibrinolytic systems that results in abnormal activation of coagulation, and this results in disseminated intravascular coagulation and deep vein thrombosis [1]. Abnormal hemostasis is a fundamental characteristic of malignant disease, and hemorrhagic and thromboembolic disorders are an important cause of morbidity and mortality in patients with cancer [2]. Tissue factor (TF) is the normal cellular activator of the coagulation system and is also an important activator of coagulation in malignant disease [3,4]. Particularly rich sources of TF include the lung, brain, and placenta, but TF is also found in epithelial cells, mononuclear cells, and many other cells and tissues [5,6]. Recently, it has
* Corresponding author. Tel.: /81-66-992-1001; fax: /81-728-333990 E-mail address: [email protected] (S. Nomura).
been suggested that TF also regulates tumor growth and angiogenesis, and Koomagi et al. reported the expression of TF and angiogenesis in non-small cell lung carcinomas (NSCLC)[7]. Sawada et al. also reported the significance of TF in NSCLC [8]. Many tumors have macrophage infiltrates that are activated by tumor cells. These macrophages have long been implicated in the coagulation abnormalities associated with solid tumors. There is increased TF activity in these tumor-associated macrophages [6,9]. Exposure of monocytes to tumor cells induces monocyte procoagulant activity [10]. Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules following stimulation by lipopolysaccharide [11]. This ability of endotoxin-stimulated monocytes to release heterogeneous monocyte-derived microparticles (MDMPs) may be an important mechanism regulating vascular cell effector functions [12]. The TF-exposing MDMPs promote prothrombinase complex assembly, thereby facilitating intravascular generation of thrombin and
0169-5002/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 9 - 5 0 0 2 ( 0 2 ) 0 0 4 4 1 - 5
S. Kanazawa et al. / Lung Cancer 39 (2003) 145 /149
146
enhanced procoagulant activity [13]. Procoagulant activity is also found in markers of platelet activation, such as platelet-bound CD62P (plt-CD62P) and PDMPs [14,15]. Thus, monocytes, platelets, and E-selectin could be very important in progression of vascular complications in lung cancer patients. To our knowledge, no other investigators have measured MDMP levels in lung cancer patients. We, therefore, measured and compared the levels of plasma MDMP and platelet activation markers [plasma PDMP, CD62P binding to platelets; plt-CD62P, CD63 binding to platelets; plt-CD63], to develop a better understanding of their potential contribution to vascular complications of lung cancer.
2. Patients and methods 2.1. Patients The study group included 30 healthy controls and 64 unrelated Japanese patients (45 men and 19 women) with lung cancer. All gave informed consent according to the Declaration of Helsinki. Table 1 shows the characteristics of patients and control subjects. The subjects ranged in age from 33 to 79 years. Twenty patients had small cell carcinoma, 16 had squamous cell carcinoma, and 28 had adenocarcinoma. Clinical staging of carcinoma was performed using the TMN classification. 2.2. Flow cytometry of activated platelets and microparticles Blood samples were collected into tubes containing 3.8% sodium citrate (9:1 vol/vol). Platelet-rich plasma (PRP) was prepared by centrifugation at 200 g for 10 min at room temperature. Washed platelets were prepared by centrifuging PRP at 1400 g for 10 min at room temperature. The resultant pellet was washed twice with washing buffer (9 mM/l Na2EDTA, 140 mM/l NaCl, and 26 mM/l Na2HPO4, pH 7.2) and resuspended in
HEPES-Tyrode’s buffer (129 mM/l NaCl, 8.9 mM/l NaHCO3, 0.8 mM/l KH2PO4, 0.8 mM/l MgCl2, 5.6 mM/ l glucose, and 10 mM/l HEPES, pH 7.4). An equal volume of 2% paraformaldehyde was added to the washed platelets, and theywere incubated for 15 min at room temperature. Platelets were then washed twice, resuspended in stock solution (9 mM/l Na2EDTA, 26.4 mM/l Na2HPO4 2H2O, 140 mM/l NaCl, 0.1% NaN3, and 2% fetal bovine serum, pH 7.2), and stored at 4 8C until analysis. PDMPs were detected with a modified version of a previously-reported method [16]. Ten microliters of platelets (3 /108/ml) added to 100 ml of HEPESTyrode’s buffer containing 5 nM/l EGTA. Both intact and aggregated platelets were removed by centrifugation at 1000 /g for 15 min to yield a supernatant containing microparticles only. Ten microliters of washed intact platelets (3 /108/ml) was then added to the supernatant, and the mixture was incubated with KMP-9 [17] (a FITC-labeled monoclonal antibody against platelet GPIX) for 30 min in the dark at room temperature. After incubation, samples were diluted 1:10 with HEPES-Tyrode’s buffer containing 5 nM/l EGTA and analyzed with an Ortho Cytoron Absolute Analyzer (Ortho Diagnostic Systems, Tokyo, Japan). Only cells and particles positive for GPIX were gated to distinguish platelets and PDMPs from electronic noise. To differentiate between platelets and PDMPs, the lower limit of the platelet gate was set at the left limit of the forward-scatter profile of resting platelets. Ten thousand FITC-positive particles in the PDMP gate were then counted to determine the number of PDMPs released per 10 000 platelets. As an index of platelet activation, CD62P expression was quantified by immunostaining with anti-CD62P monoclonal antibody (CLB-thromb/6, Immunotech, Marseille, France). Platelet expression of GPIIb/IIIa and of GPIb was analyzed with anti-GPIIb/ IIIa (NNKY1-32) [16] and anti-GPIb (NNKY5-5)[18] monoclonal antibodies, respectively.
2.3. Assessment of MDMP Table 1 Clinical characteristics of patients and healthy controls
Men/Women (no.) Age (years) WBC (/ml) PLT ( 104/ml) SCLC (no.) NSCLC (no.)
Control (n 30)
Lung cancer (n 64)
19/11 5195 46309502 22.494.9 0 0
48/16 5698 56509688 31.396.2 20 44
Control, non-lung cancer. Data are shown as mean9S.E.M. WBC, white blood cell count; PLT, platelet count; SCLC, small cell lung carcinoma; NSCLC, non-small cell lung carcinoma (squamous cell carcinoma 16; adenocarcinoma 28).
MDMP was detected by a previously reported method with some modifications [19]. Ten microliters of washed intact platelets (3 /108/ml) was added to the plasma, and the mixture was incubated with FITClabeled Annexin V (FITC-Ann V) and phycoerythrin (PE)-labeled CD14 (PE-CD14) for 30 min in the dark at room temperature. Samples were diluted 1:10 with HEPES-Tyrode’s buffer containing 5 nM/l EGTA and analyzed with an Ortho Cytoron Absolute Analyzer (Ortho Diagnostics). Flow cytometry was set to detect only particles bound to FITC-Ann V and PE-CD14 (Fig. 1).
S. Kanazawa et al. / Lung Cancer 39 (2003) 145 /149
147
Table 2 Microparticles, platelet activation markers, and sE-selectin in control subjects and lung cancer patients
4
MDMP (/10 plt) PDMP (/104plt) plt-gpIIb/IIIa (%) plt-gpIb (%) plt-CD62P (%) plt-CD63 (%) sE-selectin (ng/ml)
Control (n 30)
Lung cancer (n 64)
4697 245911 95.993.4 93.393.9 9.290.9 8.990.6 42.5916.1
79917* 543949* 95.793.0 94.194.1 24.391.9** 25.191.6** 84.7913.2*
MDMP, monocyte-derived microparticle; PDMP, platelet-derived microparticle; Other abbreviation, see Table 1. Values are shown as mean9S.E. *P B 0.01; **P B 0.001.
Fig. 1. Assessment of MDMP. Washed intact platelets was added to the plasma, and the mixture was incubated with FITC-labeled Annexin V and PE-labeled CD14 for 30 min in the dark at room temperature. Samples were diluted 1:10 with HEPES-Tyrode’s and analyzed with Flow cytometry. Only cells and particles positive for Annexin V were gated to distinguish platelets and microparticles from electronic noise (G1 gate). To differentiate between platelets and MPs, the lower limit of the platelet gate was set at the left limit of the forward-scatter profile of resting platelets. FITC-positive particles in the G2 gate were then counted to determine the number of MDMPs. Only particles bound to FITC-labeled Annexin V and PE-labeled CD14 were detected as MDMP.
2.4. Measurement of sE-selectin Blood samples from patients and healthy controls were collected into tubes with sodium citrate or tubes without anticoagulant. Blood was allowed to clot at room temperature for a minimum of 1 h serum or citrated plasma was isolated by centrifugation for 20 min at 1000 /g at 4 8C and then stored at /30 8C until analysis. As positive controls, in each assay we used the recombinant products and standard solutions provided with the commercial kits. Levels of serum sEselectin were measured with a monoclonal antibodybased ELISA kit from R & D Systems according to the manufacturer’s instructions. 2.5. Statistics Statistical analysis was performed with the paired or unpaired t-test, and P -values less than 0.05 were considered statistically significant.
than that in normal subjects. When levels of platelet activation markers were compared between the control and lung cancer groups, no between-group differences were detected in binding of anti-GPIIb/IIIa and antiGPIb monoclonal antibodies (Table 2). Levels of pltCD62P and plt-CD63, however, were significantly higher (P B/0.001 for each) in lung cancer patients than in controls (Table 2). Levels of sE-selectin were higher in lung cancer patients than in control subjects (Table 2). MDMP correlated positively with plt-CD62P, pltCD63, and PDMP with its relation to PDMP being particularly significant (Table 3). The number of MDMPs and PDMPs are patients who are NSCLC were significantly higher than that in SCLC (Table 4). In addition, levels of sE-selectin were higher in NSCLC than in SCLC patients (Table 4). The sE-selectin levels correlated with MDMP, PDMP, plt-CD62P, and plt-CD63 in NSCLC (Table 5).
4. Discussion The present study shows that levels of plt-CD62P, pltCD63, PDMP, and MDMP are elevated in patients with lung cancer, and that these elevations are the highest in NSCLC. Hyperactive platelets may lead to capillary microembolization because of the formation of microaggreTable 3 The correlation between MDMP and platelet activation markers (pltCD62P, plt-CD63, and PDMP) MDMP
3. Results We compared the results of MDMP and PDMP in normal subjects with those in patients with lung cancer (Table 2). The concentration of MDMP and PDMP in lung cancer patients was significantly higher (P B/0.001)
plt-CD62P plt-CD63 PDMP
r
P value
0.429 0.418 0.588
B 0.05 B 0.05 B 0.001
MDMP correlated positively with the platelet activation markers.
S. Kanazawa et al. / Lung Cancer 39 (2003) 145 /149
148
Table 4 Microparticles and sE-selectin in SCLC and NSCLC patients
MDMP (/104plt) PDMP (/104plt) sE-selectin (ng/ml)
SCLC (n 20)
NSCLC (n 44)
58913 422939 69.5910.2
99911* 601953* 101.4914.6*
Abbreviation, see Table 1. Values are shown as mean9S.E. *P B 0.01. Table 5 Relation between microparticles, platelet activation markers, and sEselectin in NSCLC sE selection
MDMP PDMP plt-CD62P plt-CD63
r
P value
0.423 0.413 0.491 0.447
B 0.05 B 0.05 B 0.01 B 0.01
n 44 patients.
gates [2]. In the present study, we measured PDMP and MDMP levels to assess coagulation in lung cancer. Levels of plasma PDMP and MDMP were both significantly higher in lung cancer patients than in control subjects. PDMPs play an important role in coagulation. Sims et al. [20] found that C5b-9 and thrombin increase the number of binding sites for factor V on PDMPs. They also showed that PDMPs play a major role in production of thrombin by the factor VaXa complex and that PDMPs are responsible for the procoagulant activity of platelets. Thus, increased levels of PDMPs may cause hypercoagulability. Increased PDMP levels are also observed in uremia and cerebral infarction [21]. In addition, increased PDMP levels occur in disease states where activated platelets have been detected in vivo [21]. Recently, White et al. [22] reported that NSCLC induce monocytes to increase expression of angiogenic activity. In the present study, we found that the levels of MDMPs in lung cancer patients are higher than those in controls. Mesri et al. [12] reported that co-culturing leukocytes with endothelium induces endothelial activation and inflammatory gene induction and that this pathway is mediated by membrane microparticles released from the activated leukocytes. Our current findings suggest that increased PDMPs and MDMPs in lung cancer patients may contribute to the development of angiogenesis. In the present study, no significant differences were detected in binding of anti-GPIIb/IIIa and anti-GPIb monoclonal antibodies between the control and lung cancer groups. Levels of CD62P- and CD63-positive platelets were higher in lung cancer patients than in control subjects. CD62P is glycoprotein located in the a -
granule membrane, and CD63 is located in the lysosomal integral membrane. When platelets are stimulated, CD62P and CD63 are rapidly redistributed to the platelet surface. This change in CD62P and CD63 localization is considered a marker of platelet activation [14]. Flow cytometric analysis of platelet CD62P and CD63 levels has been standardized [14,23]. With this technique, it has been shown that circulating levels of CD62P- and CD63-positive platelets are elevated in patients with thrombotic disease [23]. Our findings are consistent with those of these previous reports. Our present results on PDMP, MDMP, CD62P, and CD63 levels also suggest that enhanced platelet activity and related procoagulant activity in lung cancer may induce hypercoagulation. In the present study, both PDMP and MDMP levels were more increased in NSCLC patients than in those with SCLC, despite the fact that SCLC is the most metastasizing type of lung cancer. Levels of sE-selectin were also significantly increased in NSCLC patients. Glycochains as ligands of lung cancer include sialyl Lea and sialyl Lex. It has been proven that sialyl Lex is the ligand of selectin, and sialyl Lex level is correlated with matastatic ability in lung cancer [24]. Sialyl Lex is utilized as sialyl Lewisx-I antigen (SLX), and its specificity for adenocarcinoma is high. The importance of cell adhesion molecules and E-selectin in NSCSC has also been reported previously. Staal-van den Brekel et al. [25] described the immunohistochemical staining patterns of E-selectin, intercellular adhesion molecules (ICAM)-1 and VCAM-1 on endothelial cells of the vessels in tumor stroma and other types of cells in NSCLC associated with inflammatory cells. Tsumatori et al. [26] suggested relationship between serum Eselectin level and prognosis in patients with NSCLCs. The increased expression of adhesion molecules was also associated with activated monocytes. These activated monocytes could bind to vascular endothelial cells, and this binding could contribute to the development of angiogenesis in lung cancer patients. PDMPs and activated platelets may stimulate the activation of monocytes and promote the production of MDMPs. In our study, both MDMPs and PDMPs showed a positive correlation with activated platelets. Because MDMP concentration was especially high in the our NSCLC, we suggest that MDMP concentration in lung cancer patients may be a useful indicator of NSCLC progression. In conclusion, we measured and compared the levels of plasma MDMP and platelet activation markers to develop a better understanding of their potential contribution to vascular complications of lung cancer. Levels of CD62P- and CD63-positive platelets, and of plasma PDMP, MDMP, and sE-selectin were significantly higher in lung cancer patients than in controls. The number of MDMPs and PDMPs are patients who
S. Kanazawa et al. / Lung Cancer 39 (2003) 145 /149
are NSCLC were significantly higher than that in SCLC. In addition, levels of sE-selectin were higher in NSCLC than in SCLC patients. These findings suggest that elevated MDMPs may be a sign of vascular complication in lung cancer patients, particularly those who suffer from NSCLC.
Acknowledgements This study was partly supported by a grant from the Japan Foundation of Neuropsychiatry and Hematology Research, A Research Grant for Advanced Medical Care from the Ministry of Health and Welfare of Japan, and a Grant (13670760 to S.N.) from the Ministry of Education, Science and Culture of Japan.
References [1] Gordon SG. Cancer cell procoagulants and their role in malignant disease. Semin Thromb Hemost 1992;18:424 /33. [2] Honn KV, Tang DG, Chen YQ. Platelet and cancer metastasis: more than an epiphenomenon. Semin Thromb Hemost 1992;18:392 /415. [3] Dvorak HF. Thrombosis and cancer. Hum Pathol 1987;18:275 / 84. [4] El-Baruni K, Taylor I, Roath S, Francis JL. Factor X-activating procoagulant in normal and malignant breast tissue. Hematol Oncol 1990;8:323 /32. [5] Prydz H, Pettersen KS. Synthesis of thromboplastin (tissue factor) by endothelial cells. Haemostasis 1988;18:215 /23. [6] Adany R, Kappelmayer J, Berenyi E, Szegedi A, Fabian E, Muszbek L. Factors of the extrinsic pathway of blood coagulation in tumour associated macrophages. Thromb Haemost 1989;62:850 /5. [7] Koomagi R, Volm M. Tissue-factor expression in human nonsmall-cell lung carcinoma measured by immunohistochemistry: correlation between tissue factor and angiogenesis. Int J Cancer 1998;79:19 /22. [8] Sawada M, Miyake S, Ohdama S, Matsubara S, Masuda S, Yakumaru K, Yoshizawa Y. Expression of tissue factor in nonsmall-cell lung cancers and its relationship to metastasis. Br J Cancer 1999;79:472 /7. [9] Semeraro N, Montemurro P, Conese M, et al. Procoagulant activity of mononuclear phagocytes from different anatomical sites in patients with gynecological malignancies. Int J Cancer 1990;45:251 /4. [10] Rambaldi A, Alessio G, Casali B, Passerini CG, Donati MB, Mantovani A, Semeraro N. Induction of monocyte-macrophage procoagulant activity by transformed cell lines. J Immunol 1986;136:3848 /55.
149
[11] Satta N, Toti F, Feugeas O, et al. Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol 1994;153:3245 /55. [12] Mesri M, Altieri DC. Endothelial cell activation by leukocyte microparticles. J Immunol 1998;161:4382 /7. [13] Robinson RA, Worfolk L, Tracy PB. Endotoxin enhances the expression of monocyte prothrombinase activity. Blood 1992;79:406 /16. [14] Murakami T, Komiyama Y, Masuda M, et al. Flow cytometric analysis of platelet activation markers CD62P and CD63 in patients with coronary artery disease. Eur J Clin Invest 1996;26:996 /1003. [15] Miyazaki Y, Nomura S, Miyake T, et al. High shear stress can initiate both platelet aggregation and shedding of procoagulant containing microparticles. Blood 1996;88:3456 /64. [16] Nomura S, Suzuki M, Kido H, et al. Differences between platelet and microparticle glycoprotein IIb/IIIa. Cytometry 1992;13:621 / 9. [17] Miyake T, Nomura S, Komiyama Y, et al. Effect of a new monoclonal anti-glycoprotein IX antibody, KMP-9, on high shear-induced platelet aggregation. Thromb Haemost 1997;78:902 /9. [18] Yanabu M, Ozaki Y, Nomura S, et al. Tyrosine phosphorylation and p72syk activation by an anti-glycoprotein Ib monoclonal antibody. Blood 1997;89:1590 /8. [19] Xie GL, Nomura S, Fukuhara S. Annexin V expression and membrane vesiculation during activation of leukemic cell lines. Haemostasis 1997;27:259 /68. [20] Sims PJ, Faioni EM, Wiedmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and expres prothrombinase activity. J Biol Chem 1988;263:18205 /12. [21] Nomura S. Function and clinical significance of platelet-derived microparticles. Int J Hematol 2001;74:397 /404. [22] White ES, Strom SR, Wys NL, Arenberg DA. Non-small cell lung cancer cells induce monocytes to increase expression of angiogenic activity. J Immunol 2001;166:7549 /55. [23] Tschope D, Spangenberg P, Esser J, Schwippert B, Kehrel B, Rosen P, Gries FA. Flow cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actins status of activated platelets. Cytometry 1990;11:652 /6. [24] Inufusa H, Kojima N, Yasutomi M, Hakomori S. Human lung adenocarcinoma cell lines with different lung colonization potential (LCP), and a correlation between expression of sialosyl dimetric Le(x)(defined by Mab FH6) and LCP. Clin Exp Metastasis 1991;9:245 /57. [25] Staal-van den Brekel AJ, Thunnissen FB, Buurman WA, Wouters EF. Expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in non-small-cell lung carcinoma. Virchows Arch 1996;428:21 /7. [26] Tsumatori G, Ozeki Y, Takagi K, Ogata T, Tanaka S. Relation between the serum E-selectin level and the survival rate of patients with resected non-small cell lung cancers. Jpn J Cancer Res 1999;90:301 /7.