Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial
thrombus may result in new ways to treat arterial
thrombosis. The distinguishing feature between these two regions is the absence of
fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of
Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on
collagen or
atherosclerotic plaque homogenate at arterial shear (1000 s-1). We show that post-perfusion of the Syk inhibitor
PRT-060318 over preformed thrombi on both surfaces enhances
thrombus breakdown and platelet detachment. The resulting loss of
thrombus stability led to a reduction in
thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of
thrombus stability was observed with
ticagrelor and
indomethacin, inhibitors of platelet
adenosine diphosphate (
ADP) receptor and
thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor,
dasatinib. In contrast, the Btk inhibitor,
ibrutinib, causes only a minor decrease in
thrombus contractile score. Weak
thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of
collagen to the stability of the platelet aggregate. These results show that Syk regulates
thrombus stability in the absence of
fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by
collagen.