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© 2003 American Society for Clinical Oncology Potential Role of Platelets in Endothelial Damage Observed During Treatment With Cisplatin, Gemcitabine, and the Angiogenesis Inhibitor SU5416
From the Department of Medical Oncology, Department of Physiology, Institute for Cardiovascular Research, and Department of Clinical Epidemiology and Biostatistics, VU Medical Center; and Department of Vascular Medicine and Department of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam; Gaubius Laboratory TNO-PG, Leiden, the Netherlands; and Imperial College, Hammersmith Hospital, London, United Kingdom. Address reprint requests to H.M. Pinedo, MD, Department of Medical Oncology, VU Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands; email: HM.Pinedo{at}vumc.nl.
Purpose: An increased incidence of thromboembolic events was observed during treatment with cisplatin-gemcitabine plus SU5416 (CG+SU5416), a tyrosine kinase inhibitor targeting the vascular endothelial growth factor (VEGF) receptor-1 and -2. Nine thromboembolic events occurred in eight of 19 patients. We performed an analysis of parameters of the coagulation cascade and vessel wall activation. Materials and Methods: Markers for thrombin generation and endothelial cell activation were measured in three patients treated with CG+SU5416, two of whom developed a thromboembolic event. The results were compared with measurements in six patients treated with CG alone, and in 17 patients treated with SU5416 alone. Results: During cycles 1 and 2 of treatment with CG+SU5416, a significant cycle-dependent activation of both the coagulation cascade and endothelial cells occurred, whereas platelet counts decreased. Change in platelet number had a significant negative predictive effect on soluble (s)-E-selectin levels. Significant activation of the coagulation cascade only was observed in the patients treated with CG alone, whereas in patients treated with SU5416 alone, significant endothelial cell activation was observed. Conclusion: We hypothesize that endothelial cells deprived of VEGF after exposure to SU5416 became activated and more susceptible to damage during treatment with CG+SU5416, which was aggravated by a transient decrease in platelets, which are, among other things, carriers of VEGF. These results suggests that VEGF, in addition to being a permeability, proliferation, and migration factor, also is a maintenance and protection factor for endothelial cells, and that platelets may have a role in maintaining vascular integrity.
THE INHIBITION of angiogenesis is an experimental treatment modality in oncology that is being investigated in many clinical trials. Several antiangiogenic strategies have been developed.1,2 One of the key factors involved in angiogenesis is vascular endothelial growth factor (VEGF), which is produced by tumor cells. The effects of VEGF are mediated via several receptors, in particular VEGF receptor-2 (VEGFR-2; kinase domain receptor human homologue and fetal liver kinase-1 murine homologue), which is located mainly on endothelial cells. Disruption of the VEGF-VEGFR pathway by blocking the intracellular tyrosine kinase domain of VEGFR-2 may represent an attractive therapy. The compound SU5416 (Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone) is a small lipophilic, highly protein bound, synthetic molecule, which is metabolized mainly via cytochrome P-450 enzymes.3 SU5416 inhibits the autophosphorylation of VEGFR-1 and -2 that follows the interaction of VEGF with its receptor, thereby blocking the intracellular signaling pathway.4,5 Because of potential synergistic effects of classic chemotherapy plus angiogenesis inhibitors and the different toxicity profiles, combinations of these treatment modalities are being studied. During a phase I study investigating the feasibility of the combination of cisplatin-gemcitabine (CG) with SU5416, nine thromboembolic events occurred in eight of 19 patients. This incidence of thromboembolic events exceeds the frequencies reported with either CG chemotherapy or SU5416 monotherapy.6 Therefore, we analyzed specific markers of activation of the coagulation cascade and vessel wall activation in patients treated with this three-drug combination. We compared the results with those obtained in two control groups: patients treated with CG alone and patients receiving SU5416 alone.
Study Group Patients and treatment schedule, CG+SU5416. Patients were candidates for the phase I trial investigating CG+SU5416 if they had solid tumors amenable to treatment with CG. Study details, including eligibility criteria, study design, drug dosage and administration, and response evaluation have been reported previously.6 Briefly, patients received cisplatin 80 mg/m2 on day 1 and gemcitabine 1,250 mg/m2 on days 1 and 8, repeated every 3 weeks. SU5416 was administered on days 4, 8, 11, 15, and 18 in an initial dose of 85 mg/m2, which was escalated to 145 mg/m2 if no unacceptable toxicity occurred. To prevent allergic reactions to Cremophor, the solvent of SU5416, dexamethasone, clemastine, and cimetidine were given before every infusion. Antiemetic treatment during cisplatin administration consisted of twice daily ondansetron 8 mg and dexamethasone 8 mg. Between October 1999 and March 2000, the phase I trial of CG plus SU5416 enrolled 19 patients. No pharmacologic drug-drug interactions were observed and antitumor activity was similar to that expected in the patient population selected for this study.6 However, eight of the 19 study patients developed thromboembolic events, including two cerebrovascular accidents (CVAs), three transient ischemic attacks (TIAs), and four deep venous thromboses (DVTs), which in two patients were complicated by pulmonary emboli. These thromboembolic events occurred at both dose levels of SU5416. Other toxic effects observed were the same as have been reported with SU5416 alone (headache and phlebitis) and with CG (nausea, vomiting, fatigue, thrombocytopenia, and neutropenia). The high incidence of thromboembolic events led to the termination of this phase I study.
After informed consent was obtained, blood samples were drawn from three patients in the CG+SU5416 group on days 1, 4, 8, and 18 of the first and second cycle. Two of the three sampled patients developed a thromboembolic event (Table 1
Control Groups Patients and treatment schedule, CG. The first control group included a total of six patients with non–small-cell lung cancer (NSCLC). Two patients had stage IIIa disease, and were treated with neoadjuvant cisplatin 100 mg/m2 on day 1 and gemcitabine 1,000 mg/m2 on days 1, 8, and 15 every 4 weeks. The other four patients included one patient with stage IIIb and three patients with stage IV disease who were treated with cisplatin 80 mg/m2 on day 1 and gemcitabine 1,250 mg/m2 on days 1 and 8 every 3 weeks (CG-alone group; Table 1  ). Antiemetic treatment during cisplatin administration was the same as described above. None of the patients in the CG-alone group developed a thromboembolic event during or after treatment. Blood samples were collected on days 1, 8, and 15 of the first and second chemotherapy cycle. Informed consent was obtained.
Patients and treatment schedule, SU5416 alone.
The second control group consisted of seventeen patients with histologically proven, advanced, progressive soft tissue sarcoma, melanoma, or renal cell carcinoma who were participating in any of three multicenter phase II trials of single-agent therapy with SU5416 145 mg/m2 twice weekly (Table 2
The SU5416-alone group was reported on elsewhere.7 Three patients developed a thromboembolic or vascular event during SU5416 treatment. Blood samples were collected on days 1, 15, and 29 after informed consent was obtained.
Processing of Blood Samples
Assays The von Willebrand antigen (vWF) was measured with an ELISA according to methods reported previously.9 Soluble tissue factor (s-TF) and soluble (s)-E-selectin were determined with their respective ELISAs (American Diagnostics, Greenwich, CT; R&D Systems, Abingdon, United Kingdom). The plasma concentrations of VEGF and basic fibroblast growth factor (bFGF) were both assayed by a quantitative sandwich enzyme immunoassay (R&D Systems).
Statistical Analysis
Thrombin Generation During the first and second cycles of CG+SU5416, all three patients exhibited similar, clearly cycle-dependent increases in the levels of TAT complexes, F1+2, and ETP as shown for F1+2 and ETP in Fig 1A  and 1B. Even though SU5416 was infused on day 18, TAT complexes, F1+2, and ETP decreased after day 18 of the first course, returning to basal values by day 1 of the second cycle. In the second cycle, a similar pattern of activation occurred.
In the SU5416-alone group, we observed no clear increases in the levels of TAT complexes (not shown) and F1+2 (Fig 1A ). The potential of the plasma to form thrombin, as measured with ETP, increased significantly during SU5416 therapy (Fig 1B).7 In the CG-alone group, a slight increase in the coagulation parameters was observed (F1+2 in Fig 1A) that turned out to be statistically significant (P < .05; Table 3).
Parameters of Endothelial Cell Activation The three parameters indicating endothelial cell activation (vWF, s-E-selectin, and s-TF) increased similarly in all three patients during the first and second cycles of CG+SU5416 as shown for s-E-selectin in Fig 1C . These parameters decreased before the start of the second cycle and increased during the second cycle, similar to the pattern noted for the coagulation markers. The parameters of cellular activation did not change during treatment with CG alone (s-E-selectin in Fig 1C). In the patients receiving SU5416 alone, a gradual increase in the levels of vWF, s-E-selectin, and s-TF occurred (s-E-selectin in Fig 1C).
We observed a strong correlation between a parameter of thrombin generation (F1+2) and a parameter of endothelial cell perturbation (s-E-selectin) during the first and second cycle of CG+SU5416 (Fig 2
Growth Factors Basal plasma VEGF levels were elevated in all patients, with no significant changes seen during any of the treatments (Fig 1D ). bFGF levels were within normal limits in all patients and no treatment effects were observed (data not shown).
Leukocytes and Platelets
Statistical Analysis
The second analysis showed a significant increase of F1+2 in the CG-alone group (P < .05) and the CG+SU5416 group (P < .001; Table 3 The third analysis showed that the change in F1+2 levels could in all three groups significantly be explained by the change in s-E-selectin levels. The significant effect of the change in platelets in the CG+SU5416 group vanishes in the presence of the predictor s-E-selectin.
The main goal of this study was to explain the alarming incidence of thromboembolic events in patients treated with CG+SU5416. Unfortunately, we had the opportunity to analyze the coagulation cascade and endothelial cell activation in only three patients treated with this combination. However, during the first and second cycles, we observed similar patterns of all parameters that reflected endothelial cell and coagulation activation in all three patients, irrespective of the development of a thromboembolic event. These results were compared with two control groups treated with CG alone and SU5416 alone. Although this is a limited data set with wide confidence intervals, and control groups differ in diagnosis, stage of the disease, and probably in risk factors for the development of thromboembolic events, our data strongly indicate the following: SU5416 alone induced significant endothelial cell activation; CG alone induced a significant activation of the coagulation cascade; CG+SU5416 induced endothelial cell activation as well as activation of the coagulation cascade; the change in s-E-selectin levels, which reflects endothelial cell activation, predicts activation of coagulation (F1+2); and the decrease and the rebound in platelet number had a significant effect on the condition of the endothelium as estimated from the endothelial cell activation parameters. These findings indicate that VEGF signaling in endothelial cells is involved in the prevention of thromboembolic events. This supports the theory that VEGF is not only a permeability, proliferation, and migration factor, but also a maintenance and protection factor for endothelial cells during adult life. Consequently, platelets, which transport VEGF along with other growth factors and proteins, may play a role in maintaining vascular integrity.12 Recently, we have shown that blocking of the VEGF-VEGFR pathway with SU5416 alone did not affect fibrinolysis, the protein C pathway, or thrombin generation, but did induce endothelial cell activation.7 This indicates that endothelial cells may need a basal degree of VEGF signaling to remain in a nonactivated state. Because VEGF stimulates the expression of antiapoptotic genes and proteins, such as bcl-2 and survivin, endothelial cells may become susceptible to apoptosis during treatment with SU5416.13 The mechanism by which the cytotoxic agents induce a shift toward a procoagulant state is not completely clear. It is unknown whether gemcitabine affects the coagulation cascade or endothelial cells. From in vitro studies it is known that cisplatin activates platelets, mononuclear cells, and endothelial cells, which together may result in a shift to a prothrombotic state.14–16 We noted an interesting contrast between the cyclic pattern of endothelial cell and coagulation activation during treatment with CG+SU5416 versus a gradual increase in endothelial cell activation without activation of coagulation during treatment with SU5416 alone. This difference was evidently associated with cyclic changes in circulating platelets and mononuclear cells caused by the myelotoxic agents. The opposite pattern of platelet and leukocyte counts versus the endothelial cell and coagulation parameters strongly indicates that, in addition to VEGF, platelets and possibly mononuclear cells also contribute to maintaining a quiescent state of the endothelium. There is evidence that platelets, which are known as transporters of many factors, including VEGF, have trophic effects on endothelial cells and possibly "nurture" the endothelium.12,17–19 In vitro experiments show that increasing numbers of nonactivated- and thrombin-activated platelets stimulate endothelial cell proliferation.20 Furthermore, a mathematical model suggested that platelets adherent to the (sub)endothelium inhibited the activity of vessel-bound enzymes such as the TF-factor VIIa complex.21,22 Because CG induces a temporary decrease in the number of platelets, which is cumulative with increasing numbers of cycles, a decrease of trophic factors for endothelial cells does occur.23 The rebound of platelets between day 18 of the first cycle and day 1 of the second cycle probably restored the amount of trophic factors, resulting in repair of damaged endothelial cells, which is reflected by the normalization of the endothelial cell and coagulation parameters by the end of the cycle in the patients treated with CG+SU5416. Thus, platelets may have an important role as regulators of endothelial cell function.24 Recently, it has been shown that human platelets have functional VEGFRs that seem to be cryptic receptors that become exposed on the platelet membrane during platelet activation.25 They probably have a role in fine-tuning of platelet activation, which is possibly also affected during treatment with SU5416. In conclusion, treatment with SU5416 induces vulnerable endothelial cells, which may undergo apoptosis when SU5416 is combined with CG. Vascular integrity might be affected by the decrease in platelets and loss of platelet function, resulting in the exposure of blood to activated and/or apoptotic endothelial cells and, subsequently, subendothelium and TF, which is the trigger for activation of the coagulation cascade. To date, no increased incidence of thromboembolic events has been observed with SU5416 in combination with other cytotoxic agents, such as fluorouracil with or without irinotecan, and adriamycin (although two catheter-related thromboses and two peripheral DVTs were observed [B. Overmoyer, personal communication, July 2002]).26–28 However, in combination with paclitaxel-carboplatin, a schedule that also induces thrombocytopenia, catheter-related thrombosis and nonfatal myocardial infarction were observed.29 This indicates that combination of SU5416 with cytotoxic agents that induce thrombocytopenia is potentially dangerous for the development of thromboembolic events, which is explained by a combined effect on the endothelium and the platelets. Close monitoring of coagulation and endothelial cell parameters is indicated during investigational combinations of angiogenesis inhibitors with cytotoxic agents. At least one parameter reflecting thrombin generation, such as TAT complexes or F1+2, and one parameter reflecting endothelial cell perturbation, such as s-E-selectin or vWF, should be monitored. Alternatively, one could measure the potential of the plasma to form thrombin with the ETP. The role of platelets has been underestimated and insufficiently investigated; therefore, additional studies investigating the role of platelets in maintaining endothelial cell function are warranted.
Supported by grant D96.021 from the Netherlands Heart Foundation (J.C.M.M.).
1. Carmeliet P, Jain RK: Angiogenesis in cancer and other diseases. Nature 407:249–257, 2000[CrossRef][Medline] 2. Levitzki A: Protein tyrosine kinase inhibitors as novel therapeutic agents. Pharmacol Ther 82:231–239, 1999[CrossRef][Medline] 3. Mendel DB, Laird AD, Smolich BD, et al: Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent. Anticancer Drug Des 15:29–41, 2000[Medline]
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7. Kuenen BC, Levi M, Meijers JCM, et al: Analysis of the coagulation cascade and endothelial cell activation during inhibition of the VEGF/VEGF-receptor pathway in cancer patients. Arterioscler Thromb Vasc Biol 22:1500–1505, 2002 8. Wielders S, Mukherjee M, Michiels J, et al: The routine determination of the endogenous thrombin potential, first results in different forms of hyper- and hypocoagulability. Thromb Haemost 77:629–636, 1997[Medline]
9. Cejka J: Performance characteristics of a commercial kit for assay of factor VIII-related antigen. Clin Chem 30:814–815, 1984 10. Laird NM, Ware JH: Random-effects models for longitudinal data. Biometrics 38:963–974, 1982[CrossRef][Medline] 11. Goldstein H, Rasbash J, Plewis I, et al: A User’s Guide to MLwiN. London, United Kingdom, Institute of Education, University of London, 1998
12. Verheul HM, Hoekman K, Luykx-de Bakker S, et al: Platelet:Transporter of vascular endothelial growth factor. Clin Cancer Res 3:2187–2190, 1997 13. Benjamin LE: The controls of microvascular survival. Cancer Metastasis Rev 19:75–81, 2000[CrossRef][Medline] 14. Togna GI, Togna AR, Franconi M, et al: Cisplatin triggers platelet activation. Thromb Res 99:503–509, 2000[CrossRef][Medline] 15. Sodhi A, Pai K: Increased production of interleukin-1 and tumor necrosis factor by human monocytes treated in vitro with cisplatin or other biological response modifiers. Immunol Lett 34:183–188, 1992[CrossRef][Medline] 16. Gan XH, Jewett A, Bonavida B: Activation of human peripheral-blood-derived monocytes by cis-diamminedichloroplatinum: Enhanced tumoricidal activity and secretion of tumor necrosis factor-alpha. Nat Immun 11:144–155, 1992[Medline]
17. Verheul HM, Hoekman K, Lupu F, et al: Platelet and coagulation activation with vascular endothelial growth factor generation in soft tissue sarcomas. Clin Cancer Res 6:166–171, 2000 18. Gimbrone MA Jr, Aster RH, Cotran RS, et al: Preservation of vascular integrity in organs perfused in vitro with a platelet-rich medium. Nature 221:33–36, 1969 19. Pinedo HM, Verheul HM, D’Amato RJ, et al: Involvement of platelets in tumour angiogenesis? Lancet 352:1775–1777, 1998[CrossRef][Medline]
20. Verheul HM, Jorna AS, Hoekman K, et al: Vascular endothelial growth factor-stimulated endothelial cells promote adhesion and activation of platelets. Blood 96:4216–4221, 2000 21. Kuharsky AL, Fogelson AL: Surface-mediated control of blood coagulation: The role of binding site densities and platelet deposition. Biophys J 80:1050–1074, 2001[Medline] 22. Diamond SL: Reaction complexity of flowing human blood. Biophys J 80:1031–1032, 2001[Medline]
23. Kroep JR, Peters GJ, van Moorsel CJ, et al: Gemcitabine-cisplatin: A schedule finding study. Ann Oncol 10:1503–1510, 1999 24. Shepro D: The American Microcirculatory Society Landis Award lecture: Endothelial cells, inflammatory edema, and the microvascular barrier—Comments by a "free radical." Microvasc Res 35:246–264, 1988[Medline] 25. Selheim F, Holmsen H, Vassbotn FS: Identification of functional VEGF receptors on human platelets. FEBS Lett 512:107–110, 2002[CrossRef][Medline] 26. Rosen PJ, Amado R, Hecht JR, et al: A phase I/II study of SU5416 in combination with 5-FU/leucovorin in patients with metastatic colorectal cancer. Proc Am Soc Clin Oncol 19:3a, 2000 (abstr 5D) 27. Rothenberg M, Berlin J, Cropp G, et al: Phase I/II study of SU5416 in combination with irinotecan/5-FU/LV (IFL) in patients with metastatic colorectal cancer. Proc Am Soc Clin Oncol 20:75a, 2001 (abstr 298) 28. Overmoyer B, Robertson K, Persons M, et al: A phase I pharmacokinetic and pharmacodynamic study of SU5416 and doxorubicin (ADR) in inflammatory breast cancer (IBC). Proc Am Soc Clin Oncol 20:99a, 2001 (abstr 391) 29. Rosen P, Kabbinavar F, Figlin R, et al: A phase I/II trial and pharmacokinetic study of SU5416 in combination with paclitaxel/carboplatin. Proc Am Soc Clin Oncol 20:98a, 2001 (abstr 389) Submitted August 6, 2002; accepted March 3, 2003.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
k + ßk log (PLit/PLi1) + 
k log (sEit/sEi1) + 
it. The index k distinguishes the three treatment groups, PL is platelets and sE is s-E-selectin. This means that a separate intercept 
, n = 3), CG alone (•, n = 6), and SU5416 alone (
, n = 17). Values between or below dashed lines are normal.
