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Ibrutinib and acalabrutinib are irreversible inhibitors of Bruton’s tyrosine kinase used in the treatment of B cell malignancies. They bind irreversibly to cysteine 481 of Bruton’s tyrosine kinase, blocking autophosphorylation on tyrosine 223 and phosphorylation of downstream substrates including phospholipase-ɣ2. In the present study, we demonstrate that concentrations of ibrutinib and acalabrutinib that block Bruton’s tyrosine kinase activity as shown by loss of phosphorylation at tyrosine 223 and phospholipase-ɣ2 delay but do not block aggregation to a maximally-effective concentration of collagen related peptide or collagen. In contrast, 10-20 fold higher concentrations of ibrutinib or acalabrutinib block platelet aggregation to Glycoprotein VI agonists. Ex vivo studies on patients treated with ibrutinib, but not acalabrutinib, show a reduction of platelet aggregation to collagen related peptide indicating that the clinical dose of ibrutinib but not acalabrutinib is supramaximal for Bruton’s tyrosine kinase blockade. Unexpectedly, low concentrations of ibrutinib inhibit aggregation to collagen related peptide in patients deficient in Bruton’s tyrosine kinase. The increased bleeding seen with ibrutinib over acalabrutinib is due to off-target actions of ibrutinib that occur because of unfavourable pharmacodynamics.

Background: Platelet GPVI binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen and occurs constitutively on resting platelets. Objective: To identify higher order oligomerisation (clustering) of pre-existing GPVI-dimers upon interaction with collagen as a mechanism to initiate GPVI-mediated signalling. Methods: GPVI was located using fluorophore conjugated GPVI-dimer-specific Fab (antigen-binding fragment. Tested substrates include Horm collagen I fibres, soluble collagen III, GPVI-specific collagen peptides and fibrinogen. GPVI-dimer clusters on the platelet surface interacting with these substrates were visualised\ud using complementary imaging techniques: Total Internal Reflection Fluorescence Microscopy (TIRFM) to monitor real time interactions and direct Stochastic Optical Reconstruction Microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI-dimer clusters, GPIb, integrin α2β1, and phosphotyrosine. Results: Upon platelet adhesion to all collagenous substrates, GPVI-dimers coalesced to form clusters; notably clusters formed along the fibres of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate,\ud collagen III being most effective. Clusters on fibrinogen-adherred platelets were much smaller and more numerous; whether these are pre-existing oligomers of GPVI-dimers or\ud fibrinogen-induced is not conclusive. Some GPVI-dimer clusters colocalized with areas of phosphotyrosine, indicative of signalling activity. Integrin α2β1 localized to collagen fibres close to GPVI-dimer clusters. GPVI-clustering depends on a dynamic actin cytoskeleton.\ud Conclusions: Platelet adhesion to collagen induces GPVI-dimer clustering. GPVI-clustering increases both avidity for collagen and proximity of GPVI-associated signalling molecules, which may be crucial for initiation and persistence of signalling.

The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome.

Summary Background The activation of platelet CLEC‐2 by podoplanin on lymphatic endothelial cells (LECs) has a critical role in prevention of mixing of lymphatic and blood vasculatures during embryonic development. Paradoxically, LECs release cAMP and cGMP‐elevating agents, prostacyclin (PGI2) and nitric oxide (NO), respectively, which are powerful inhibitors of platelet activation. This raises the question of how podoplanin is able to activate CLEC‐2 in the presence of the inhibitory cyclic nucleotides. Objectives We investigated the influence of cyclic nucleotides on CLEC‐2 signaling in platelets. Methods We used rhodocytin, CLEC‐2 monoclonal antibody, LECs and recombinant podoplanin as CLEC‐2 agonists on mouse platelets. The effects of the cyclic nucleotide‐elevating agents PGI2, forskolin and the NO‐donor GSNO were assessed with light transmission aggregometry, flow cytometry, protein phosphorylation and fluorescent imaging of platelets on LECs. Results We show that platelet aggregation induced by CLEC‐2 agonists is resistant to GSNO but inhibited by PGI2. The effect of PGI2 is mediated through decreased phosphorylation of CLEC‐2, Syk and PLCγ2. In contrast, adhesion and spreading of platelets on recombinant podoplanin, CLEC‐2 antibody and LECs is not affected by PGI2 and GSNO. Consistent with this, CLEC‐2 activation of Rac, which is required for platelet spreading, is not altered in the presence of PGI2. Conclusions The present results demonstrate that platelet adhesion and activation on CLEC‐2 ligands or LECs is maintained in the presence of PGI2 and NO.

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.