http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–8 ! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2013.870332
ORIGINAL ARTICLE
The integrin antagonist, cilengitide, is a weak inhibitor of aIIbb3 mediated platelet activation and inhibits platelet adhesion under flow Sascha Meyer dos Santos1 Karina Kuczka1, Bettina Picard-Willems1, Karen Nelson2, Ute Klinkhardt3, & Sebastian Harder1
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Institute for Clinical Pharmacology, J.W. Goethe University Hospital, Frankfurt, Germany, 2Department of Vascular and Endovascular Surgery, J.W. Goethe University Hospital, Frankfurt, Germany, and 3Merck KGaA, Darmstadt, Germany
Abstract
Keywords
The RGD cyclic pentapetide, cilengitide, is a selective inhibitor of avb3 and avb5 integrins and was developed for antiangiogenic therapy. Since cilengitide interacts with platelet aIIbb3 and platelets express av integrins, the effect of cilengitide on platelet pro-coagulative response and adhesion is of interest. Flow-based adhesion assays were performed to evaluate platelet adhesion and rolling on von Willebrand factor (vWf), on fibrinogen and on human umbilical vein endothelial cells (HUVECs). Flow cytometry was used to detect platelet activation (PAC1) and secretion (CD62P) by cilengitide and light transmission aggregometry was used to detect cilengitide-dependent platelet aggregation. Cilengitide inhibited platelet adhesion to fibrinogen at concentrations above 250 mM [which is the Cmax in human studies] and adhesion to vWf and HUVECs at higher concentrations under physiologic flow conditions. Platelet aggregation was already impaired at cilengitide concentrations 410 mM. Activation of aIIbb3 integrin was inhibited by 250 mM cilengitide, whereas platelet secretion was unaffected by cilengitide. No evidence of cilengitide-induced platelet activation was found at all tested concentrations (0.01–1500 mM). At higher concentrations, platelet activation was inhibited, predominantly due to aIIbb3 inhibition.
Alpha-v beta-3 Integrin, angiogenesis inhibitors, cilengitide, platelet membrane glycoproteins receptors, thrombosis
Introduction Thromboembolic events and pulmonary embolism are side effects of the VEGF-neutralizing antibody bevacizumab, which interferes with endothelial VEGF receptors and inhibits angiogenesis [1, 2]. Disturbance of vascular homeostasis by blocking VEGF may lead to endothelial dysfunction and adverse vascular effects [3], and it has been speculated that thromboembolism may be a general feature of anti-angiogenic chemotherapy [4] since thromboembolism often occurs in cancer patients. Venous thromboembolism risk is particularly high in patients with glioma with about 12% in the first three months after diagnosis, which is more than twice that of patients with other malignancies [5]. Integrins are a family of cell-extracellular matrix adhesion molecules, playing important roles in tumor angiogenesis. ab3-integrin has received much attention as a potential anticancer target because it is highly expressed on both tumor cells and in new vasculature of diverse tumors. ab3 also plays a role in cell migration and extravasation, which occurs during metastasis. Two currently approved integrin antagonists are available, though not intended for oncologic indications. Several compounds are under clinical evaluation for treating malignancy. Cilengitide, a cyclic RGD pentapeptide, is a selective inhibitor of avb3 and
Correspondence: Sascha Meyer dos Santos, Institute for Clinical Pharmacology, J.W. Goethe University Hospital, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Tel: +49 69 6301 7622; Fax: +49 69 6301 83921. E-mail: [email protected]
History Received 24 July 2013 Revised 23 October 2013 Accepted 25 November 2013 Published online 16 January 2014
avb5 integrins, with IC50 values in the low nanomolar range in solid phase assays. Cilengitide has shown considerably lower affinity for other integrins (avb1, avb6, and avb8), with IC50 concentrations 10- to 100-fold higher in receptor binding assays. Cilengitide has demonstrated anti-angiogenic as well as direct anti-tumor and anti-invasive effects in various animal models [6–8]. It has recently been investigated in a large Phase III trial (CENTRIC), including patients with newly diagnosed glioblastoma with a methylated O6-methylguanine-DNA methyltransferase (MGMT) gene promoter. A phase I/II study with cilengitide is currently ongoing in non-small cell lung cancer (NSCLC) and further trials are being conducted by the U.S. National Cancer Institute (NCI). Despite promising clinical Phase I and II results, cilengitide did not meet its primary endpoint of significantly increasing overall survival, when added to a current standard chemo-radiotherapy regimen or temozolomide and radiotherapy in this pivotal trial [9]. Development of cilengitide by Merck was stopped, but academic research investigating its use for other indications is still ongoing (e.g. NCT01118676, NCT01517776). Since b3 integrins are expressed on platelets, and since a weak interaction of cilengitide with aIIbb3 has been reported [10] platelet interaction with avb3 integrin antagonists is conceivable. This might lead to platelet activation via outside-in signaling, or even platelet inhibition via blockage of the aIIbb3 b3 integrin, which is the main binding site for fibrinogen. The aim of this study was to evaluate the in vitro profile of cilengitide on various parameters of platelet function and platelet-endothelial interaction under static and flow conditions.
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Methods The study protocol regarding blood withdrawal for scientific purposes was approved by the Ethics Committee of the University Hospital of Frankfurt. Prior to inclusion all subjects gave written, informed consent for blood donation.
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Preparation of blood samples for flow-based adhesion assays Suspensions of calcein-AM labeled platelets in washed blood were prepared as has previously been described [11, 12], with minor modifications. Briefly, blood was centrifuged at 3300 g for 5 minutes, washed three times with Tyrode’s buffer1 and then centrifuged at 102 g for 15 minutes. Platelets in the supernatant were collected and fluorescently labeled with calcein-AM for 30 minutes in the dark. They were then added back to the red blood cells, which were washed once more in Tyrode’s buffer. The cells were finally resuspended in the original volume. Incubation with cilengitide Pharmacokinetic studies revealed steady-state plasma peak levels of 169 21 mg/ml cilengitide after a standard 2000 mg infusion, which corresponds to a concentration of 286 mM. However, individual peak/trough concentrations may vary and therefore a supra- as well as sub-therapeutic concentration range was employed. Final concentrations were 0, 0.01, 10, 100, 250, 750, and 1500 mM cilengitide. Platelet aggregation assay The platelet aggregation assay was performed in citrated Platelet Rich Plasma (PRP) with a turbidimetric, light-transmittance device (APACT) within 1 hour after blood sampling. Aggregation inducers were collagen (SKF Horm, Nycomed) at a final concentration of 1 mg/ml and ADP (Sigma) at a final concentration of 5 mM. Flow cytometry assay The flow cytometric determination of platelet activation status focused on parameters that represented different platelet functions: CD62P expression is a marker of platelet degranulation, whereas PAC-1-binding reflects the activation induced conformational change of the aIIbb3 receptor. The following antibodies and reagents were used: anti-CD62P-PE (Immunotech), and PAC-1-FITC (BD Biosciences). After the first incubation step of PRP with cilengitide or vehicle, about 2 106 platelets were resuspended in PBS with Ca2þ/Mg2þ at a final volume of 100 ml. The samples were activated with 5 mM TRAP-6. Baseline samples were not activated. After 10 minutes incubation, 20 ml antiCD62P-PE and 20 ml PAC-1-FITC were added. After another incubation period of 20 minutes, 2 ml PBS with Ca2þ/Mg2þ were added to each sample tube. The samples were centrifuged (300 g for 5 min at room temperature), decanted and resuspended in 500 ml Cellfix (1:10 dilution with double distilled water; Becton Dickinson). All measurements were carried out using a FACScanTM flow cytometer (Becton Dickinson), data acquisition with CellQuest Pro software (Becton Dickinson). A total of 4–6 experiments were carried out for each cilengitide concentration. Platelet adhesion to selected subendothelial matrix components To determine the effect of cilengitide on adhesion to vWf, platelets reconstituted with red blood cells containing the desired cilengitide concentration, 0.15 mM osteopontin or abciximab were
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perfused at wall shear rates of 150 , 600, and 1800 s1 for 5 minutes each. Adhesion assays were performed in transparent chambers designed for microscopic observation of cells or cell cultures under flow conditions (m-slide I 0.4 mm in height; Ibidi, Munich, Germany). The chambers were filled with a solution containing 13.3 mg rvWf (recombinant von Willebrand factor) or 100 mg fibrinogen and left overnight at 4 C in a humid environment to coat the chambers. The flow chambers were blocked with 2% BSA for 2 hours, washed and placed under an epifluorescent microscope. Perfusion experiments were carried out as has been described [11]. Platelet adhesion to human endothelial cells To study platelet adhesion to endothelial cells, HUVECs (Lonza) were cultivated to confluency in flow chambers in the presence of the desired cilengitide concentration for 18 hours. The flow chambers were placed under the epifluorescent microscope and the reconstituted blood containing the corresponding cilengitide concentration was aspirated through the chambers and platelet interaction with endothelial cells was visualized in real time. The entire period of perfusion with at least three randomly selected fields of view was recorded and processed. Calculations Data for all assays were tabulated and subjected to descriptive statistics. Aggregometry Data obtained after cilengitide addition was compared with that from untreated controls by one-way ANOVA and corrected for multiple comparisons by Dunnett’s test. A p value 0.05 was considered significant. For FACS-experiments, expression of PAC1 or CD62P (given in mean fluorescence intensity) was converted to percent of the value of the untreated control, which was set to 100%. Cilengitide data was compared with control by one-sample t-test and a p value 0.05 was considered significant. Flow based adhesion assays For each shear rate at least three randomly selected fields of view were recorded and the accumulation rate of adherent platelets within each observation period was computed in Graph Pad Prism and set to 100% for untreated control blood. Cilengitide data was compared to control by one-sample t-test and a p value 0.05 was considered significant.
Results Platelet adhesion onto human endothelial cells An in vitro flow chamber model was used to evaluate the effects of the anti-integrin drug cilengitide on platelet adhesion mechanisms. To mimic the situation in blood vessels, we used confluent HUVECs cultured in flow chambers as a model to determine cilengitide effects on platelet-endothelial interactions. After 18 hours incubation, cilengitide was added to the perfusion medium and the confluent endothelial cell monolayer was perfused with blood containing the corresponding cilengitide concentration. The interaction of fluorescently labeled platelets with HUVECs was recorded in real time and the number of cilengitide treated platelets sticking firmly to the cilengitide treated HUVECs was measured. Platelet accumulation on HUVECs was inhibited concentration-dependent at all shear rates. However, the effect remained non-significant at clinically relevant concentrations. Application of cilengitide at
DOI: 10.3109/09537104.2013.870332
supratherapeutic concentrations of 1500 mM weakly inhibited the accumulation of platelets on the endothelial cells at all shear rates. At elevated shear rates (1800 s1), a weak but significant inhibition of platelet adhesion was also seen at a concentration of 750 mM (Figure 1). Thus, these experiments prove that high cilengitide concentrations tend to reduce platelet adhesion to the endothelium. None of the tested concentrations stimulated the adhesion of platelets.
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Platelet aggregation The following experiments were designed to investigate the role of b3 integrins on the cilengitide-dependent inhibition of platelet adhesion. The influence of cilengitide on platelet aggregation was evaluated by light transmission aggregometry. PRP was activated by threshold doses of 5 mM ADP and 1 mg/ml collagen in the presence of 0–1500 mM cilengitide. Platelet aggregation was unaffected by low nanomolar concentrations of cilengitide, however, inhibition began to appear between 1 and 10 mM. At cilengitide concentrations 100 mM, platelet aggregation induced by either ADP or collagen was almost completely absent (Figure 2). Cilengitide, therefore, showed no proaggregatory properties, but instead, inhibited the platelet–platelet interaction. Often, such homotypic interaction is driven by bridging aIIbb3 on platelets. Flow cytometry For non-activated samples, only cilengitide concentrations of 1500 mM showed inhibitory effects. No activating effects on
Cilengitide has no pro-thrombotic properties
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resting platelets were observed for any concentration of cilengitide tested. However, flow cytometry revealed a strong inhibitory influence of cilengitide on the expression of activated aIIbb3 after stimulation with 5 mM TRAP-6 (peptide fragment (residues 42–47) of protease-activated receptor 1 (PAR1) that acts as a PAR1 agonist) [13] at concentrations 250 mM (Figure 3A). To test whether cilengitide competes with fibrinogen binding to aIIbb3, fibrinogen binding to activated platelets in the presence of cilengitide was measured. Cilengitide physically interfered with fibrinogen binding to aIIbb3 at all tested concentrations (Figure 3B). Cilengitide had no effect on the expression of platelet activation and secretion marker CD62P at all concentrations tested, neither in resting platelets nor in TRAP stimulated platelets (Figure 3C). Platelet adhesion at subendothelial matrix components Cilengitide inhibited the adhesion of platelets to endothelial cells at supratherapeutic doses. Aggregometry and flow-cytometric data show that cilengitide dose-dependently down-regulated the expression of activated aIIbb3. To test the contribution of different platelet b3 integrins and shear stress on cilengitide induced effects, platelets in reconstituted blood were perfused over vWf in the presence of b3 inhibitors and the adhesion to cilengitide treated platelets was compared. Osteopontin was used as a selective ab3 ligand, since Hu and coworkers [14] have reported that osteopontin does not bind to integrin aIIbb3 with measurable affinity. Measurements of
Figure 1. Fluorescence-labeled platelets in reconstituted human blood containing cilengitide (0.01, 250, 750, or 1500 mM) or buffer alone (0 mM) was perfused over confluent HUVEC monolayers at the indicated wall shear rate. The number of adherent platelets was determined by microscopic image processing and the adhesion of control blood (0 mM cilengitide) was set to 100%. The results are the mean SEM of six independent experiments with blood from different donors. *p50.05, **p50.01 in one-sample t-test vs. 100% (the untreated control).
Figure 2. Platelet aggregation in PRP was induced by 5 mM ADP, 1 mg/ml collagen, and 20 mM TRAP and monitored by turbidimetric aggregometry. Shown is the aggregation response as percentage of maximal light transmission Amax of six experiments in untreated PRP. In one-way ANOVA corrected for multiple comparisons by Dunnett’s test, all concentrations 10 mM cilengitide had a p value 5 0.0001 compared to untreated controls.
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Figure 3. (A) Flow cytometric determination of the activated aIIbb3-receptor expression by means of PAC-1 binding in the presence of cilengitide at the indicated concentrations or control buffers (0 mM) in PRP. (B) Flow cytometric determination of fibrinogen binding to platelets in the presence of cilengitide in PRP. (C) Flow cytometric determination of the activation and secretion marker CD62P in the presence of cilengitide in PRP. The response is given as mean fluorescence intensity of unstimulated samples (left panel) or after stimulation with 5 mM TRAP (right panel). The results are the mean SEM of three to six independent experiments with PRP from different donors. Expression of untreated control was set to 100%. abciximab denotes the presence of 5 mg abciximab/ml PRP.
Cilengitide has no pro-thrombotic properties
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binding constants between human osteopontin and purified ab3 showed a cation-dependent affinity with Kd values ranging from 0.43 to 11 nM. An osteopontin concentration of 0.15 mM was chosen to far exceed platelet aVb3 saturation. The presence of cilengitide inhibited the adhesion of platelets to vWf at low shear rates (150 s1), commonly found in venous circulation. This effect was seen at the therapeutic dose of 250 mM cilengitide and at supra-therapeutic doses up to the highest tested dose of 1500 mM (Figure 4). The tendency of cilengitide to inhibit platelet adhesion to vWf remained at elevated shear rates of 600 s1 within the same dose range between 250 and 1500 mM cilengitide as was observed at 150 s1 (Figure 4). When the shear rate was further elevated to 1800 s1, platelet adhesion to vWf was still reduced at 750 and 1500 mM (Figure 4). For all shear rates, platelet adhesion to vWf in the presence of 20 mg/ml abciximab was unaffected. In contrast to stable adhesion, cilengitide did not affect the number of platelets rolling on vWf at any concentration up to 1500 mM (data not shown). This shows that cilengitide does not influence activation-independent capturing of platelets by GPIb, supporting the assumption that cilengitide primarily impacted aIIbb3 mediated stable platelet arrest on vWf, a process that requires the activation of platelets after initial tethering and during rolling on vWf. Previous investigations have shown that the presence of aIIbb3 inhibitor abciximab does not significantly block firm platelet adhesion to vWf [12]. Therefore, the next step was to determine whether the adhesion to a classic aIIbb3 ligand is inhibited by increasing cilengitide concentrations. Platelets in reconstituted blood were allowed to settle on fibrinogen for 4 minutes at low shear (150 s1), then without interrupting flow the wall shear rate was instantly increased to 1800 s1. The sudden increase in wall shear rate opposes the binding forces between fibrinogen and the platelets, a decrease of adherent platelets in the presence of cilengitide would reflect a weakened adhesion force of platelets to fibrinogen. Platelet accumulation at 150 s1 was unaffected by the presence of cilengitide, the same was observed in the presence of abciximab. However, at elevated shear rates, the presence of cilengitide in the blood did inhibit the stable adhesion to fibrinogen in a dose proportional manner. The effect was already significant at therapeutic doses. No effect was seen with osteopontin at an avb3-saturating concentration of 0.15 mM (Figure 5; Supplemental Video). Blocking platelet aIIbb3 (and avb3) with abciximab almost completely abolished platelet adhesion to fibrinogen. Complete aIIbb3 receptor blockade by abciximab is reached at approximately 5 mg/mL (100 nM) [15]. These experiments prove that high cilengitide concentrations
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negatively interfere with aIIbb3-depedent stable platelet adhesion, corroborating observed dose- and activation-dependent inhibitory effects on ligand binding properties of aIIbb3, seen in flow-cytometry and aggregometry. ab3 antagonizing properties of cilengitide did not impact platelet adhesion.
Discussion Several antagonists of av, avb3, and/or avb5 integrins are being evaluated for treatment of malignancy in clinical trials. These integrin-blocking molecules may potentially affect platelet function by altering interaction with platelet b3 receptors, thereby contributing to an increased risk of thromboembolic and/or bleeding events. The aim of this investigation was to evaluate the in vitro profile of the ab3 and ab5 inhibitor, cilengitide, on various parameters of platelet function and platelet-endothelial interactions under static and under flow conditions. Sub- as well as supra-therapeutic concentrations were employed for the following reasons. Very large concentrations of cilengitide may exhibit off-target effects affecting aIIbb3 receptors. Low concentrations of cilengitide might induce an outside-in signaling on avb3 or the aIIbb3receptor, leading to platelet activation by ligand mimetic activity. The latter mechanism has been postulated for small molecule peptides containing an RGD sequence, which interferes with aIIbb3 [16]. Thromboembolic events can be ascribed to two possible distinct pathways: (1) activation of circulating platelets or (2) activation of endothelial cells with subsequent platelet attraction and thrombus formation [17]. The investigation presented here indicates no activating properties of cilengitide on platelets and platelet–vessel–wall interaction. In fact, cilengitide concentrations at the upper range of those attained in clinical studies (250 mM) confer moderate inhibition of platelet aggregation and inhibit formation of aIIbb3activation. The effect became even stronger at supra-therapeutic concentrations up to 1500 mM. No effects were seen on platelet degranulation, neither in unstimulated nor in stimulated samples, as demonstrated by a lack of effect on CD62P expression under cilengitide. For some aIIbb3 antagonists, dissociation between antiaggregatory and secretory effects has been reported [18–20]. While binding of fibrinogen to activated aIIbb3 was very effectively inhibited by abciximab, global activation signaling in platelets, and secretion upon activation was unaffected and aIIbb3 neutralizing treatment even potentiated ADP-induced expression of P-selectin, possibly via an outside-in signaling mechanism [21].
Figure 4. Fluorescence-labeled platelets in reconstituted human blood containing cilengitide (0.01 mM to 1500 mM) or buffer alone (0 mM) was perfused over von Willebrand factor (vWf) at a wall shear rate of 150 s1 (left), 600 second1 (middle), and 1800 second1 (right). The number of adherent platelets was determined by microscopic image processing and the adhesion of control blood (0 mM cilengitide) was set to 100%. The results are the mean SEM of six independent experiments with blood from different donors. *p50.05, **p50.01, ***p50.001 in one-sample t-test vs. 100% (the untreated control). OP denotes osteopontin and abciximab the presence of 5 mg abciximab/ml blood.
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Figure 5. Fluorescence-labeled platelets in reconstituted human blood containing cilengitide (250, 750,or 1500 mM) or buffer alone (0 mM) was perfused over fibrinogen. Platelets were allowed to accumulate at a wall shear rate of 150 s1 for 4 minutes. The surface was then populated by adherent platelets and the shear rate instantly increased to 1800 s1. The number of platelets remaining attached to fibrinogen was determined by microscopic image processing (left panel, given is the mean of four experiments with blood from different donors). For statistical evaluation the adhesion of control blood (0 mM cilengitide) at 1800 s1 was set to 100%. The results are the mean SEM of four independent experiments with blood from different donors. ***p50.001 in one-sample t-test vs. 100% (the untreated control). OP denotes 0.15 mM osteopontin and abiximab the presence of 5 mg/ml abciximab in the perfused blood.
It can be speculated that ab3 and/or ab5 integrin antagonists might also affect the adhesive properties of the endothelium. Cilengitide has been shown to induce activation of endothelial cell surface avb3, thereby eliciting downstream signaling events [22]. It is also conceivable that stimulation of endothelial cells by cilengitide might induce expression of endothelial adhesive ligands (such as ICAM, VCAM, fractalkine etc.) that may in turn enhance platelet adhesion and eventually lead to pathological vessel occlusion and fatal thrombosis. Cilengitide did not promote platelet adhesion to endothelial cells at all concentrations tested. Platelet adhesion was inhibited by cilengitide at supratherapeutic concentrations of 750 or 1500 mM, depending on the shear rate. Flow based adhesion assays with immobilized matrix proteins show that cilengitide dose-dependent exerted its inhibitory properties, primarily through interaction with aIIbb3, thereby affecting activationdependent platelet adhesion. Platelet adhesion to fibrinogen was already impaired at Cmax concentrations of cilengitide, very similar to abciximab inhibition at elevated shear. It might appear parodoxical that platelet adhesion to an aIIbb3 ligand at 150 second1 is neither inhibited by cilengitide nor by abciximab, but it has recently been demonstrated that platelet adhesion rates are sensitive to testing device variation or coating conditions [23], similar to variations encountered in collagen-dependent adhesion [24]. aIIbb3-dependent platelet adhesion to fibrinogen surfaces appears to be extremely sensitive to fibrinogen immobilization procedures [25], which might explain results in the present investigation deviating from previous reports. However, cilengitide does act identically to abciximab in this investigation, making it a classical antagonist at micromolar concentration. Thus, cilengitide has some aIIbb3 inhibitory properties at higher concentrations but does not exert pro-adhesive effects. This finding does not come unexpectedly for a compound interfering with avb3 [26]. avb3 and aIIbb3 share the same b3 subunit and the av subunit is highly homologous to aIIb in aIIbb3 [27]. Some avb3 is expressed on platelets and it has been estimated that approximately 100 copies of avb3 receptors are expressed per platelet, compared to approximately 100,000 copies of aIIbb3 [28]. Abciximab displays nearly identical affinity to aIIbb3 and avb3 [29]. In the concentration used, osteopontin acts as a selective avb3 ligand, without affecting aIIbb3 [14]. Binding the soluble RGD ligand to platelet avb3 did not affect platelet
adhesion to proteins such as vWf and fibrinogen. Therefore, cilengitide anti-avb3 properties responsible for the antiangiogenic effects are not considered to affect platelet interaction with the vessel wall, either by direct receptor occupancy or downstream mechanisms. However, it is reasonable to assume that at very high supratherapeutic concentrations, cilengitide’s anti-aIIbb3 properties become predominant. This is in agreement with flow cytometry data: PAC-1 is dose-dependently reduced. It is known that cilengitide has a more than 1000-fold higher affinity for avb3 than for aIIbb3 [10, 30] and that cilengitide binds to av in the nanomolar range. It is, therefore, plausible that higher micro-molar concentrations predominantly exert effects through aIIbb3 inhibition in aggregation and fibrinogen binding, all of which rely on aIIbb3 activation. Different apparent potencies of fibrinogen–aIIbb3 interactions in aggregometry, fibrinogen binding in FACS and fibrinogen binding under flow in adhesion assays might be related to differences in fibrinogen activity between soluble and solid phases [31]. Even the potency of platelet–fibrinogen interaction on immobilized fibrinogen varies considerably, depending on coating procedures [25, 32]. Even if cilengitide did show some aIIbb3 inhibitory properties at high concentrations, it is likely that its receptor binding and signaling properties, including intrinsic receptor activity, may be substantially different from those reported for abciximab. For other small peptides, containing or mimicking the RGD sequence, it is known that the characteristics of aIIbb3-binding and blocking of fibrinogen binding differ widely from those of the large chimeric Fab fragment, abciximab. Since platelets are considered to play an important role in tumor-growth, -metastasis, and -angiogenesis, any anti-platelet properties of an ab3 integrin inhibitor, such as cilengitide, may provide beneficial anti-tumor activity. Platelets are involved in key steps of malignancy progression and are recognized as dynamic reservoirs of proangiogenic and anti-angiogenic proteins [33]. Experimental blockade of key platelet receptors such as aIIbb3 has been shown to attenuate the progression and metastatic potential of various tumor types. Abciximab has successfully been used to inhibit angiogenesis and tumor growth in the mouse [34]. This illustrates the bridging of classic platelet function (hemostasis) and angiogenesis, via the integrin inhibitors [35]. It has been argued that cilengitide at low concentrations can activate avb3 integrin [36, 37]. Such activation might lead to
Cilengitide has no pro-thrombotic properties
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stimulation of endothelial cells and increased platelet interaction with activated endothelium or direct platelet activation. Furthermore, thromboembolism has been noted as a side effect for a variety of agents interfering with the VEGF pathway, e.g., bevacizumab [38, 39]. In a physiologically robust system, cilengitide in low concentrations did not activate HUVECs to result in higher platelet adhesion to them. In contrast, high cilengitide concentrations inhibited platelet adhesion to HUVECs and adhesion ligands, such as fibrinogen, through the down-regulated expression of activated aIIbb3. This is in line with safety data from clinical trials with cilengitide having been applied to a range of different solid tumors. Data did not point to an increased risk of thromboembolic events in cilengitide treated patients [9, 40]. In conclusion, cilengitide showed a weak inhibitory effect on blood platelets, suggesting an antiaggregatory effect at higher concentrations. There was no evidence for any pro-thrombotic effect in terms of platelet activation and adhesion.
Acknowledgements
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The authors thank Simon Goodman for helpful hints, fruitful discussion and continuous support of the project. 18.
Declaration of interest This study has been supported by a grant from Merck KGaA. SH has received scientific grants from Merck KGaA and The Medicines Company and has received honoraria for lectures from Merck KGaA and LEO Pharmaceuticals. UK is employed by Merck KGaA. All other authors have no disclosures.
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References 1. Taugourdeau-Raymond S, Rouby F, Default A, Jean-Pastor MJ. Bevacizumab-induced serious side-effects: A review of the French pharmacovigilance database. Eur J Clin Pharmacol 2012;68: 1103–1107. 2. Nalluri SR, Chu D, Keresztes R, Zhu X, Wu S. Risk of venous thromboembolism with the angiogenesis inhibitor bevacizumab in cancer patients: A meta-analysis. JAMA 2008;300:2277–2285. 3. Ferroni P, Formica V, Roselli M, Guadagni F. Thromboembolic events in patients treated with anti-angiogenic drugs. Curr Vasc Pharmacol 2010;8:102–113. 4. Elice F, Rodeghiero F, Falanga A, Rickles FR. Thrombosis associated with angiogenesis inhibitors. Best Pract Res Clin Haematol 2009;22:115–128. 5. Jenkins EO, Schiff D, Mackman N, Key NS. Venous thromboembolism in malignant gliomas. J Thromb Haemost 2010;8: 221–227. 6. Buerkle MA, Pahernik SA, Sutter A, Jonczyk A, Messmer K, Dellian M. Inhibition of the alpha-nu integrins with a cyclic RGD peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. Br J Cancer 2002;86:788–795. 7. MacDonald TJ, Stewart CF, Kocak M, Goldman S, Ellenbogen RG, Phillips P, Lafond D, Poussaint TY, Kieran MW, Boyett JM, et al. Phase I clinical trial of cilengitide in children with refractory brain tumors: Pediatric Brain Tumor Consortium Study PBTC-012. J Clin Oncol 2008;26:919–924. 8. Yamada S, Bu XY, Khankaldyyan V, Gonzales-Gomez I, McComb JG, Laug WE. Effect of the angiogenesis inhibitor Cilengitide (EMD 121974) on glioblastoma growth in nude mice. Neurosurgery 2006;59:1304–1312; discussion 1312. 9. Stupp R, Hegi ME, Gorlia T, Erridge S, Grujicic D, Steinbach JP, Wick W, Tarnawski R, Nam D-H, Weyerbrock A, et al. Cilengitide combined with standard treatment for patients with newly diagnosed glioblastoma and methylated O6-methylguanine-DNA methyltransferase (MGMT) gene promoter: Key results of the multicenter, randomized, open-label, controlled, phase III CENTRIC study. J Clin Oncol 2013;13:(suppl; abstr LBA2009). 10. Dechantsreiter MA, Planker E, Matha B, Lohof E, Holzemann G, Jonczyk A, Goodman SL, Kessler H. N-Methylated cyclic RGD
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
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peptides as highly active and selective alpha(V)beta(3) integrin antagonists. J Med Chem 1999;42:3033–3040. Meyer Dos Santos S, Klinkhardt U, Schneppenheim R, Harder S. Using ImageJ for the quantitative analysis of flow-based adhesion assays in real-time under physiologic flow conditions. Platelets 2010;21:60–66. Meyer dos Santos S, Klinkhardt U, Scholich K, Nelson K, Monsefi N, Deckmyn H, Kuczka K, Zorn A, Harder S. The CX3C chemokine fractalkine mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib. Blood 2011;117:4999–5008. Vassallo Jr RR, Kieber-Emmons T, Cichowski K, Brass LF. Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. J Biol Chem 1992;267: 6081–6085. Hu DD, Hoyer JR, Smith JW. Ca2þ suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin alpha v beta 3. J Biol Chem 1995;270:9917–9925. Valgimigli M, Campo G, Tebaldi M, Carletti R, Arcozzi C, Ferrari R, Percoco G. Abciximab: A reappraisal of its use in coronary care. Biol Targets Ther 2008;2:29–39. Armstrong PC, Peter K. GPIIb/IIIa inhibitors: From bench to bedside and back to bench again. Thromb Haemost 2012;107: 808–814. Fuentes QE, Fuentes QF, Andres V, Pello OM, de Mora JF, Palomo GI. Role of platelets as mediators that link inflammation and thrombosis in atherosclerosis. Platelets 2013;24:255–262. Tsao PW, Forsythe MS, Mousa SA. Dissociation between the antiaggregatory & anti-secretory effects of platelet integrin alpha IIb beta 3 (GPIIb/IIIa) antagonists, c7E3 and DMP728. Thromb Res 1997;88:137–146. Schneider DJ, Taatjes DJ, Sobel BE. Paradoxical inhibition of fibrinogen binding and potentiation of alpha-granule release by specific types of inhibitors of glycoprotein IIb-IIIa. Cardiovasc Res 2000;45:437–446. Peter K, Schwarz M, Ylanne J, Kohler B, Moser M, Nordt T, Salbach P, Kubler W, Bode C. Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (alphaIIbbeta3) inhibitors. Blood 1998;92: 3240–3249. Klinkhardt U, Graff J, Harder S. Clopidogrel, but not abciximab, reduces platelet leukocyte conjugates and P-selectin expression in a human ex vivo in vitro model. Clin Pharmacol Ther 2002;71: 176–185. Alghisi GC, Ponsonnet L, Ruegg C. The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells. PLoS One 2009;4:e4449. Roest M, Reininger A, Zwaginga JJ, King MR, Heemskerk JW. Flow chamber-based assays to measure thrombus formation in vitro: Requirements for standardization. J Thromb Haemost: JTH 2011;9: 2322–2324. Heemskerk JW, Sakariassen KS, Zwaginga JJ, Brass LF, Jackson SP, Farndale RW. Collagen surfaces to measure thrombus formation under flow: Possibilities for standardization. J Thromb Haemost 2011;9:856–858. Jirouskova M, Jaiswal JK, Coller BS. Ligand density dramatically affects integrin alpha IIb beta 3-mediated platelet signaling and spreading. Blood 2007;109:5260–5269. Nurden AT. Is there a redundancy of beta3 and other platelet receptors in the brain and central nervous system? Platelets 2013;24: 170–172. Coller BS. Binding of abciximab to alpha V beta 3 and activated alpha M beta 2 receptors: With a review of platelet-leukocyte interactions. Thromb Haemost 1999;82:326–336. Coller BS, Cheresh DA, Asch E, Seligsohn U. Platelet vitronectin receptor expression differentiates Iraqi-Jewish from Arab patients with Glanzmann thrombasthenia in Israel. Blood 1991;77:75–83. Tam SH, Sassoli PM, Jordan RE, Nakada MT. Abciximab (ReoPro, chimeric 7E3 Fab) demonstrates equivalent affinity and functional blockade of glycoprotein IIb/IIIa and alpha(v)beta3 integrins. Circulation 1998;98:1085–1091. Mas-Moruno C, Beck JG, Doedens L, Frank AO, Marinelli L, Cosconati S, Novellino E, Kessler H. Increasing alphavbeta3 selectivity of the anti-angiogenic drug cilengitide by N-methylation. Angew Chem Int Ed Engl 2011;50:9496–9500.
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31. Bonnefoy A, Liu Q, Legrand C, Frojmovic MM. Efficiency of platelet adhesion to fibrinogen depends on both cell activation and flow. Biophys J 2000;78:2834–2843. 32. Cook BC. Reactivity of human platelets with immobilized fibrinogen is dictated by the chemical character of the surface. Thromb Res 2001;104:39–48. 33. Peterson JE, Zurakowski D, Italiano Jr JE, Michel LV, Fox L, Klement GL, Folkman J. Normal ranges of angiogenesis regulatory proteins in human platelets. Am J Hematol 2010;85:487–493. 34. Varner JA, Nakada MT, Jordan RE, Coller BS. Inhibition of angiogenesis and tumor growth by murine 7E3, the parent antibody of c7E3 Fab (abciximab; ReoPro). Angiogenesis 1999;3:53–60. 35. Trikha M, Zhou Z, Timar J, Raso E, Kennel M, Emmell E, Nakada MT. Multiple roles for platelet GPIIb/IIIa and alphavbeta3 integrins in tumor growth, angiogenesis, and metastasis. Cancer Res 2002;62:2824–2833. 36. Hodivala-Dilke KM, Reynolds AR, Reynolds LE. Integrins in angiogenesis: Multitalented molecules in a balancing act. Cell Tissue Res 2003;314:131–144.
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37. Reynolds AR, Hart IR, Watson AR, Welti JC, Silva RG, Robinson SD, Da Violante G, Gourlaouen M, Salih M, Jones MC, et al. Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors. Nat Med 2009;15:392–400. 38. Narita Y. Drug review: Safety and efficacy of bevacizumab for glioblastoma and other brain tumors. Jpn J Clin Oncol 2013;43: 587–595. 39. Costagliola C, Agnifili L, Arcidiacono B, Duse S, Fasanella V, Mastropasqua R, Verolino M, Semeraro F. Systemic thromboembolic adverse events in patients treated with intravitreal antiVEGF drugs for neovascular age-related macular degeneration. Expert Opin Biol Ther 2012;12:1299–1313. 40. Nabors LB, Mikkelsen T, Hegi ME, Ye X, Batchelor T, Lesser G, Peereboom D, Rosenfeld MR, Olsen J, Brem S, et al. A safety run-in and randomized phase 2 study of cilengitide combined with chemoradiation for newly diagnosed glioblastoma (NABTT 0306). Cancer 2012;118:5601–5607.
ORIGINAL ARTICLE
The integrin antagonist, cilengitide, is a weak inhibitor of aIIbb3 mediated platelet activation and inhibits platelet adhesion under flow Sascha Meyer dos Santos1 Karina Kuczka1, Bettina Picard-Willems1, Karen Nelson2, Ute Klinkhardt3, & Sebastian Harder1
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Institute for Clinical Pharmacology, J.W. Goethe University Hospital, Frankfurt, Germany, 2Department of Vascular and Endovascular Surgery, J.W. Goethe University Hospital, Frankfurt, Germany, and 3Merck KGaA, Darmstadt, Germany
Abstract
Keywords
The RGD cyclic pentapetide, cilengitide, is a selective inhibitor of avb3 and avb5 integrins and was developed for antiangiogenic therapy. Since cilengitide interacts with platelet aIIbb3 and platelets express av integrins, the effect of cilengitide on platelet pro-coagulative response and adhesion is of interest. Flow-based adhesion assays were performed to evaluate platelet adhesion and rolling on von Willebrand factor (vWf), on fibrinogen and on human umbilical vein endothelial cells (HUVECs). Flow cytometry was used to detect platelet activation (PAC1) and secretion (CD62P) by cilengitide and light transmission aggregometry was used to detect cilengitide-dependent platelet aggregation. Cilengitide inhibited platelet adhesion to fibrinogen at concentrations above 250 mM [which is the Cmax in human studies] and adhesion to vWf and HUVECs at higher concentrations under physiologic flow conditions. Platelet aggregation was already impaired at cilengitide concentrations 410 mM. Activation of aIIbb3 integrin was inhibited by 250 mM cilengitide, whereas platelet secretion was unaffected by cilengitide. No evidence of cilengitide-induced platelet activation was found at all tested concentrations (0.01–1500 mM). At higher concentrations, platelet activation was inhibited, predominantly due to aIIbb3 inhibition.
Alpha-v beta-3 Integrin, angiogenesis inhibitors, cilengitide, platelet membrane glycoproteins receptors, thrombosis
Introduction Thromboembolic events and pulmonary embolism are side effects of the VEGF-neutralizing antibody bevacizumab, which interferes with endothelial VEGF receptors and inhibits angiogenesis [1, 2]. Disturbance of vascular homeostasis by blocking VEGF may lead to endothelial dysfunction and adverse vascular effects [3], and it has been speculated that thromboembolism may be a general feature of anti-angiogenic chemotherapy [4] since thromboembolism often occurs in cancer patients. Venous thromboembolism risk is particularly high in patients with glioma with about 12% in the first three months after diagnosis, which is more than twice that of patients with other malignancies [5]. Integrins are a family of cell-extracellular matrix adhesion molecules, playing important roles in tumor angiogenesis. ab3-integrin has received much attention as a potential anticancer target because it is highly expressed on both tumor cells and in new vasculature of diverse tumors. ab3 also plays a role in cell migration and extravasation, which occurs during metastasis. Two currently approved integrin antagonists are available, though not intended for oncologic indications. Several compounds are under clinical evaluation for treating malignancy. Cilengitide, a cyclic RGD pentapeptide, is a selective inhibitor of avb3 and
Correspondence: Sascha Meyer dos Santos, Institute for Clinical Pharmacology, J.W. Goethe University Hospital, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Tel: +49 69 6301 7622; Fax: +49 69 6301 83921. E-mail: [email protected]
History Received 24 July 2013 Revised 23 October 2013 Accepted 25 November 2013 Published online 16 January 2014
avb5 integrins, with IC50 values in the low nanomolar range in solid phase assays. Cilengitide has shown considerably lower affinity for other integrins (avb1, avb6, and avb8), with IC50 concentrations 10- to 100-fold higher in receptor binding assays. Cilengitide has demonstrated anti-angiogenic as well as direct anti-tumor and anti-invasive effects in various animal models [6–8]. It has recently been investigated in a large Phase III trial (CENTRIC), including patients with newly diagnosed glioblastoma with a methylated O6-methylguanine-DNA methyltransferase (MGMT) gene promoter. A phase I/II study with cilengitide is currently ongoing in non-small cell lung cancer (NSCLC) and further trials are being conducted by the U.S. National Cancer Institute (NCI). Despite promising clinical Phase I and II results, cilengitide did not meet its primary endpoint of significantly increasing overall survival, when added to a current standard chemo-radiotherapy regimen or temozolomide and radiotherapy in this pivotal trial [9]. Development of cilengitide by Merck was stopped, but academic research investigating its use for other indications is still ongoing (e.g. NCT01118676, NCT01517776). Since b3 integrins are expressed on platelets, and since a weak interaction of cilengitide with aIIbb3 has been reported [10] platelet interaction with avb3 integrin antagonists is conceivable. This might lead to platelet activation via outside-in signaling, or even platelet inhibition via blockage of the aIIbb3 b3 integrin, which is the main binding site for fibrinogen. The aim of this study was to evaluate the in vitro profile of cilengitide on various parameters of platelet function and platelet-endothelial interaction under static and flow conditions.
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Methods The study protocol regarding blood withdrawal for scientific purposes was approved by the Ethics Committee of the University Hospital of Frankfurt. Prior to inclusion all subjects gave written, informed consent for blood donation.
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Preparation of blood samples for flow-based adhesion assays Suspensions of calcein-AM labeled platelets in washed blood were prepared as has previously been described [11, 12], with minor modifications. Briefly, blood was centrifuged at 3300 g for 5 minutes, washed three times with Tyrode’s buffer1 and then centrifuged at 102 g for 15 minutes. Platelets in the supernatant were collected and fluorescently labeled with calcein-AM for 30 minutes in the dark. They were then added back to the red blood cells, which were washed once more in Tyrode’s buffer. The cells were finally resuspended in the original volume. Incubation with cilengitide Pharmacokinetic studies revealed steady-state plasma peak levels of 169 21 mg/ml cilengitide after a standard 2000 mg infusion, which corresponds to a concentration of 286 mM. However, individual peak/trough concentrations may vary and therefore a supra- as well as sub-therapeutic concentration range was employed. Final concentrations were 0, 0.01, 10, 100, 250, 750, and 1500 mM cilengitide. Platelet aggregation assay The platelet aggregation assay was performed in citrated Platelet Rich Plasma (PRP) with a turbidimetric, light-transmittance device (APACT) within 1 hour after blood sampling. Aggregation inducers were collagen (SKF Horm, Nycomed) at a final concentration of 1 mg/ml and ADP (Sigma) at a final concentration of 5 mM. Flow cytometry assay The flow cytometric determination of platelet activation status focused on parameters that represented different platelet functions: CD62P expression is a marker of platelet degranulation, whereas PAC-1-binding reflects the activation induced conformational change of the aIIbb3 receptor. The following antibodies and reagents were used: anti-CD62P-PE (Immunotech), and PAC-1-FITC (BD Biosciences). After the first incubation step of PRP with cilengitide or vehicle, about 2 106 platelets were resuspended in PBS with Ca2þ/Mg2þ at a final volume of 100 ml. The samples were activated with 5 mM TRAP-6. Baseline samples were not activated. After 10 minutes incubation, 20 ml antiCD62P-PE and 20 ml PAC-1-FITC were added. After another incubation period of 20 minutes, 2 ml PBS with Ca2þ/Mg2þ were added to each sample tube. The samples were centrifuged (300 g for 5 min at room temperature), decanted and resuspended in 500 ml Cellfix (1:10 dilution with double distilled water; Becton Dickinson). All measurements were carried out using a FACScanTM flow cytometer (Becton Dickinson), data acquisition with CellQuest Pro software (Becton Dickinson). A total of 4–6 experiments were carried out for each cilengitide concentration. Platelet adhesion to selected subendothelial matrix components To determine the effect of cilengitide on adhesion to vWf, platelets reconstituted with red blood cells containing the desired cilengitide concentration, 0.15 mM osteopontin or abciximab were
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perfused at wall shear rates of 150 , 600, and 1800 s1 for 5 minutes each. Adhesion assays were performed in transparent chambers designed for microscopic observation of cells or cell cultures under flow conditions (m-slide I 0.4 mm in height; Ibidi, Munich, Germany). The chambers were filled with a solution containing 13.3 mg rvWf (recombinant von Willebrand factor) or 100 mg fibrinogen and left overnight at 4 C in a humid environment to coat the chambers. The flow chambers were blocked with 2% BSA for 2 hours, washed and placed under an epifluorescent microscope. Perfusion experiments were carried out as has been described [11]. Platelet adhesion to human endothelial cells To study platelet adhesion to endothelial cells, HUVECs (Lonza) were cultivated to confluency in flow chambers in the presence of the desired cilengitide concentration for 18 hours. The flow chambers were placed under the epifluorescent microscope and the reconstituted blood containing the corresponding cilengitide concentration was aspirated through the chambers and platelet interaction with endothelial cells was visualized in real time. The entire period of perfusion with at least three randomly selected fields of view was recorded and processed. Calculations Data for all assays were tabulated and subjected to descriptive statistics. Aggregometry Data obtained after cilengitide addition was compared with that from untreated controls by one-way ANOVA and corrected for multiple comparisons by Dunnett’s test. A p value 0.05 was considered significant. For FACS-experiments, expression of PAC1 or CD62P (given in mean fluorescence intensity) was converted to percent of the value of the untreated control, which was set to 100%. Cilengitide data was compared with control by one-sample t-test and a p value 0.05 was considered significant. Flow based adhesion assays For each shear rate at least three randomly selected fields of view were recorded and the accumulation rate of adherent platelets within each observation period was computed in Graph Pad Prism and set to 100% for untreated control blood. Cilengitide data was compared to control by one-sample t-test and a p value 0.05 was considered significant.
Results Platelet adhesion onto human endothelial cells An in vitro flow chamber model was used to evaluate the effects of the anti-integrin drug cilengitide on platelet adhesion mechanisms. To mimic the situation in blood vessels, we used confluent HUVECs cultured in flow chambers as a model to determine cilengitide effects on platelet-endothelial interactions. After 18 hours incubation, cilengitide was added to the perfusion medium and the confluent endothelial cell monolayer was perfused with blood containing the corresponding cilengitide concentration. The interaction of fluorescently labeled platelets with HUVECs was recorded in real time and the number of cilengitide treated platelets sticking firmly to the cilengitide treated HUVECs was measured. Platelet accumulation on HUVECs was inhibited concentration-dependent at all shear rates. However, the effect remained non-significant at clinically relevant concentrations. Application of cilengitide at
DOI: 10.3109/09537104.2013.870332
supratherapeutic concentrations of 1500 mM weakly inhibited the accumulation of platelets on the endothelial cells at all shear rates. At elevated shear rates (1800 s1), a weak but significant inhibition of platelet adhesion was also seen at a concentration of 750 mM (Figure 1). Thus, these experiments prove that high cilengitide concentrations tend to reduce platelet adhesion to the endothelium. None of the tested concentrations stimulated the adhesion of platelets.
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Platelet aggregation The following experiments were designed to investigate the role of b3 integrins on the cilengitide-dependent inhibition of platelet adhesion. The influence of cilengitide on platelet aggregation was evaluated by light transmission aggregometry. PRP was activated by threshold doses of 5 mM ADP and 1 mg/ml collagen in the presence of 0–1500 mM cilengitide. Platelet aggregation was unaffected by low nanomolar concentrations of cilengitide, however, inhibition began to appear between 1 and 10 mM. At cilengitide concentrations 100 mM, platelet aggregation induced by either ADP or collagen was almost completely absent (Figure 2). Cilengitide, therefore, showed no proaggregatory properties, but instead, inhibited the platelet–platelet interaction. Often, such homotypic interaction is driven by bridging aIIbb3 on platelets. Flow cytometry For non-activated samples, only cilengitide concentrations of 1500 mM showed inhibitory effects. No activating effects on
Cilengitide has no pro-thrombotic properties
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resting platelets were observed for any concentration of cilengitide tested. However, flow cytometry revealed a strong inhibitory influence of cilengitide on the expression of activated aIIbb3 after stimulation with 5 mM TRAP-6 (peptide fragment (residues 42–47) of protease-activated receptor 1 (PAR1) that acts as a PAR1 agonist) [13] at concentrations 250 mM (Figure 3A). To test whether cilengitide competes with fibrinogen binding to aIIbb3, fibrinogen binding to activated platelets in the presence of cilengitide was measured. Cilengitide physically interfered with fibrinogen binding to aIIbb3 at all tested concentrations (Figure 3B). Cilengitide had no effect on the expression of platelet activation and secretion marker CD62P at all concentrations tested, neither in resting platelets nor in TRAP stimulated platelets (Figure 3C). Platelet adhesion at subendothelial matrix components Cilengitide inhibited the adhesion of platelets to endothelial cells at supratherapeutic doses. Aggregometry and flow-cytometric data show that cilengitide dose-dependently down-regulated the expression of activated aIIbb3. To test the contribution of different platelet b3 integrins and shear stress on cilengitide induced effects, platelets in reconstituted blood were perfused over vWf in the presence of b3 inhibitors and the adhesion to cilengitide treated platelets was compared. Osteopontin was used as a selective ab3 ligand, since Hu and coworkers [14] have reported that osteopontin does not bind to integrin aIIbb3 with measurable affinity. Measurements of
Figure 1. Fluorescence-labeled platelets in reconstituted human blood containing cilengitide (0.01, 250, 750, or 1500 mM) or buffer alone (0 mM) was perfused over confluent HUVEC monolayers at the indicated wall shear rate. The number of adherent platelets was determined by microscopic image processing and the adhesion of control blood (0 mM cilengitide) was set to 100%. The results are the mean SEM of six independent experiments with blood from different donors. *p50.05, **p50.01 in one-sample t-test vs. 100% (the untreated control).
Figure 2. Platelet aggregation in PRP was induced by 5 mM ADP, 1 mg/ml collagen, and 20 mM TRAP and monitored by turbidimetric aggregometry. Shown is the aggregation response as percentage of maximal light transmission Amax of six experiments in untreated PRP. In one-way ANOVA corrected for multiple comparisons by Dunnett’s test, all concentrations 10 mM cilengitide had a p value 5 0.0001 compared to untreated controls.
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Figure 3. (A) Flow cytometric determination of the activated aIIbb3-receptor expression by means of PAC-1 binding in the presence of cilengitide at the indicated concentrations or control buffers (0 mM) in PRP. (B) Flow cytometric determination of fibrinogen binding to platelets in the presence of cilengitide in PRP. (C) Flow cytometric determination of the activation and secretion marker CD62P in the presence of cilengitide in PRP. The response is given as mean fluorescence intensity of unstimulated samples (left panel) or after stimulation with 5 mM TRAP (right panel). The results are the mean SEM of three to six independent experiments with PRP from different donors. Expression of untreated control was set to 100%. abciximab denotes the presence of 5 mg abciximab/ml PRP.
Cilengitide has no pro-thrombotic properties
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DOI: 10.3109/09537104.2013.870332
binding constants between human osteopontin and purified ab3 showed a cation-dependent affinity with Kd values ranging from 0.43 to 11 nM. An osteopontin concentration of 0.15 mM was chosen to far exceed platelet aVb3 saturation. The presence of cilengitide inhibited the adhesion of platelets to vWf at low shear rates (150 s1), commonly found in venous circulation. This effect was seen at the therapeutic dose of 250 mM cilengitide and at supra-therapeutic doses up to the highest tested dose of 1500 mM (Figure 4). The tendency of cilengitide to inhibit platelet adhesion to vWf remained at elevated shear rates of 600 s1 within the same dose range between 250 and 1500 mM cilengitide as was observed at 150 s1 (Figure 4). When the shear rate was further elevated to 1800 s1, platelet adhesion to vWf was still reduced at 750 and 1500 mM (Figure 4). For all shear rates, platelet adhesion to vWf in the presence of 20 mg/ml abciximab was unaffected. In contrast to stable adhesion, cilengitide did not affect the number of platelets rolling on vWf at any concentration up to 1500 mM (data not shown). This shows that cilengitide does not influence activation-independent capturing of platelets by GPIb, supporting the assumption that cilengitide primarily impacted aIIbb3 mediated stable platelet arrest on vWf, a process that requires the activation of platelets after initial tethering and during rolling on vWf. Previous investigations have shown that the presence of aIIbb3 inhibitor abciximab does not significantly block firm platelet adhesion to vWf [12]. Therefore, the next step was to determine whether the adhesion to a classic aIIbb3 ligand is inhibited by increasing cilengitide concentrations. Platelets in reconstituted blood were allowed to settle on fibrinogen for 4 minutes at low shear (150 s1), then without interrupting flow the wall shear rate was instantly increased to 1800 s1. The sudden increase in wall shear rate opposes the binding forces between fibrinogen and the platelets, a decrease of adherent platelets in the presence of cilengitide would reflect a weakened adhesion force of platelets to fibrinogen. Platelet accumulation at 150 s1 was unaffected by the presence of cilengitide, the same was observed in the presence of abciximab. However, at elevated shear rates, the presence of cilengitide in the blood did inhibit the stable adhesion to fibrinogen in a dose proportional manner. The effect was already significant at therapeutic doses. No effect was seen with osteopontin at an avb3-saturating concentration of 0.15 mM (Figure 5; Supplemental Video). Blocking platelet aIIbb3 (and avb3) with abciximab almost completely abolished platelet adhesion to fibrinogen. Complete aIIbb3 receptor blockade by abciximab is reached at approximately 5 mg/mL (100 nM) [15]. These experiments prove that high cilengitide concentrations
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negatively interfere with aIIbb3-depedent stable platelet adhesion, corroborating observed dose- and activation-dependent inhibitory effects on ligand binding properties of aIIbb3, seen in flow-cytometry and aggregometry. ab3 antagonizing properties of cilengitide did not impact platelet adhesion.
Discussion Several antagonists of av, avb3, and/or avb5 integrins are being evaluated for treatment of malignancy in clinical trials. These integrin-blocking molecules may potentially affect platelet function by altering interaction with platelet b3 receptors, thereby contributing to an increased risk of thromboembolic and/or bleeding events. The aim of this investigation was to evaluate the in vitro profile of the ab3 and ab5 inhibitor, cilengitide, on various parameters of platelet function and platelet-endothelial interactions under static and under flow conditions. Sub- as well as supra-therapeutic concentrations were employed for the following reasons. Very large concentrations of cilengitide may exhibit off-target effects affecting aIIbb3 receptors. Low concentrations of cilengitide might induce an outside-in signaling on avb3 or the aIIbb3receptor, leading to platelet activation by ligand mimetic activity. The latter mechanism has been postulated for small molecule peptides containing an RGD sequence, which interferes with aIIbb3 [16]. Thromboembolic events can be ascribed to two possible distinct pathways: (1) activation of circulating platelets or (2) activation of endothelial cells with subsequent platelet attraction and thrombus formation [17]. The investigation presented here indicates no activating properties of cilengitide on platelets and platelet–vessel–wall interaction. In fact, cilengitide concentrations at the upper range of those attained in clinical studies (250 mM) confer moderate inhibition of platelet aggregation and inhibit formation of aIIbb3activation. The effect became even stronger at supra-therapeutic concentrations up to 1500 mM. No effects were seen on platelet degranulation, neither in unstimulated nor in stimulated samples, as demonstrated by a lack of effect on CD62P expression under cilengitide. For some aIIbb3 antagonists, dissociation between antiaggregatory and secretory effects has been reported [18–20]. While binding of fibrinogen to activated aIIbb3 was very effectively inhibited by abciximab, global activation signaling in platelets, and secretion upon activation was unaffected and aIIbb3 neutralizing treatment even potentiated ADP-induced expression of P-selectin, possibly via an outside-in signaling mechanism [21].
Figure 4. Fluorescence-labeled platelets in reconstituted human blood containing cilengitide (0.01 mM to 1500 mM) or buffer alone (0 mM) was perfused over von Willebrand factor (vWf) at a wall shear rate of 150 s1 (left), 600 second1 (middle), and 1800 second1 (right). The number of adherent platelets was determined by microscopic image processing and the adhesion of control blood (0 mM cilengitide) was set to 100%. The results are the mean SEM of six independent experiments with blood from different donors. *p50.05, **p50.01, ***p50.001 in one-sample t-test vs. 100% (the untreated control). OP denotes osteopontin and abciximab the presence of 5 mg abciximab/ml blood.
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Figure 5. Fluorescence-labeled platelets in reconstituted human blood containing cilengitide (250, 750,or 1500 mM) or buffer alone (0 mM) was perfused over fibrinogen. Platelets were allowed to accumulate at a wall shear rate of 150 s1 for 4 minutes. The surface was then populated by adherent platelets and the shear rate instantly increased to 1800 s1. The number of platelets remaining attached to fibrinogen was determined by microscopic image processing (left panel, given is the mean of four experiments with blood from different donors). For statistical evaluation the adhesion of control blood (0 mM cilengitide) at 1800 s1 was set to 100%. The results are the mean SEM of four independent experiments with blood from different donors. ***p50.001 in one-sample t-test vs. 100% (the untreated control). OP denotes 0.15 mM osteopontin and abiximab the presence of 5 mg/ml abciximab in the perfused blood.
It can be speculated that ab3 and/or ab5 integrin antagonists might also affect the adhesive properties of the endothelium. Cilengitide has been shown to induce activation of endothelial cell surface avb3, thereby eliciting downstream signaling events [22]. It is also conceivable that stimulation of endothelial cells by cilengitide might induce expression of endothelial adhesive ligands (such as ICAM, VCAM, fractalkine etc.) that may in turn enhance platelet adhesion and eventually lead to pathological vessel occlusion and fatal thrombosis. Cilengitide did not promote platelet adhesion to endothelial cells at all concentrations tested. Platelet adhesion was inhibited by cilengitide at supratherapeutic concentrations of 750 or 1500 mM, depending on the shear rate. Flow based adhesion assays with immobilized matrix proteins show that cilengitide dose-dependent exerted its inhibitory properties, primarily through interaction with aIIbb3, thereby affecting activationdependent platelet adhesion. Platelet adhesion to fibrinogen was already impaired at Cmax concentrations of cilengitide, very similar to abciximab inhibition at elevated shear. It might appear parodoxical that platelet adhesion to an aIIbb3 ligand at 150 second1 is neither inhibited by cilengitide nor by abciximab, but it has recently been demonstrated that platelet adhesion rates are sensitive to testing device variation or coating conditions [23], similar to variations encountered in collagen-dependent adhesion [24]. aIIbb3-dependent platelet adhesion to fibrinogen surfaces appears to be extremely sensitive to fibrinogen immobilization procedures [25], which might explain results in the present investigation deviating from previous reports. However, cilengitide does act identically to abciximab in this investigation, making it a classical antagonist at micromolar concentration. Thus, cilengitide has some aIIbb3 inhibitory properties at higher concentrations but does not exert pro-adhesive effects. This finding does not come unexpectedly for a compound interfering with avb3 [26]. avb3 and aIIbb3 share the same b3 subunit and the av subunit is highly homologous to aIIb in aIIbb3 [27]. Some avb3 is expressed on platelets and it has been estimated that approximately 100 copies of avb3 receptors are expressed per platelet, compared to approximately 100,000 copies of aIIbb3 [28]. Abciximab displays nearly identical affinity to aIIbb3 and avb3 [29]. In the concentration used, osteopontin acts as a selective avb3 ligand, without affecting aIIbb3 [14]. Binding the soluble RGD ligand to platelet avb3 did not affect platelet
adhesion to proteins such as vWf and fibrinogen. Therefore, cilengitide anti-avb3 properties responsible for the antiangiogenic effects are not considered to affect platelet interaction with the vessel wall, either by direct receptor occupancy or downstream mechanisms. However, it is reasonable to assume that at very high supratherapeutic concentrations, cilengitide’s anti-aIIbb3 properties become predominant. This is in agreement with flow cytometry data: PAC-1 is dose-dependently reduced. It is known that cilengitide has a more than 1000-fold higher affinity for avb3 than for aIIbb3 [10, 30] and that cilengitide binds to av in the nanomolar range. It is, therefore, plausible that higher micro-molar concentrations predominantly exert effects through aIIbb3 inhibition in aggregation and fibrinogen binding, all of which rely on aIIbb3 activation. Different apparent potencies of fibrinogen–aIIbb3 interactions in aggregometry, fibrinogen binding in FACS and fibrinogen binding under flow in adhesion assays might be related to differences in fibrinogen activity between soluble and solid phases [31]. Even the potency of platelet–fibrinogen interaction on immobilized fibrinogen varies considerably, depending on coating procedures [25, 32]. Even if cilengitide did show some aIIbb3 inhibitory properties at high concentrations, it is likely that its receptor binding and signaling properties, including intrinsic receptor activity, may be substantially different from those reported for abciximab. For other small peptides, containing or mimicking the RGD sequence, it is known that the characteristics of aIIbb3-binding and blocking of fibrinogen binding differ widely from those of the large chimeric Fab fragment, abciximab. Since platelets are considered to play an important role in tumor-growth, -metastasis, and -angiogenesis, any anti-platelet properties of an ab3 integrin inhibitor, such as cilengitide, may provide beneficial anti-tumor activity. Platelets are involved in key steps of malignancy progression and are recognized as dynamic reservoirs of proangiogenic and anti-angiogenic proteins [33]. Experimental blockade of key platelet receptors such as aIIbb3 has been shown to attenuate the progression and metastatic potential of various tumor types. Abciximab has successfully been used to inhibit angiogenesis and tumor growth in the mouse [34]. This illustrates the bridging of classic platelet function (hemostasis) and angiogenesis, via the integrin inhibitors [35]. It has been argued that cilengitide at low concentrations can activate avb3 integrin [36, 37]. Such activation might lead to
Cilengitide has no pro-thrombotic properties
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DOI: 10.3109/09537104.2013.870332
stimulation of endothelial cells and increased platelet interaction with activated endothelium or direct platelet activation. Furthermore, thromboembolism has been noted as a side effect for a variety of agents interfering with the VEGF pathway, e.g., bevacizumab [38, 39]. In a physiologically robust system, cilengitide in low concentrations did not activate HUVECs to result in higher platelet adhesion to them. In contrast, high cilengitide concentrations inhibited platelet adhesion to HUVECs and adhesion ligands, such as fibrinogen, through the down-regulated expression of activated aIIbb3. This is in line with safety data from clinical trials with cilengitide having been applied to a range of different solid tumors. Data did not point to an increased risk of thromboembolic events in cilengitide treated patients [9, 40]. In conclusion, cilengitide showed a weak inhibitory effect on blood platelets, suggesting an antiaggregatory effect at higher concentrations. There was no evidence for any pro-thrombotic effect in terms of platelet activation and adhesion.
Acknowledgements
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The authors thank Simon Goodman for helpful hints, fruitful discussion and continuous support of the project. 18.
Declaration of interest This study has been supported by a grant from Merck KGaA. SH has received scientific grants from Merck KGaA and The Medicines Company and has received honoraria for lectures from Merck KGaA and LEO Pharmaceuticals. UK is employed by Merck KGaA. All other authors have no disclosures.
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References 1. Taugourdeau-Raymond S, Rouby F, Default A, Jean-Pastor MJ. Bevacizumab-induced serious side-effects: A review of the French pharmacovigilance database. Eur J Clin Pharmacol 2012;68: 1103–1107. 2. Nalluri SR, Chu D, Keresztes R, Zhu X, Wu S. Risk of venous thromboembolism with the angiogenesis inhibitor bevacizumab in cancer patients: A meta-analysis. JAMA 2008;300:2277–2285. 3. Ferroni P, Formica V, Roselli M, Guadagni F. Thromboembolic events in patients treated with anti-angiogenic drugs. Curr Vasc Pharmacol 2010;8:102–113. 4. Elice F, Rodeghiero F, Falanga A, Rickles FR. Thrombosis associated with angiogenesis inhibitors. Best Pract Res Clin Haematol 2009;22:115–128. 5. Jenkins EO, Schiff D, Mackman N, Key NS. Venous thromboembolism in malignant gliomas. J Thromb Haemost 2010;8: 221–227. 6. Buerkle MA, Pahernik SA, Sutter A, Jonczyk A, Messmer K, Dellian M. Inhibition of the alpha-nu integrins with a cyclic RGD peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. Br J Cancer 2002;86:788–795. 7. MacDonald TJ, Stewart CF, Kocak M, Goldman S, Ellenbogen RG, Phillips P, Lafond D, Poussaint TY, Kieran MW, Boyett JM, et al. Phase I clinical trial of cilengitide in children with refractory brain tumors: Pediatric Brain Tumor Consortium Study PBTC-012. J Clin Oncol 2008;26:919–924. 8. Yamada S, Bu XY, Khankaldyyan V, Gonzales-Gomez I, McComb JG, Laug WE. Effect of the angiogenesis inhibitor Cilengitide (EMD 121974) on glioblastoma growth in nude mice. Neurosurgery 2006;59:1304–1312; discussion 1312. 9. Stupp R, Hegi ME, Gorlia T, Erridge S, Grujicic D, Steinbach JP, Wick W, Tarnawski R, Nam D-H, Weyerbrock A, et al. Cilengitide combined with standard treatment for patients with newly diagnosed glioblastoma and methylated O6-methylguanine-DNA methyltransferase (MGMT) gene promoter: Key results of the multicenter, randomized, open-label, controlled, phase III CENTRIC study. J Clin Oncol 2013;13:(suppl; abstr LBA2009). 10. Dechantsreiter MA, Planker E, Matha B, Lohof E, Holzemann G, Jonczyk A, Goodman SL, Kessler H. N-Methylated cyclic RGD
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
7
peptides as highly active and selective alpha(V)beta(3) integrin antagonists. J Med Chem 1999;42:3033–3040. Meyer Dos Santos S, Klinkhardt U, Schneppenheim R, Harder S. Using ImageJ for the quantitative analysis of flow-based adhesion assays in real-time under physiologic flow conditions. Platelets 2010;21:60–66. Meyer dos Santos S, Klinkhardt U, Scholich K, Nelson K, Monsefi N, Deckmyn H, Kuczka K, Zorn A, Harder S. The CX3C chemokine fractalkine mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib. Blood 2011;117:4999–5008. Vassallo Jr RR, Kieber-Emmons T, Cichowski K, Brass LF. Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. J Biol Chem 1992;267: 6081–6085. Hu DD, Hoyer JR, Smith JW. Ca2þ suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin alpha v beta 3. J Biol Chem 1995;270:9917–9925. Valgimigli M, Campo G, Tebaldi M, Carletti R, Arcozzi C, Ferrari R, Percoco G. Abciximab: A reappraisal of its use in coronary care. Biol Targets Ther 2008;2:29–39. Armstrong PC, Peter K. GPIIb/IIIa inhibitors: From bench to bedside and back to bench again. Thromb Haemost 2012;107: 808–814. Fuentes QE, Fuentes QF, Andres V, Pello OM, de Mora JF, Palomo GI. Role of platelets as mediators that link inflammation and thrombosis in atherosclerosis. Platelets 2013;24:255–262. Tsao PW, Forsythe MS, Mousa SA. Dissociation between the antiaggregatory & anti-secretory effects of platelet integrin alpha IIb beta 3 (GPIIb/IIIa) antagonists, c7E3 and DMP728. Thromb Res 1997;88:137–146. Schneider DJ, Taatjes DJ, Sobel BE. Paradoxical inhibition of fibrinogen binding and potentiation of alpha-granule release by specific types of inhibitors of glycoprotein IIb-IIIa. Cardiovasc Res 2000;45:437–446. Peter K, Schwarz M, Ylanne J, Kohler B, Moser M, Nordt T, Salbach P, Kubler W, Bode C. Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (alphaIIbbeta3) inhibitors. Blood 1998;92: 3240–3249. Klinkhardt U, Graff J, Harder S. Clopidogrel, but not abciximab, reduces platelet leukocyte conjugates and P-selectin expression in a human ex vivo in vitro model. Clin Pharmacol Ther 2002;71: 176–185. Alghisi GC, Ponsonnet L, Ruegg C. The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells. PLoS One 2009;4:e4449. Roest M, Reininger A, Zwaginga JJ, King MR, Heemskerk JW. Flow chamber-based assays to measure thrombus formation in vitro: Requirements for standardization. J Thromb Haemost: JTH 2011;9: 2322–2324. Heemskerk JW, Sakariassen KS, Zwaginga JJ, Brass LF, Jackson SP, Farndale RW. Collagen surfaces to measure thrombus formation under flow: Possibilities for standardization. J Thromb Haemost 2011;9:856–858. Jirouskova M, Jaiswal JK, Coller BS. Ligand density dramatically affects integrin alpha IIb beta 3-mediated platelet signaling and spreading. Blood 2007;109:5260–5269. Nurden AT. Is there a redundancy of beta3 and other platelet receptors in the brain and central nervous system? Platelets 2013;24: 170–172. Coller BS. Binding of abciximab to alpha V beta 3 and activated alpha M beta 2 receptors: With a review of platelet-leukocyte interactions. Thromb Haemost 1999;82:326–336. Coller BS, Cheresh DA, Asch E, Seligsohn U. Platelet vitronectin receptor expression differentiates Iraqi-Jewish from Arab patients with Glanzmann thrombasthenia in Israel. Blood 1991;77:75–83. Tam SH, Sassoli PM, Jordan RE, Nakada MT. Abciximab (ReoPro, chimeric 7E3 Fab) demonstrates equivalent affinity and functional blockade of glycoprotein IIb/IIIa and alpha(v)beta3 integrins. Circulation 1998;98:1085–1091. Mas-Moruno C, Beck JG, Doedens L, Frank AO, Marinelli L, Cosconati S, Novellino E, Kessler H. Increasing alphavbeta3 selectivity of the anti-angiogenic drug cilengitide by N-methylation. Angew Chem Int Ed Engl 2011;50:9496–9500.
8
S. M. dos Santos et al.
Platelets Downloaded from informahealthcare.com by University of Ulster at Jordanstown on 12/29/14 For personal use only.
31. Bonnefoy A, Liu Q, Legrand C, Frojmovic MM. Efficiency of platelet adhesion to fibrinogen depends on both cell activation and flow. Biophys J 2000;78:2834–2843. 32. Cook BC. Reactivity of human platelets with immobilized fibrinogen is dictated by the chemical character of the surface. Thromb Res 2001;104:39–48. 33. Peterson JE, Zurakowski D, Italiano Jr JE, Michel LV, Fox L, Klement GL, Folkman J. Normal ranges of angiogenesis regulatory proteins in human platelets. Am J Hematol 2010;85:487–493. 34. Varner JA, Nakada MT, Jordan RE, Coller BS. Inhibition of angiogenesis and tumor growth by murine 7E3, the parent antibody of c7E3 Fab (abciximab; ReoPro). Angiogenesis 1999;3:53–60. 35. Trikha M, Zhou Z, Timar J, Raso E, Kennel M, Emmell E, Nakada MT. Multiple roles for platelet GPIIb/IIIa and alphavbeta3 integrins in tumor growth, angiogenesis, and metastasis. Cancer Res 2002;62:2824–2833. 36. Hodivala-Dilke KM, Reynolds AR, Reynolds LE. Integrins in angiogenesis: Multitalented molecules in a balancing act. Cell Tissue Res 2003;314:131–144.
Platelets, Early Online: 1–8
37. Reynolds AR, Hart IR, Watson AR, Welti JC, Silva RG, Robinson SD, Da Violante G, Gourlaouen M, Salih M, Jones MC, et al. Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors. Nat Med 2009;15:392–400. 38. Narita Y. Drug review: Safety and efficacy of bevacizumab for glioblastoma and other brain tumors. Jpn J Clin Oncol 2013;43: 587–595. 39. Costagliola C, Agnifili L, Arcidiacono B, Duse S, Fasanella V, Mastropasqua R, Verolino M, Semeraro F. Systemic thromboembolic adverse events in patients treated with intravitreal antiVEGF drugs for neovascular age-related macular degeneration. Expert Opin Biol Ther 2012;12:1299–1313. 40. Nabors LB, Mikkelsen T, Hegi ME, Ye X, Batchelor T, Lesser G, Peereboom D, Rosenfeld MR, Olsen J, Brem S, et al. A safety run-in and randomized phase 2 study of cilengitide combined with chemoradiation for newly diagnosed glioblastoma (NABTT 0306). Cancer 2012;118:5601–5607.
