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Structural hallmarks of stroke thrombi
is composed of rather homogeneously distributed RBC that are densely packed and embedded in thin fibrin strands, with no other main structural components.
Platelet-rich areas consist of dense structures of fibrin, von Willebrand Factor and platelets
Platelet-rich thrombus material appeared light pink on H&E staining, without clear cellular elements (Figure 4A). MSB staining showed pink, dense fibrin structures that were clearly visible throughout the platelet-rich regions (Figure 4B). To gain more detailed insight into the microstructural organization of these platelet-rich areas, both immunohistochemical and immunofluorescent co- stainings were performed combining fibrin(ogen) with vWF and/or platelets. In accordance with the MSB stain- ing, fluorescence microscopy for fibrin(ogen) confirmed the dense fibrin structures that demarcate platelet-rich zones (Figure 4C and D). Remarkably, co-staining with vWF showed that these dense fibrin structures are also positive for vWF (Figure 4C and D), suggesting an interac- tion between vWF and fibrin. Together, fibrin and vWF delineate substructures that are filled with platelets. Indeed, co-staining of platelets and fibrin(ogen) or vWF shows the presence of such platelet islands within these structures of fibrin and vWF. Hence, in contrast to the
RBC-rich zones, the platelet-rich zones contain no RBC, but are mainly composed of dense formations of fibrin and vWF, packed with platelets. Figure 5 shows a typical overview picture of both zones in one thrombus, illustrat- ing on the one hand platelet-rich areas surrounded by dense fibrin with little fibrin between platelets and on the other hand RBC-rich areas with densely packed RBC (not stained) entangled in a thin fibrin network.
Leukocytes and DNA are mainly present on the interface between red blood cell-rich and platelet-rich areas
We and others have previously shown the abundant presence of leukocytes in stroke thrombi, but apart from their presence, not much is known about their specific cel- lular or molecular distribution.11,13 Remarkably, when staining for RBC-rich and platelet-rich areas, we observed that leukocytes are primarily found at the interface between RBC-rich and platelet-rich areas (Figure 6A-D and Online Supplementary Figure S5). Besides their specific presence in these boundary zones, leukocytes are also abundantly present within the platelet-rich zones. In con- trast, leukocytes are not commonly found in RBC-rich areas, where, if present, they are homogenously distrib- uted throughout the RBC (Figure 6E).
Figure 5. Immunofluorescent overview picture of red blood cell (RBC)-rich and platelet-rich areas. Immunofluorescent analysis was used to visualize fibrin(ogen) (green), platelets (red), and nuclei (blue). RBC-rich areas consist of thin fibrin strands and RBC (not stained), whereas platelet-rich areas consist of dense fibrin structures packed with platelets. Nucleated cells were mainly found near platelet-rich areas. Scale bar = 20 μm. P: platelet-rich area; R: RBC-rich area.
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