Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients

ORIGINAL RESEARCH published: 16 October 2018 doi: 10.3389/fcvm.2018.00142 Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients Bibian M. E. Tullemans 1 , Magdolna Nagy 1 , Siamack Sabrkhany 2 , Arjan W. Griffioen 3 , Mirjam G. A. oude Egbrink 2 , Maureen Aarts 4 , Johan W. M. Heemskerk 1 and Marijke J. E. Kuijpers 1* 1 Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands, Department of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands, 3 Angiogenesis Laboratory, Department of Medical Oncology, VU Medical Center, Amsterdam, Netherlands, 4 Department of Medical Oncology, Maastricht University Medical Center, Maastricht, Netherlands 2 Edited by: Anna Falanga, Ospedale Papa Giovanni XXIII, Italy Reviewed by: Ulrich Walter, Universitätsmedizin Mainz, Germany Marie Lordkipanidzé, Université de Montréal, Canada *Correspondence: Marijke J. E. Kuijpers Specialty section: This article was submitted to Atherosclerosis and Vascular Medicine, a section of the journal Frontiers in Cardiovascular Medicine Received: 08 May 2018 Accepted: 24 September 2018 Published: 16 October 2018 Citation: Tullemans BME, Nagy M, Sabrkhany S, Griffioen AW, oude Egbrink MGA, Aarts M, Heemskerk JWM and Kuijpers MJE (2018) Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients. Front. Cardiovasc. Med. 5:142. doi: 10.3389/fcvm.2018.00142 Pazopanib is an angiostatic tyrosine kinase inhibitor (TKI) presently used for cancer treatment, particularly in patients with renal cell carcinoma (RCC). This treatment can be accompanied by mild bleeding as an adverse effect. Given the role of protein tyrosine kinases in platelet activation processes, we investigated whether and how pazopanib can affect platelet functions in purified systems and during treatment of advanced RCC patients. In isolated platelets from healthy volunteers, pazopanib dose-dependently reduced collagen-induced integrin activation and secretion, as well as platelet aggregation. Pazopanib addition diminished glycoprotein (GP) VI-dependent tyrosine phosphorylation of multiple platelet proteins, including the tyrosine kinase Syk. Furthermore, pazopanib inhibited GPVI-induced Ca2+ elevation, resulting in reduced exposure of the procoagulant phospholipid phosphatidylserine (PS). Upon perfusion of control blood over a collagen surface, pazopanib inhibited thrombus size as well as PS exposure. Blood samples from 10 RCC patients were also analyzed before and after 14 days of pazopanib treatment as monotherapy. This treatment caused an overall lowering in platelet count, with 3 out of 10 patients experiencing mild bleeding. Platelets isolated from pazopanib-treated patients showed a significant lowering of PS exposure upon activation. In addition, platelet procoagulant activity was inhibited in thrombi formed under flow conditions. Control experiments indicated that higher pazopanib concentrations were required to inhibit GPVI-mediated PS exposure in the presence of plasma. Together, these results indicated that pazopanib suppresses GPVI-induced platelet activation responses in a way partly antagonized by the presence of plasma. In treated cancer patients, pazopanib effects were confined to a reduction in GPVI-dependent PS exposure. Together with the reduced platelet count, this may explain the mild bleeding tendency observed in pazopanib-treated patients. Keywords: platelets, thrombus, tyrosine kinase inhibitor, phosphatidylserine, pazopanib, procoagulant activity Frontiers in Cardiovascular Medicine | www.frontiersin.org 1 October 2018 | Volume 5 | Article 142 Tullemans et al. Pazopanib Inhibits Platelet Procoagulant Activity INTRODUCTION fibroblast growth factor receptor, IL-2 receptor inducible T-cell kinase (Itk), leukocyte-specific protein tyrosine kinase (Lck), and the glycoprotein receptor c-Fms (24). In addition, other in vitro kinase targets of pazopanib have been described, of which Abl1, Abl2, Fgr, Src, Fyn, and Lck are present in platelets (26, 27). Patients are commonly treated with a high daily doses of pazopanib (800 mg), resulting in a steady-state plasma concentration of up to 45 µg/mL after several weeks (24). This treatment regimen can reduce the platelet count and lead to bleeding events (24). Since effects of pazopanib on platelet function have not been reported, we aimed to investigate this in vitro and ex vivo, using blood from RCC patients and control subjects. Tyrosine kinase inhibitors (TKIs) are widely approved drugs, aiming to target tyrosine kinase signaling pathways that regulate uncontrolled cellular growth and proliferation. Currently, several TKIs are in clinical use for the treatment of malignancies, such as lung, breast, kidney, and neuro-endocrine pancreatic cancers as well as gastro-intestinal stromal tumors and chronic myeloid leukemia (1–3). Their common way of action is by competition with adenosine triphosphate (ATP) in the conserved catalytic binding site in the protein tyrosine kinase superfamily. In spite of this action mechanism, individual TKIs can target partially different spectra of intracellular tyrosine kinases, can have different pharmacokinetics, and vary in their adverse effects (1). Commonly, TKIs are clinically applied as a multi-target therapy to intervene in tumor proliferation (4, 5). Specific targets are the receptors for vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), which are all involved in (tumor) angiogenesis (3, 6). The expected effects are to reduce tumor lesions, delay disease development, and thus prolong the progression-free survival of patients (7). Platelets contain several protein tyrosine kinases as key signal transducers, which regulate the function of platelets in hemostasis (8). Downstream of glycoprotein (GP)VI (collagen receptor), GPIb-IX-V (von Willebrand receptor) and CLEC-2 (podoplanin receptor), Src family tyrosine kinases control the signaling routes to most platelet responses (8–10). Activation of GPVI also implies tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) present on the Fc-receptor γ-chain, which is co-expressed with GPVI (11). This results in activation of the protein tyrosine kinase Syk (10, 12), and further downstream Bruton tyrosine kinase (Btk), culminating in the phosphorylation and activation of phospholipase Cγ2 (PLCγ2), an event required for integrin activation and granule secretion. A similar set of protein tyrosine kinases (Src-family kinases, Syk and JAK isoforms) is known to play a critical role in megakaryocyte development and thrombocytopoiesis (13). Considering the role of protein tyrosine kinases in both platelet activation and platelet formation, it can be expected that treatment of patients with (...truncated)