Abstract
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.
Highlights
The outer shell of an arterial thrombus is composed of aggregated platelets held together by the interaction of fibrinogen with integrin αIIbβ3, with activation of the integrin mediated by the autocrine feedback mediators, adenosine diphosphate (ADP) and thromboxane A2 (TxA2) [18,19]
Platelets in the shell region of aggregates formed in vivo are held together by the binding of fibrinogen to the major platelet integrin αIIbβ3, with the diffusion of secondary mediators from the thrombus core region mediating inside-out activation of the integrin and binding to fibrinogen
We demonstrate a critical role for Src and Syk tyrosine kinases in supporting the stability of preformed shell-type thrombi on collagenous surfaces in the absence of fibrin formation, and show that this is mediated in concert with the signals from
Summary
Platelet-rich thrombi formed in vivo in mice have been shown to be composed of a core and shell region in both the arterial and venous microcirculation [1]. Platelets and fibrin are densely packed, and permeability is heavily restricted [2], necessitating the use of a fibrinolytic to dissolve the platelet aggregate [3,4]. The outer, more permeable, shell region consists of weakly activated and loosely packed platelets and does not contain fibrin [2], suggesting that a different strategy is needed to promote disruption of this region and help to prevent the build-up of an occlusive thrombus. In vitro flow studies have shown that GPVI contributes to the stability of newly formed platelet aggregates on collagen at high shear through use of the blocking anti-GPVI Fab ACT017, which has more recently been named glenzocimab [4]. Since the activation of GPVI by fibrinogen is dependent on integrin αIIbβ, with the interplay of the two receptors driving platelet adhesion and activation [7]; this suggests that blocking signalling pathways common to both receptors may have a greater antithrombotic effect than blocking GPVI alone
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
