Background
Thrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the Von Willebrand factor (VWF) cleaving protease ADAMTS13. Endothelial injury is believed to be a key initiating event in the pathogenesis leading to platelet activation and formation of platelet-rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and seems to differ among the microvascular territory. Especially, a cardiac injury has recently been identified and hypothesized as a major cause of death in TTP.
Methods
We used an animal model in which some TTP-like symptoms have been triggered in ADAMTS13 knockout mice (B6.129-ADAMTS13tm1Dgi ) by challenge with 2000 units/kg body weight of recombinant human VWF containing ultra large VWF (rhVWF, Baxter, Vienna Austria). rhVWF was also injected in 6 wild type mice (C57/bl6-B129 WT+VWF) while seven other wild type mice received placebo (WT). Cardiac involvement was assessed by transthoracic echocardiography using validated parameters i.e left ventricular ejection fraction (LVEF), fractional shortening (FS) and cardiac index (CI). Cardiac perfusion was determined by arterial spin labeling MRI (Biospec 47/40 advanced II, Brucker, Ettlingen, Germany). Ex vivo endothelial function analysis in isolated coronary, mesenteric and renal segments were performed using a myograph. Arteries were precontracted by phenylephrine or serotonin and the relaxations induced by increasing concentrations of acetylcholine (receptor-mediated, endothelium-dependent relaxation) or the nitric oxide (NO) donor sodium nitroprusside (SNP; endothelium-independent- relaxation) were assessed. Arteries were also incubated with the NO synthase-inhibitor N-Nitro L-Arginine (LNNA) to assess the contribution of NO to the relaxing responses to acetylcholine (endothelium-dependent NO-independent-relaxation).
Results
On day 1, VWF-challenged ADAMTS13 KO mice rapidly developed symptoms and severe thrombocytopenia (41.8G/L ± 6.9G/L vs 480.7G/L ± 86.5G/L (WT+VWF) and 1118.6G/L ± 160.8G/L (WT); p<0.001), schistocytosis and a decrease in hematocrit levels (40.9% ± 0.8% vs WT+VWF: 51.3% ± 3,8%; p< 0.05 and WT: 56.1% ± 3%; p< 0.001). On day 2, necropsy revealed that macroscopic lesions were restricted to the heart and consisted of acute myocardial hemorrhage and necrosis. Myocardial necrosis was accompanied by infiltration of lymphocytes and interstitial edema. Minimal, acute tubular necrosis in the kidneys was seen in a few animals. Injection of rhVWF in ADAMTS13 KO mice resulted in a decrease in LVEF (73.8% ± 2.7% vs WT+VWF: 86.6% ± 1.3% and WT: 86.8% ± 1.3%; p< 0.01), in FS (37.3% ± 2.1% vs WT+VWF: 50.1% ± 1.6%; p<0.05 and WT: 50.9% ± 12.8%; p< 0.01) and in CI (1.21 ml/min/g ± 0.09 vs WT+VWF: 1.70 ml/min/g ± 0.14 and WT: 1.66 ml/min/g ± 0.1; p< 0.05) suggesting an early systolic dysfunction. However, cardiac perfusion was not significantly affected on day 2. Administration of rhVWF in ADAMTS13KO mice decreased the NO-mediated relaxing responses to acetylcholine in mesenteric (51.1% ± 14.8% vs WT+VWF: 94.4% ± 1.6%; p< 0.05 and WT: 96.5% ± 2.3%; p< 0.001) and coronary arteries (61.7% ± 12.4% vs WT+VWF: 89.9% ± 6.3% and WT: 96.8% ± 0.9%; p< 0.05) but not in renal arteries. Interestingly, under LNNA, endothelium-dependent relaxations were also markedly affected in coronary arteries (19.3% ± 3.7% vs WT+VWF: 70.2% ± 4.7%; p<0.001) in ADAMTS13KO mice suggesting the role of other endothelium-derived vasoactive factors. In parallel, the relaxing responses to SNP were slightly affected (47.8% ± 13.8% vs WT+VWF: 79.1% ± 13.3% and WT: 80.2% ± 7.2%; p< 0.05), suggesting a strong coronary vascular dysfunction on day 2.
Conclusions
This experimental study reproduced an animal model in which TTP like symptoms are triggered in ADAMTS13 KO mice by administration of VWF and demonstrated that the heart is the most sensitive target organ with myocardial necrosis and systolic dysfunction. We showed for the first time a strong mesenteric and coronary endothelial dysfunction in an induced-TTP model. It may represent an important trigger of the systemic organs failure occurring in this disease, strengthening the need to define the mechanisms involved in this early vascular injury.
Disclosures:
Schiviz: Baxter: Employment.
Morbidities and Mortality in Patients with Hereditary Thrombotic Thrombocytopenic Purpura
