Abstract
Primary hemostasis results in a platelet-rich thrombus that has long been assumed to form a solid plug. Unexpectedly, our 3-dimensional (3D) electron microscopy of mouse jugular vein puncture wounds revealed that the resulting thrombi were structured about localized, nucleated platelet aggregates, pedestals and columns, that produced a vaulted thrombus capped by extravascular platelet adherence. Pedestal and column surfaces were lined by procoagulant platelets. Furthermore, early steps in thrombus assembly were sensitive to P2Y12 inhibition and late steps to thrombin inhibition. Based on these results, we propose a Cap and Build, puncture wound paradigm that should have translational implications for bleeding control and hemostasis.
Highlights
Primary hemostasis results in a platelet-rich thrombus that has long been assumed to form a solid plug
Interpretation has been dominated by the outcomes seen by intravital microscopy[1,11] in which an initial platelet deposition associated with the damaged vessel wall is followed by α-granule secretion[12] in many of the platelets and cell-surface exposure of p-selectin, an α-granule membrane protein
In the Core and Shell paradigm [for review, see 17], it has been assumed that the formation of a solid, platelet plug is the final result of primary hemostasis, a process in which bleeding cessation is produced in a stepwise manner through platelet adhesion to the exposed vessel wall, subsequent platelet activation and plug formation[18]
Summary
Primary hemostasis results in a platelet-rich thrombus that has long been assumed to form a solid plug. The hemostatic thrombus when viewed at early stages showed a pebbly distribution of platelet aggregates suggestive of nucleated platelet accumulation rather than the smooth layers that would follow from a Core and Shell paradigm The outgrowth of these aggregates could lead to a vaulted structure rather than a solid platelet plug. Taking an initial cue from nucleated platelet aggregate formation, we tested the possibility that thrombus formation within a nominal 300 μm diameter, jugular vein, puncture hole forms a vaulted structure in which platelet activation patterns follow from the underlying structure To achieve this goal, we visualized the interior and overall structure of the forming puncture wound thrombus fully in 3D at subplatelet level resolution using serial block face scanning electron microscopy (SBF-SEM) to give a “coarse grain” full thrombus structure supported by more detailed higher resolution wide area transmission electron microscopy (WA-TEM). We suggest that our mothers’ repeated admonitions to not play with the scab have a much stronger basis than once suspected
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