Plasminogen activator-coated nanobubbles targeting cellbound β2-glycoprotein I as a novel thrombus-specific thrombolytic strategy

Abstract

β2-glycoprotein I (β2-GPI) is a serum protein widely recognized as the main target of antibodies present in patients with antiphospholipid syndrome (APS). β2-GPI binds to activated endothelial cells, platelets and leukocytes, key players in thrombus formation. We developed a new targeted thrombolytic agent consisting of nanobubbles (NB) coated with recombinant tissue plasminogen activator (rtPA) and a recombinant antibody specific for cell-bound β2-GPI. The therapeutic efficacy of targeted NB was evaluated in vitro, using platelet-rich blood clots, and in vivo in three different animal models: i) thrombosis developed in a rat model of APS; ii) ferric chloride-induced mesenteric thrombosis in rats, and iii) thrombotic microangiopathy in a mouse model of atypical hemolytic uremic syndrome (C3-gain-of-function mice). Targeted NB bound preferentially to platelets and leukocytes within thrombi and to endothelial cells through β2-GPI expressed on activated cells. In vitro, rtPA-targeted NB (rtPA-tNB) induced greater lysis of platelet-rich blood clots than untargeted NB. In a rat model of APS, administration of rtPA-tNB caused rapid dissolution of thrombi and, unlike soluble rtPA that induced transient thrombolysis, prevented new thrombus formation. In a rat model of ferric chloride triggered thrombosis, rtPA-tNB, but not untargeted NB and free rtPA, induced rapid and persistent recanalization of occluded vessels. Finally, treatment of C3-gain-of-function mice with rtPA-tNB, that target β2-GPI deposited in kidney glomeruli, decreased fibrin deposition, and improved urinalysis data with a greater efficiency than untargeted NB. Our findings suggest that targeting cell-bound β2-GPI may represent an efficient and thrombus-specific thrombolytic strategy in both APS-related and APS-unrelated thrombotic conditions.

Figure 1.

Detection of β2-GPI on thrombi by immunofluorescence analysis. Clot sections were double stained with rabbit antibody to β2 glycoprotein 1 (β2-GPI) and either antibody to fibrin or to CD9, to investigate the localization of β2-GPI on fibrin, platelets and leukocytes. DAPI was used to stain cell nuclei. The thrombi were obtained from 2 different sources: (A) 3 patients undergoing surgical thrombectomy; (B) in vitro blood clots generated under static (platelet-poor) or flow (platelet-rich) conditions (see Methods for additional details). Representative images of thrombus section from 1 patient showing absence of co-staining of β2-GPI and fibrin. Arrows highlight the co-localization of β2-GPI with CD9-positive structures and arrowheads show the co-localization of β2-GPI with DAPI-positive nucleated cells.

Figure 2.

Physico-chemical characteristics and binding of nanobubbles to thrombi. (A) Transmission electron microspcopy (TEM) images showing similar morphology of untargeted nanobubbles (NB) and targeted NB (tNB); (B) average size, size distribution (polydispersity index) and particle charge (Z potential); MBB2DCH2 denotes the recombinant CH2-deleted scFv-Fc miniantibody against the DI domain of (32-GPI. (C) In vitro binding of tNB to patient’s thrombus sections and inhibition by soluble MBB2DCH2. Tissue sections were pre-incubated either with MBB2DCH2 or an unrelated recombinant antibody (unrelated MB) (100 mg/mL) for 15 minutes (min) prior to exposure to tNB containing 10 mg/mL MBB2DCH2 for further 60 min; (D) in vivo co-localization of platelets and leukocytes (stained in red with rhodamine 6G) and NB loaded with coumarin 6 (green) on thrombi induced in rats by administration of antibodies to (32-GPI. SD: standard deviation.

Figure 3.

Physico-chemical characteristics and functional activity of nanobubbles coated with recombinant tissue plasminogen activator. (A) Size and characteristicsof rtPAtNB and rtPA-NB (see legend of Figure 2 for further details). (B) Thrombolytic activity of recombinant tissue plasminogen activator-bound (rtPA-bound) nanobubbles (NB) and soluble rtPA on blood clot formed under flow conditions (Chandler loop). NB (500 ng/mL bound rtPA) and soluble rtPA (500 ng/mL) were added to the plasma surrounding the clot and the percent lysis was determined at the indicated intervals as detailed in the Methods. The results are presented as mean ± standard deviation (SD) of 3 different experiments. *P<0.05 using one-way ANOVA followed by Student-Newman-Keuls test.

Figure 4.

Effect of targeted and untargeted nanobubbles coated with recombinant tissue plasminogen activator (rtPA) (0,1 mg/g body weight) and of soluble rtPA (1 mg/g body weight) on thrombus dissolution and vascular occlusion in the rat antiphospholipd syndrome model. Thrombosis was induced by administration of anti(32 glycoprotein 1 (anti-(32-GPI) antibodies and treatment with thrombolytic agents was started after thrombus formation as detailed in the Methods. (A) Changes of fluorescence intensity of thrombi shown in the Online Supplementary Videos S3 to S5 during the first 15 minutes after thrombolytic treatment. Note the rapid and persistent decrease of fluorescence intensity in rats receiving targeted nanobubbles (rtPA-tNB), whereas soluble rtPA produced only a transient thrombolysis and untargeted NB (rtPA-NB) were ineffective. (B) Effect of NB and soluble rtPA on vascular occlusion during a 90-minute follow-up, as assessed by blood flow measurement. Consistent with the data of (A), only rtPA-tNB caused a marked and significant reduction of occluded vessels at all time points. The results in (B) are presented as mean ± standard deviation (SD) of experiments conducted in 3 rats. *P<0.05, **P<0.005 using one-way ANOVA followed by Student-Newman-Keuls test.

Figure 5.

Localization of targeted nanobubbles on endothelium and vascular thrombi during thrombolytic treatment in a rat model of antiphospolipid syndrome. Thrombus formation and nanobubble (NB) deposits were followed by intravital microscopy and the images were collected 90 minutes after injection of NB. Residual intravascular thrombi are visualized in red by in vivo staining with rhodamine 6G and NB loaded with coumarin 6 in green. Arrows show the co-localization of rtPA-tNB and residual vascular thrombi and arrowheads highlight the localization of rtPA-tNB on activated endothelium. Note the absence of untargeted NB and the presence of occluded vessels in rtPA-NB-treated animal. TNB: targeted NB; rtPA: recombinant tissue plasminogen activator.

Figure 6.

Localization of targeted and untargeted nonobubbles (tNB and NB) (without recombinant tissue plasminogen activator [rtPA]) on vascular thrombi induced by ferric chloride and effect of rtPA-NB, rtPA-tNB and soluble rtPA on vascular thrombotic occlusion. (A) Intravascular thrombi are visualized in red by in vivo staining with rhodamine 6G and in green by coumarin 6-loaded targeted nanobubbles (tNB). The images were collected 30 minutes after injection of NB. Note the absence of thrombus green staining in rats that received untargeted NB. (B) Time course of vascular occlusion after treatment with rtPA-coated tNB or untargeted NB (0,2 mg/g body weight) or soluble recombinant tissue plasminogen activator (rtPA) (2 pg/g body weight), as assessed by intravascular microscopy analysis. A significant reduction in the number of occluded vessels was seen in rats treated with targeted NB (rtPA-tNB) but not in those treated with untargeted NB or soluble rtPA. The results are presented as mean ± standard deviation of experiments conducted in 3 rats. *P<0.05, **P< 0.005 using one-way ANOVA followed by Student-New-man-Keuls test.

Figure 7.

Recombinant tissue plasminogen activator targeted nanobubbles dissolve clots in the C3 gain-of function mouse model of atypical hemolytic uremic syndrome. (A) Representative image of glomerular fibrin deposition in saline-treated atypical hemolytic uremic syndrome (aHUS) mice (n=3), aHUS mice treated with rtPA-NB, (0,5 mg/g body weight; n=6) or aHUS mice treated with recombinant tissue plasminogen activator targeted nanobubbles (rtPA-tNB) (0,5 mg/g body weight; n=5). (B) Densitometry analysis of glomerular fibrin deposition, 87 glomeruli scored in saline-treated C3 gain of function (GOF), 358 glomeruli scored in rtPA-NB, 459 scored in rtPA-tNB. *P<0.05, **P<0.005, ***P<0.0001 using one-way ANOVA followed by Student-New-