Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical 
roles in platelet physiology, facilitating intracellular immunoreceptor 
tyrosine-based activation motif (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 antineoplastic 
and anti-inflammatory therapeutics and have also gained interest as antiplatelet 
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 cross-linked collagen-related peptide 
(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. Although all TKIs tested inhibited phospholipase C γ2 (PLCγ2) 
phosphorylation following GPVI-mediated activation, other downstream signaling 
events proximal to phosphoinositide 3-kinase (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.