PB0348 - High shear rate thrombi occlude blood flow by a cable of VWF and platelets

Background: The structure and ultrastructure of freshly occlusive arterial thrombi remain ill defined. Using a combination of light microscopy, fluorescent antibodies, and high-resolution scanning electron microscopy (SEM), we discover the complex architecture of VWF-platelet thrombi.

Aims: To reveal the structure and ultrastructure of occlusive clots formed by shear-induced platelet aggregation (SIPA).

Methods: Thrombi were formed by flowing whole heparinized human blood at high shear rate (>6,000/s) through a collagen-coated stenotic chamber (80x1000 µm). VWF and fibrinogen presence were assessed by immunohistochemistry (IHC). Thrombi were fixed with glutaraldehyde and formalin for SEM and IHC, respectively. Light microscopy (10x), confocal microscopy (20x) and SEM (2,000-10,000x) were used to image thrombi.

Results: Relatively large thrombi 200x1000µm in size formed within 5 minutes at the upstream end of the stenotic test section. The formation of thrombotic patches throughout the test section was easily seen by light microscopy (Fig. 1A). SEM revealed the occlusive mass to have the structure of a large, transverse cable spanning 1000 µm (Fig. 1B). The cable was 100-150-µm wide and concave. The shape and orientation of the thrombotic mass are consistent with a structure formed under tension resisting upstream pressure. The thrombotic cable was anchored by 10-50-µm elongated islets both upstream and downstream. Non-coalescing islets were scattered further downstream but were not occlusive. Higher resolution showed the cable to be formed from hundreds of 1-3-µm-wide chains of platelets connected in series by strands of fibers 0.5-1-µm wide (Fig. 2A). IHC identified the fibers to be made of VWF, not fibrin (Fig. 2B).

Conclusion(s): SEM images unveil a new ultrastructure for high shear rate thrombi. Islets of platelet aggregates are bridged to a cable of platelets wrapped by strands of VWF. The cable orientation perpendicular to the blood flow can provide strength for arterial occlusive thrombi, like a suspension bridge.