Thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial VWF from cleavage by ADAMTS13

Abstract

The function of thrombospondin-1 (TSP-1) in hemostasis was investigated in wild-type (WT) and Tsp1-/- mice, via dynamic platelet interaction studies with A23187-stimulated mesenteric endothelium and with photochemically injured cecum subendothelium. Injected calcein-labeled WT platelets tethered or firmly adhered to almost all A23187-stimulated blood vessels of WT mice, but Tsp1-/- platelets tethered to 45% and adhered to 25.8% of stimulated Tsp1-/- vessels only. Stimulation generated temporary endothelium-associated ultralarge von Willebrand factor (VWF) multimers, triggering platelet string formation in 48% of WT versus 20% of Tsp1-/- vessels. Injection of human TSP-1 or thrombotic thrombocytopenic purpura (TTP) patient-derived neutralizing anti-ADAMTS13 antibodies corrected the defective platelet recruitment in Tsp1-/- mice, while having a moderate effect in WT mice. Photochemical injury of intestinal blood vessels induced thrombotic occlusions with longer occlusion times in Tsp1-/- venules (1027 +/- 377 seconds) and arterioles (858 +/- 289 seconds) than in WT vessels (559 +/- 241 seconds, P < .001; 443 +/- 413 seconds, P < .003) due to defective thrombus adherence, resulting in embolization of complete thrombi, a defect restored by both human TSP-1 and anti-ADAMTS13 antibodies. We conclude that in a shear field, soluble or local platelet-released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by plasma ADAMTS13, thus securing platelet tethering and thrombus adherence to inflamed and injured endothelium, respectively.

Figure 1.

General morphology and composition of Tsp1-null platelets. (A) Transmission electron microscopy of murine WT platelets and Tsp1^-/- platelets. The scale bar represents 5 μm. (B) SDS-PAGE under reducing conditions and Western blot analysis of the platelet content in TSP-1, fibrinogen (Fg), and fibronectin (Fn), as indicated. (C) Multimer analysis via agarose gel electrophoresis of von Willebrand factor (VWF) in WT and Tsp1^-/- plasma and platelets, with representative scans of the multimer distribution patterns. AU indicates arbitrary units.

Figure 2.

Platelet recruitment on A23187-stimulated endothelium in vivo. (A) Dot plot representation of the time in seconds (s) until initiation of platelet tethering in vivo on endothelium of murine mesenteric venules stimulated with A23187 for the indicated number of mice (m). Means are represented as short horizontal lines; indicated 2-tailed P values were calculated by unpaired nonparametric Mann-Whitney test with Welch correction for nonequal variances. (B) Distribution of numbers of mesenteric vessels, reactive (▪) and nonreactive (□) with platelets, during analysis of tethering and firm adhesion, as indicated for the mentioned number of mice (m); indicated P values were calculated by 2 × 2 contingency table analysis and Fisher exact test with 2-sided P value. C indicates nontreated controls; +TSP-1, bolus of 80 μg/kg + infusion of 80 μg/kg/h; +4N1K, injection with calcein-labeled platelets preactivated with 100 μM of the CD47 activating peptide 4N1K; and +Tsp1^-/- platelets and +WT platelets, cross-design experiments with platelet injection in WT and Tsp1^-/- mice, respectively.

Figure 3.

Kinetics of platelet adhesion on A23187-stimulated mesenteric blood vessels. Mean number (± SEM) of firmly adhering platelets after start of A23187 stimulation of mesenteric endothelium as a function of time upon injection with homologous calcein-labeled platelets in WT (WT) and Tsp1-null mice (Tsp1^-/-) in nontreated controls (Control) and in mice treated with irrelevant human IgG (23 mg/kg + 23 mg/kg/h; h-IgG), anti-ADAMTS13 IgG (23 mg/kg + 23 mg/kg/h; p-IgG), and human TSP-1 (80 μg/kg + 80 μg/kg/h); number of mice varied from 1 (h-IgG treatment) to 5 (Tsp1-treated TSP-1-null mice); v indicates number of vessels analyzed; P values were calculated via nonparametric Mann-Whitney test, with 2-tailed P-value (^*P < .001 vs Tsp1^-/- Control analyzed at minutes 1 and 10, respectively; #P < .05 vs WT Control, analyzed at minute 10).

Figure 4.

Platelet string formation on stimulated human endothelial cells. Platelet adhesion to resting and A23187 (30 μM)-stimulated human EA.hy926 endothelial cells during perfusion of calcein-labeled human platelets (20 × 10⁹ platelets/L) in reconstituted blood. (A) Platelet strings form on A23187-stimulated endothelial cells; they are prevented in the presence of the VWF-neutralizing antibody AJvW-2. Objective, 10×/long working distance numerical aperture (B) Mean numbers (± SEM) of platelets adhering per field and of strings formed, before and after endothelial cell stimulation, followed by perfusion with reconstituted blood, supplemented with human plasma, containing anti-ADAMTS13 antibodies (p-IgG; 250 μg/mL) or human TSP-1 at 6 or 3 μg/mL, as indicated. (C) Mean numbers (± SD) of waving strings formed per field after endothelial cell stimulation, followed by perfusion with reconstituted blood containing calcein-labeled human platelets (20 × 10⁹ platelets/L) and subsequent perfusion with human recombinant ADAMTS13 (2 nM) supplemented with human TSP-1 at 3 μg/mL, as indicated. The white arrow (length = 50 μm) represents the direction of the blood flow (^*P < .01; #P < .001 vs A23187 stimulation).

Figure 5.

Platelet string formation on stimulated murine endothelium. (A-C) Individual examples of strings of adherent platelets on endothelium of exposed mesenteric venules following stimulation with exogenously administered A23187. (D) Example of progressive string resorption over an interval of 15 seconds. The black arrow (length = 50 μm) represents the direction of the blood flow. (E) Distribution of numbers of mesenteric vessels, demonstrating platelet string formation (▪) or not (□), following vessel stimulation by A23187, for the indicated number of mice (m); indicated P values were calculated by 2 × 2 contingency table analysis and Fisher exact test with 2-tailed P value. C indicates nontreated controls; +TSP-1, bolus of 80 μg/kg + infusion of 80 μg/kg/h; +4N1K, injection with calcein-labeled platelets preactivated with 100 μM of the CD47-activating peptide 4N1K; and +Tsp1^-/- platelets and +WT platelets, cross-design experiments with platelet injection in WT and TSP-1^-/- mice, respectively.

Figure 6.

Photochemically injured intestinal vessel thrombosis. Distribution of occlusion time and mean (horizontal bars) upon injury induction in venules (top panel) and arterioles (bottom panel) of WT and Tsp1-null mice, without (Tsp1^-/-) or after pretreatment with human TSP-1 or anti-ADAMTS13 IgGs for the indicated number of mice (m); thin parallel lines reflect the time to first massive thrombus formation in Tsp1-null mice; C indicates nontreated controls; +TSP-1, bolus of 80 μg/kg + infusion of 80 μg/kg/h; and +p-IgG, bolus of anti-ADAMTS13 IgG of 23 mg/kg + infusion of 23 mg/kg/h. P values were calculated via nonparametric Mann-Whitney test with 2-tailed P value.

Figure 7.

Embolization of Tsp1^-/- platelet thrombi. (A) Examples of thrombus formation and sudden embolization (as indicated by the time in seconds) of large thrombi after intestinal venule damage in Tsp1-null mice. Scale bars represent 50 μm. (B) Mean number (± SEM) of embolization cycli before occurrence of occlusion in venules and arterioles of WT and Tsp1-null mice for the number of vessels indicated (v). P values were calculated via nonparametric Mann-Whitney test, with 2-tailed P value (^*P < .001 between mean number of embolizations in WT and TSP-1-null mice).

Figure 8.

Intestinal vessel thrombosis during hirudin treatment. Distribution of occlusion time and mean (horizontal bars) upon injury induction in venules (top panel) and arterioles (bottom panel) of WT or Tsp1-null hirudin-treated mice, for the indicated number of mice (m), in control conditions (C), and after treatment with human TSP-1, either with a bolus of 240 μg/kg + infusion of 240 μg/kg/h or 2400 μg/kg + infusion of 2400 μg/kg/h; P values were calculated via nonparametric Mann-Whitney test, with 2-tailed P value.