Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating ITAM-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as anti-neoplastic and anti-inflammatory therapeutics and have also gained interest as anti-platelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include, four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659, four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib), and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist CRP-XL. Similarly, these TKIs reduced the percentage of activated integrin αIIbβ3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. While all TKIs tested inhibited PLCγ2 phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to PI3K and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

Small molecule tyrosine kinase inhibitors (TKIs) serve increasingly important roles in the treatment of hematologic malignancies and also have shown efficacy in a range of immunological and inflammatory conditions. However, many successful TKI therapies are associated with problematic effects - particularly on platelets - as bleeding complications have been reported for many TKIs, both on and off clinical trials. Bleeding risk is especially apparent for patients treated with ibrutinib and other irreversible inhibitors of Bruton's tyrosine kinase (BTK), several of which now effectively treat multiple B cell malignancies. Intriguingly, other therapeutic TKIs targeting mechanistic partners of BTK are not associated with platelet-related complications. For instance, inhibitors against the BTK-activating kinase Syk - both with selective (i.e., entospletinib) as well as promiscuous (i.e., fostamatinib) target profiles - are not associated with clinically increased bleeding, despite some reported antiplatelet effects. To gain insight into the multifaceted effects of Syk and BTK inhibitors on platelet function, we analyzed the effects of a panel of eight different clinically relevant Syk- and BTK-directed TKIs (i.e., fostamatinib/R406, entospletinib, ibrutinib, acalabrutinib, AVL-292 and others) on essential platelet responses. We first assessed the effects of TKI treatment on Syk and BTK-mediated signaling events downstream of the platelet immunoreceptor tyrosine-based activation motif (ITAM) receptor GPVI. All Syk and BTK TKIs tested inhibited the phosphorylation of phospholipase C (PLCγ2) Y1217, protein kinase Cδ (PKCδ) Y311 and Akt T308, as well as Akt substrate phosphorylation following platelet stimulation with the GPVI receptor agonist, crosslinked collagen-related peptide (CRP-XL). Independent of TKI target profile, Syk and BTK TKIs differentially inhibited the phosphorylation of DAPP1 Y132, phosphoinositide 3-kinase (PI3K) p85α Y458, Akt S473 and PKC substrates. Syk and BTK TKIs also inhibited platelet adhesion to immobilized CRP-XL in a manner matching the effects of each TKI on ITAM-mediated signaling events. However, platelet signaling and adhesion phenotypes did not match the effects of these same TKIs on GPVI-triggered dense granule secretion, which was inhibited by all TKIs tested with the exception of R406. In contrast, all Syk and BTK inhibitors tested inhibited platelet spreading on immobilized fibrinogen. Based on observations above, we hypothesized that TKIs that irreversibly bind to BTK may disrupt critical molecular interactions around BTK within the PI3K signalosome essential to the proper orchestration of platelet activation programs. To test the effects of Syk and BTK inhibitors on the organization of PI3K signaling in activating platelets, platelets were pretreated with TKIs prior to incubation on immobilized fibrinogen and processing for immunofluorescence microscopy. We found that under control conditions, PI3K p85α regulatory subunit localized to phospholipid PI(3,4)P2-rich regions in adherent platelets associated with active PKC signaling. While Syk and PI3K inhibitors abrogated platelet spreading on fibrinogen, PI3K p85α staining remained strong around undeveloped, nascent adhesions in platelets treated with Syk and PI3K inhibitors. In contrast, PI3K p85α staining was diffuse, diminished or absent in platelets adherent to fibrinogen in the presence of all irreversible BTK inhibitors tested. Our results show that clinically effective kinase inhibitors that target Syk-BTK-PI3K signaling systems have varying effects on platelet function that may offer insights to adverse effects of therapeutic TKIs in different contexts. Moreover, our findings suggest that in addition inhibiting protein kinase activities, the discrepant effects of some TKIs may be attributed to, in part, altered protein relations around BTK, PLCγ2 and other components of the PI3K signalosome in activating platelets. Ongoing studies aim to specify protein:protein interactions affected by reversible as well as irreversible TKIs; to examine patient samples for evidence of PI3K mislocalization - especially in cases of BTK inhibitor-associated bleeding; and, to determine if PI3K signalosome disorganization has roles in other physiological, therapeutic and toxic effects of TKIs. Disclosures Shatzel: Aronora, Inc.: Consultancy.