*3.1. Animals*

LA free wall samples from 39 cats were used for quantification of endocardial vWF. Thrombi were also retrieved from the LA of 3 (out of the total 39 cats) for immunohistological investigation. The LAA thrombus in situ was acquired from an extra cat that was collected later in the timeline thus not included in the quantification of endocardial vWF (Cat ATE 12 in supplementary material Tables S3 and S4). The inclusion and exclusion criteria for the 4 separate groups of cats are shown in Table 1. The cats in the control group were generally younger but this was not statistically significant (*p* = 0.137). Male cats were overrepresented in cats with cardiomyopathy (*p* = 0.009). Further demographic information about the cats used in the study are given in Table 2.


**Table 2.** Demographics of the cats enrolled.

See supplementary material Tables S3 and S4 for details of clinical presentation, cardiac imaging and histopathological diagnosis for each cat used in this study. One cat in the CHF group (Cat CHF 7 in Tables S3 and S4) had been given clopidogrel prior to presentation.

#### *3.2. Localisation of vWF Protein in the Left Atrial Samples*

In all cats, vWF could be observed to a variable degree in the vascular endothelium (Figure 1A,C). In the majority of cats with cardiomyopathy and clinical signs (groups ATE and CHF), microthrombi, defined as thrombi only visible on microscopic examination, were identified on the vascular endothelium and occasionally on the endocardium. Conversely, microthrombi were rarely seen in the control and subclinical groups. Identifiable components of these microthrombi using immunohistofluorescence were vWF (green), platelets (reddish orange), RBC (mild autofluorescence), and leucocytes (blue) (Figure 1).

**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.

#### *3.3. Quantification of Endocardial vWF Expression in Left Atrial Samples*

The intensity of vWF fluorescence at the endocardium (Figure 2) was quantified using ImageJ for comparison between groups. To avoid quantifying the fluorescence signals from platelets where vWF can also be detected, the slides were double immunostained for vWF and the platelet marker integrin αIIb (Figure 3).

**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.

**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).

Representative images of vWF immunolabelling in different groups of cats are shown in Figure 4. Medians of the detected fluorescence intensity of endocardial vWF in the left atrial endocardium from the four groups from left to right as shown in Figure 5 were Control group: 30.8 (IQR 28.1–34.2), Subclinical 39.6 (IQR 31.8–59.6), CHF group: 46.0 (IQR 36.6–56.8), and ATE group: 44.7 (IQR 34.9–54.6). The fluorescence intensity was significantly higher in the ATE and CHF groups compared to the control group.

**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).

**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.

#### *3.4. Characterisation of vWF in Thrombi Obtained from LA and LAA*

Thrombi were found in the LA or LAA in three cats (CHF 8, ATE 9, and ATE 12 in Tables S3 and S4) at postmortem. The necropsy images of the thrombi and the hearts can be found in Figure S1. The microscopic images of the thrombi, two retrieved from the LA and one remaining in situ in the LAA, are shown in Figure 6. These thrombi were immunostained for vWF (green) and platelets (reddish orange) and were counterstained with the nucleus stain (DAPI). WBC can be identified by the blue round-shaped nucleus stained by DAPI. RBC can be easily identified by their autofluorescence in the reagen<sup>t</sup> controls (Figure 6, autofluorescence column) [41]. The autofluorescence of RBC was markedly weaker compared to the fluorophores bound to the antibodies used for labelling vWF, platelets and nuclei and thus appeared relatively dark in Figure 1, Figure 6, and Figure 7. The composition of each thrombi was very different in terms of relative proportions of the main components and how different components were organised. An enlarged image of the upper right quadrant of the LA thrombus from CHF 8 (Figure 6) is used as an example to show the various patterns of organisation (Figure 7A). Different patterns of vWF expression were also observed and enlarged images of these different patterns of expression are described in Figure 7B.

**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.

(**A**)

**Figure 7.** *Cont.*

(**B**)

**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.
