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Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear. [ABSTRACT FROM AUTHOR]

GPVI and CLEC-2 have emerged as promising targets for long-term prevention of both arterial thrombosis and thrombo-inflammation with a decreased bleeding risk relative to current drugs. However, while there are potent blocking antibodies of both receptors, their protein nature comes with decreased bioavailability, making formulation for oral medication challenging. Small molecules are able to overcome these limitations, but there are many challenges in developing antagonists of nanomolar potency, which is necessary when considering the structural features that underlie the interaction of CLEC-2 and GPVI with their protein ligands. In this review, we describe current small-molecule inhibitors for both receptors and strategies to overcome such limitations, including considerations when it comes to in silico drug design and the importance of complex compound library selection. [ABSTRACT FROM AUTHOR]