Endothelial alterations in a canine model of immune thrombocytopenia

Abstract

Bleeding heterogeneity amongst patients with immune thrombocytopenia (ITP) is poorly understood. Platelets play a role in maintaining endothelial integrity, and variable thrombocytopenia-induced endothelial changes may influence bleeding severity. Platelet-derived endothelial stabilizers and markers of endothelial integrity in ITP are largely underexplored. We hypothesized that, in a canine ITP model, thrombocytopenia would lead to alterations in the endothelial ultrastructure and that the Von Willebrand factor (vWF) would serve as a marker of endothelial injury associated with thrombocytopenia. Thrombocytopenia was induced in healthy dogs with an antiplatelet antibody infusion; control dogs received an isotype control antibody. Cutaneous biopsies were obtained prior to thrombocytopenia induction, at platelet nadir, 24 hours after nadir, and on platelet recovery. Cutaneous capillaries were assessed by electron microscopy for vessel thickness, the number of pinocytotic vesicles, the number of large vacuoles, and the number of gaps between cells. Pinocytotic vesicles are thought to represent an endothelial membrane reserve that can be used for repair of damaged endothelial cells. Plasma samples were assessed for vWF. ITP dogs had significantly decreased pinocytotic vesicle numbers compared to control dogs (P = 0.0357) and the increase in plasma vWF from baseline to 24 hours correlated directly with the endothelial large vacuole score (R = 0.99103; P < 0.0001). This direct correlation between plasma vWF and the number of large vacuoles, representing the vesiculo-vacuolar organelle (VVO), a permeability structure, suggests that circulating vWF could serve as a biomarker for endothelial alterations and potentially a predictor of thrombocytopenic bleeding. Overall, our results indicate that endothelial damage occurs in the canine ITP model and variability in the degree of endothelial damage may account for differences in the bleeding phenotype among patients with ITP.

Keywords: Dog; ITP; endothelium.

Figure 1A.. Demonstration of determination of endothelial cell thickness.

A grid was superimposed on endothelial cells and endothelial cell thickness measured each place the grid crossed the vessel. Average thickness was calculated from each individual measurement. Magnification x 5,000. Definition of ultrastructural features: B. Pinocytotic vesicles are 0.07–0.08 μm in diameter (arrow), magnification x20,000; C. Space between cells (arrow), magnification x20,000; D. Large vacuoles are > 0.1 μm (arrows); x20,000.

Figure 1A.. Demonstration of determination of endothelial cell thickness.

A grid was superimposed on endothelial cells and endothelial cell thickness measured each place the grid crossed the vessel. Average thickness was calculated from each individual measurement. Magnification x 5,000. Definition of ultrastructural features: B. Pinocytotic vesicles are 0.07–0.08 μm in diameter (arrow), magnification x20,000; C. Space between cells (arrow), magnification x20,000; D. Large vacuoles are > 0.1 μm (arrows); x20,000.

Figure 1A.. Demonstration of determination of endothelial cell thickness.

A grid was superimposed on endothelial cells and endothelial cell thickness measured each place the grid crossed the vessel. Average thickness was calculated from each individual measurement. Magnification x 5,000. Definition of ultrastructural features: B. Pinocytotic vesicles are 0.07–0.08 μm in diameter (arrow), magnification x20,000; C. Space between cells (arrow), magnification x20,000; D. Large vacuoles are > 0.1 μm (arrows); x20,000.

Figure 1A.. Demonstration of determination of endothelial cell thickness.

A grid was superimposed on endothelial cells and endothelial cell thickness measured each place the grid crossed the vessel. Average thickness was calculated from each individual measurement. Magnification x 5,000. Definition of ultrastructural features: B. Pinocytotic vesicles are 0.07–0.08 μm in diameter (arrow), magnification x20,000; C. Space between cells (arrow), magnification x20,000; D. Large vacuoles are > 0.1 μm (arrows); x20,000.

Figure 2.. Platelet counts in 2F9 and control antibody-treated dogs.

Counts are shown as for individual ITP dogs and as median and range for control dogs. Time zero is when the platelet count first fell into the target platelet range of 5,000–30,000 platelets/μl or 1 hour after control antibody infusion.

Figure 3.. Normal cutaneous capillary.

The vessel is enveloped by the basement membrane (BM). Two pericytes are visible (P). Pinocytotic vesicles are numerous (V). There are no spaces between endothelial cells and a well-apposed junction (J) is present connecting two adjacent endothelial cells adjacent to the nucleus (N). A red blood cell (RBC) is present in the vessel lumen. Magnification = 10,000 x.

Figure 4.

Representative micrograph demonstrating reduction in number of pinocytotic vesicles observed in thrombocytopenic dogs (A). A RBC is shown in the vessel lumen. Note that vesicles are almost absent compared to the normal vessel of comparable diameter shown in B. B; x 10,000.

Figure 4.

Representative micrograph demonstrating reduction in number of pinocytotic vesicles observed in thrombocytopenic dogs (A). A RBC is shown in the vessel lumen. Note that vesicles are almost absent compared to the normal vessel of comparable diameter shown in B. B; x 10,000.

Figure 5.. Effect of ITP on endothelial ultrastructure.

A) Vessel thickness, (B) number of endothelial pinocytotic vesicle score, (C) large vacuole score, and (D) number of spaces between cells. **There is a significant decrease in number pinocytotic vesicles from baseline to 24 hours of thrombocytopenia compared to time-matched controls (P<0.0357). See text for vacuole and vesicle scoring system. Symbols represent the mean value for all capillaries evaluated in each dog at that time point. Bars represent overall treatment group mean. n=5 2F9 treated dogs; n = 3 control dogs.

Figure 6.. Ecchymoses occurred in two dogs.

A. Photograph of the large abdominal bruise (8.5 × 13 cm) that formed at 48 hours in one dog. B. Electron micrograph from the dog in A after 24 hours of thrombocytopenia, 24 hours prior to the development of the bruise. A large gap between adjacent endothelial cells is marked with an arrow. RBCs are shown in lumen; x 20,000. C. Pale, swollen endothelial cells consistent with necrotic cells and inter-endothelial cell gaps (arrows) present in an electron micrograph from the bruised region of another dog that developed an ecchymosis; x 20,000. D. For comparison, normal closely apposed adjacent endothelial cells connected by an intact junction (arrow) in a dog at baseline are shown; x 20,000.

Figure 6.. Ecchymoses occurred in two dogs.

A. Photograph of the large abdominal bruise (8.5 × 13 cm) that formed at 48 hours in one dog. B. Electron micrograph from the dog in A after 24 hours of thrombocytopenia, 24 hours prior to the development of the bruise. A large gap between adjacent endothelial cells is marked with an arrow. RBCs are shown in lumen; x 20,000. C. Pale, swollen endothelial cells consistent with necrotic cells and inter-endothelial cell gaps (arrows) present in an electron micrograph from the bruised region of another dog that developed an ecchymosis; x 20,000. D. For comparison, normal closely apposed adjacent endothelial cells connected by an intact junction (arrow) in a dog at baseline are shown; x 20,000.

Figure 6.. Ecchymoses occurred in two dogs.

A. Photograph of the large abdominal bruise (8.5 × 13 cm) that formed at 48 hours in one dog. B. Electron micrograph from the dog in A after 24 hours of thrombocytopenia, 24 hours prior to the development of the bruise. A large gap between adjacent endothelial cells is marked with an arrow. RBCs are shown in lumen; x 20,000. C. Pale, swollen endothelial cells consistent with necrotic cells and inter-endothelial cell gaps (arrows) present in an electron micrograph from the bruised region of another dog that developed an ecchymosis; x 20,000. D. For comparison, normal closely apposed adjacent endothelial cells connected by an intact junction (arrow) in a dog at baseline are shown; x 20,000.

Figure 6.. Ecchymoses occurred in two dogs.

A. Photograph of the large abdominal bruise (8.5 × 13 cm) that formed at 48 hours in one dog. B. Electron micrograph from the dog in A after 24 hours of thrombocytopenia, 24 hours prior to the development of the bruise. A large gap between adjacent endothelial cells is marked with an arrow. RBCs are shown in lumen; x 20,000. C. Pale, swollen endothelial cells consistent with necrotic cells and inter-endothelial cell gaps (arrows) present in an electron micrograph from the bruised region of another dog that developed an ecchymosis; x 20,000. D. For comparison, normal closely apposed adjacent endothelial cells connected by an intact junction (arrow) in a dog at baseline are shown; x 20,000.

Figure 7.. Platelet reactivity in two dogs that developed large ecchymoses when platelet hemostatic function was adequate.

Unfilled symbols show time at which new bleeding was occurring. A. EC50 thrombin for P-selectin expression and platelet count in one dog. Dashed line indicates baseline EC50 in that dog. B. Coated-platelet levels and platelet count in the same dog. Dashed line indicates baseline coated-platelets in that dog. C. EC50 thrombin and platelet count in the second dog. D. Coated-platelet levels and platelet count in the second dog.

Figure 8.. Alterations of von Willebrand factor over time in thrombocytopenic and control dogs.

Similar increases in vWF:Ag in both groups of dogs likely reflect vWF’s induction as an acute phase reactant secondary to biopsies (P<0.0001 for change in vWF:Ag over time). However, vWF may also play a role as a marker of thrombocytopenic endothelial damage as demonstrated by its correlation with large vacuole score. Plasma vWF:Ag concentration is reported as percent compared to a normal canine standard (median, range).