Immunohistological Evaluation of Von Willebrand Factor in the Left Atrial Endocardium and Atrial Thrombi from Cats with Cardiomyopathy

Figures

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  Figure 1. Localisation of vWF in the LA samples. (A) vWF localised to the endothelium of a vessel (wide arrow) in a control cat where there was minimal immunostaining of vWF at the left atrial endocardium. 100× magnification. (B) In this microthrombus from a cardiomyopathic cat with clinical signs, vWF appeared to form a scaffold outside (open arrows) and within the microthrombus. Red blood cells (RBC) were relatively fluorescence-lucent (thick arrow). vWF also localised to the leucocytes, (closed arrow heads) and platelets (PLTs) within the microthrombus. Some leucocytes did not immunostain for vWF (open arrow heads). Endocardial endothelial cells (thin arrows) also expressed vWF. (C) vWF localised to the endothelium of a vessel in a cat with cardiomyopathy and clinical signs. The intravascular microthrombus had vWF (green) encompassing the thrombus (open arrows) and contacting the vascular endothelium. Platelets and vWF co-localised in the centre of the microthrombus (orange, or yellow). (D) vWF (green), leucocytes, or WBC (blue) and some platelets (orange or yellow) attached on the endocardium from a cardiomyopathic cat with clinical signs. 400× magnification.

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  Figure 2. Localisation of vWF in the endocardium. Immunostaining of vWF in the endocardium. On the left, endocardial vWF (green) in a cat with cardiomyopathy and clinical signs. On the right, minimal endocardial immuostaining of vWF in a control cat. 400× magnification, Bar = 50 µm.

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  Figure 3. Double immunostaining for vWF and integrin αIIb. In addition to vWF, each slide was also stained for Integrin αIIb, a platelet marker, so the quantification of endocardial vWF would not include any platelets which are known to also express vWF. Images were displayed as split channels with the merged image on the bottom right. 400× magnification; bar = 20 µm; Blue—Nuclei; Green—vWF; Red—Integrin αIIb (CD41).

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  Figure 4. Comparison of the vWF immunostaining at the endocardium. (A) Image illustrating the LA anatomical structures and the region of interest where the intensity of immunofluorescence was quantified (dotted overlay). (B) Representative images showed the variation in immunolabelling of vWF (green) at the endocardium in the different groups of cats. (C) Isotype control immunostaining was performed using rabbit IgG. Reagent control was performed with the primary antibody omitted. 100× magnification; Bar = 100 µm; LA (Left atrium), L (Lumen), E (Endocardium), M (Myocardium).

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  Figure 5. Quantification of the endocardial fluorescence from vWF: Cats with cardiomyopathy and clinical signs showed higher expression of vWF at the endocardium. The fluorescence signals detected in the ATE and CHF group were significantly higher compared to that of control group. No difference was detected between the rest of the groups. “ns” denotes non-significant. The number of cats in each group was shown in brackets. Data were analysed using Kruskal-Wallis test with Dunn’s multiple comparison test. Bars represent median and quartiles.

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  Figure 6. Microscopic images of thrombi immunostained for vWF and integrin αIIb: Images from Cats ATE 9, CHF 8: Images on the left shows the merged channels of vWF (green), platelet marker integrin αIIb (reddish orange) and nuclei (blue). In the merged channel, bright orange colour (closed arrow heads) represented clumps of platelets and vWF, while the RBC appeared dark and non-fluorescent (open arrow heads) compared to the immunolabelled vWF and platelets. WBCs were identified based on the blue colour of their nuclei and could be observed with platelets in clumps (arrows). Images were collated to show the whole thrombus. Images from Cat ATE 12: Split channels from left to right showed vWF in green, platelet marker integrin αIIb in red and the merged of the two channels. This thrombus remained in situ in the LAA. vWF and platelets encompassed RBC and connected to the endocardium. 50× magnification; bar = 200 µm. RBC, red blood cells; PLT, platelets; WBC, white blood cells; LAA, left atrial appendage.

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  Figure 7. Enlarged image of the LA thrombus from Cat CHF 8. The clump of WBCs and platelets denoted by a thick white arrow in (A) is the same area of the thrombus from CAT CHF 8 shown in the middle left hand image of Figure 6. Different patterns of vWF staining and the variety of blood cell types in different areas of the thrombus are shown in (B) 1–5: 1—dots interspersed in clumps of platelets; 2—beads interspersed within RBC; 3—web surrounding RBC; 4—strands surrounding RBC; 5—mesh with minimal platelets, RBC, or WBC.