Background Treatment of chronic myelogenous leukemia (CML) with the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib significantly improves patient outcomes. As some patients are unresponsive to imatinib, next generation BCR-ABL inhibitors such as nilotinib have been developed to treat patients with imatinib-resistant CML. The use of some BCR-ABL inhibitors has been associated with bleeding diathesis, and these inhibitors have been shown to inhibit platelet functions, which may explain the hemostasis impairment. Surprisingly, a new TKI, ponatinib, has been associated with a high incidence of severe acute ischemic cardiovascular events. The mechanism of this unexpected adverse effect remains undefined. Objective and Methods This study used biochemical and functional assays to evaluate whether ponatinib was different from the other BCR-ABL inhibitors with respect to platelet activation, spreading, and aggregation. Results and Conclusions Our results show that ponatinib, similar to other TKIs, acts as a platelet antagonist. Ponatinib inhibited platelet activation, spreading, granule secretion, and aggregation, likely through broad spectrum inhibition of platelet tyrosine kinase signaling, and also inhibited platelet aggregate formation in whole blood under shear. As our results indicate that pobatinib inhibits platelet function, the adverse cardiovascular events observed in patients taking ponatinib may be the result of the effect of ponatinib on other organs or cell types or disease-specific processes, such as BCR-ABL+ cells undergoing apoptosis in response to chemotherapy, or drug-induced adverse effects on the integrity of the vascular endothelium in ponatinib-treated patients.

Rigg RA, Healy LD, Nowak MS, Mallet J, Thierheimer ML, Pang J, McCarty OJ, Aslan JE. Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation. Molecular chaperones that support protein quality control, including heat shock protein 70 (Hsp70), participate in diverse aspects of cellular and physiological function. Recent studies have reported roles for specific chaperone activities in blood platelets in maintaining hemostasis; however, the functions of Hsp70 in platelet physiology remain uninvestigated. Here we characterize roles for Hsp70 activity in platelet activation and function. In vitro biochemical, microscopy, flow cytometry, and aggregometry assays of platelet function, as well as ex vivo analyses of platelet aggregate formation in whole blood under shear, were carried out under Hsp70-inhibited conditions. Inhibition of platelet Hsp70 blocked platelet aggregation and granule secretion in response to collagen-related peptide (CRP), which engages the immunoreceptor tyrosine-based activation motif-bearing collagen receptor glycoprotein VI (GPVI)-Fc receptor-␥ chain complex. Hsp70 inhibition also reduced platelet integrin-␣IIb␤3 activation downstream of GPVI, as Hsp70-inhibited platelets showed reduced PAC-1 and fibrinogen binding. Ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear. Biochemical studies supported a role for Hsp70 in maintaining the assembly of the linker for activation of T cells signalosome, which couples GPVI-initiated signaling to integrin activation, secretion, and platelet function. Together, our results suggest that Hsp70 regulates platelet activation and function by supporting linker for activation of T cells-associated signaling events downstream of platelet GPVI engagement, suggesting a role for Hsp70 in the intracellular organization of signaling systems that mediate platelet secretion, "inside-out" activation of platelet integrin-␣IIb␤3, platelet-platelet aggregation, and, ultimately, hemostatic plug and thrombus formation. chaperones; hemostasis; integrin; platelets; thrombosis HEAT SHOCK PROTEIN (Hsp) 70 is an ATP-powered, 70-kDa molecular chaperone that regulates a myriad of protein qualitycontrol processes, including folding of nascent polypeptides, trafficking of proteins across membranes, prevention of protein aggregation, and protein complex assembly and disassembly (15,34,46). Hsp70 is an abundantly expressed and highly conserved protein with 50% sequence homology between mammals and prokaryotes, highlighting the importance of Hsp family members in protein homeostasis throughout evolution (10). In human cells, the significance and complexity of Hsp70 function are evident in the functions of the 13 members of the human Hsp70 family, including the cytosolic, stress-inducible Hsp70 (or Hsp72) and its constitutively expressed heat shock cognate 71-kDa protein (Hsc70), the endoplasmic reticulum (ER)-localized 78-kDa glucose-regulated protein (Grp78, or bindi...

Summary Background and Objectives The reversible acetylation of protein lysine ε-amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in the orchestration of diverse cellular activities. Here, we aimed to investigate the role of lysine acetyltransfer in platelet function. Methods and Results Proteomics methods identified 552 acetyllysine (acK) modifications on 273 platelet proteins that serve as candidate substrates for lysine acetyltransferases. Bioinformatics analyses of identified acK-modified platelet proteins supported roles for the lysine acetyltransferase p300 in the regulation of actin-mediated platelet processes. Biochemical experiments found that platelets express p300, which is activated in a Src kinase-dependent manner upon platelet stimulation with the platelet glycoprotein VI (GPVI) agonist CRP. Inhibition of platelet p300 abrogated CRP-stimulated lysine acetylation of actin, filamin and cortactin as well as F-actin polymerization, integrin activation and platelet aggregation. Super resolution visualization of platelet actin-rich adhesion structures revealed abundant acetyllysine protein content colocalized with platelet actin cytoskeletal proteins. Inhibition of p300 blocked platelet filopodia formation and the spreading of platelets on fibrinogen and collagen surfaces. In whole blood, p300 inhibition prevented the formation of platelet aggregates under shear, suggesting a physiological role for lysine acetyltransferase activity in platelet function. Conclusion Together, our findings uncover lysine acetyltransfer as a potential regulator of platelet actin dynamics and reveal potential roles for lysine acetylation in the molecular coordination of platelet activation and function.