HDL and HDL-Mimetic Nanoparticles Prevent Induced TTP and Interrupt the Course of Ongoing Episodes of the Disease

Background: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening condition caused by a severe deficiency of the von Willebrand factor (VWF)–cleaving protease ADAMTS13 accompanied by inflammatory stimuli that activate endothelial cells. This results in widespread microvascular thrombosis and organ injury. We previously reported that high-density lipoprotein (HDL) can prevent VWF self-association and reduce thrombocytopenia induced by VWF infusion in ADAMTS13-deficient (KO) mice (PMID: 26552698). This suggests that HDL might be a useful primary or adjunct therapy to interrupt active episodes of the disease.

Aim: To investigate the potential utility of HDL and HDL-mimetic nanoparticles in preventing and reversing TTP in mouse models.

Methods: We induced microvascular thrombosis in two ways: 1) systemically by intravenous injection of VWF, and 2) locally by ionophore treatment of mesenteric microvessels. 1) To provoke systemic TTP, we injected ADAMTS13-KO mice (C57BL/6 background) intravenously with recombinant human VWF (rVWF, 2000 U/kg). This is a well-established model of congenital TTP triggered by the high concentration of plasma VWF (PMID: 22529289). We evaluated whether HDL infusion could both prevent (prophylaxis) or interrupt (therapy) an ongoing episode. For this, we treated the mice with HDL (100 mg/kg) either 30 min before or 1 hour after rVWF challenge. We assessed clinical signs of TTP and blood cell counts at 0, 3, 24, 48, 72, and 96 hours post rVWF challenge. In a subset of mice challenged with rVWF with or without HDL treatment (10 min prior to rVWF challenge), we monitored and recorded platelet-VWF strings/thrombi in arterioles and venules of the cremaster muscle. 2) We also assessed the effect of HDL and nanoparticles containing the HDL-mimetic peptide DF4N on platelet-VWF thrombi in activated mesenteric venules (by calcium ionophore applied locally) in ADAMTS13-KO mice (PMID: 31340669, PMID: 21266777).

Results: ADAMTS13-KO mice challenged with rVWF rapidly developed signs of behavioral depression, hunched posture, and immobility, associated with severe thrombocytopenia, hemolytic anemia, and schistocytosis. These changes lasted for up to 3 days. Prophylactic HDL treatment prevented the development of TTP-like signs and thrombocytopenia. Therapeutic administration of HDL 1 hour after the rVWF injection effectively stopped the ongoing platelet clearance and reversed signs of TTP. Microscopic examination of the cremaster microcirculation after the rVWF challenge revealed platelet adhesion to endothelium-associated VWF fibers and the formation of long platelet-decorated strings on the walls of arterioles and venules. Many microvessels were obstructed completely and the obstructions persisted over 90 minutes. Prophylactic HDL treatment reduced the number of VWF-platelet strings by more than 50%, and microvessel occlusion was almost completely prevented.

In the ionophore model, the ionophore-treated mesenteric vessels of the ADAMTS13-KO mice evinced robust platelet adhesion, platelet-VWF string/thrombus formation and embolization, and small vessel occlusion. In contrast, pretreatment of the mice with HDL (100 mg/kg) or HDL-mimetic nanoparticles DF4N (300 μg/mL) significantly reduced platelet adhesion and platelet-VWF string/thrombus formation, and completely preventing microvessel occlusion. HDL pretreatment also reduced the time to thrombus resolution in ADAMTS13-KO mice by 40%, about the same as resolution takes in WT mice.

Summary: HDL and HDL-like nanoparticles can not only prevent TTP if given prophylactically, but they are also effective in interrupting the thrombotic process after it has been initiated. This suggests that HDL or HDL-like particles may provide another, possibly safer, tool to treat TTP.