Thrombosis underlies numerous life-threatening cardiovascular syndromes. Development of thrombosis-specific molecular imaging agents to detect and monitor thrombogenesis and fibrinolysis in vivo could improve the diagnosis, risk stratification, and treatment of thrombosis syndromes. To this end, we have synthesized efficient multimodal nanoagents targeted to two different constituents of thrombi, namely, fibrin and activated factor XIII. These agents are targeted via the conjugation of peptide-targeting ligands to the surface of fluorescently labeled magnetic nanoparticles. As demonstrated by in vitro and in vivo studies, both nanoagents possess high affinities for thrombi, and enable mutimodal fluorescence and magnetic resonance imaging.

Aims Current thrombolytic therapies rely upon exogenous plasminogen activators (PA) to effectively lyse clots, thereby restoring blood flow and preventing tissue and organ death. Yet, these PAs may also impair normal hemostasis which may lead to life-threatening bleeding, including intracerebral hemorrhage. Thus, the aim of this current study is to develop new thrombus-targeted fibrinolytic agents that harness the multifunctional theranostic capabilities of nanomaterials, potentially allowing for the generation of efficacious thrombolytics while minimizing deleterious side effects. Materials and Methods A thrombus-targeted nano-fibrinolytic agent (CLIO-FXIII-PEG-tPA) was synthesized using a magnetofluorescent crosslinked dextran-coated iron oxide (CLIO) nanoparticle platform that was conjugated to recombinant tissue plasminogen activator (tPA). Thrombus-targeting was achieved by derivatizing the nanoparticle with an activated factor XIII (FXIIIa)-sensitive peptide based on the amino terminus of α2-antiplasmin. Human plasma clot binding ability of the targeted and control agents was assessed by fluorescence reflectance imaging. Next, the in vitro enzymatic activity of the agents was assessed by S2288-based amidolytic activity, and an ELISA D-dimer assay for fibrinolysis. In vivo targeting of the nanoagent was next examined by intravital fluorescence microscopy of murine arterial and venous thrombosis. The fibrinolytic activity of the targeted nanoagent compared to free tPA was then evaluated in vivo in murine pulmonary embolism. Results In vitro, the targeted thrombolytic nanoagent demonstrated binding to fresh frozen plasma (FFP) clots superior to control nanoagents (ANOVA p < 0.05). On a weight (mg) basis, the S2288 amidolytic efficiency of the targeted nanoagent was approximately 15% reduced compared to free tPA. When normalized by S2288-based activity, targeted, control, and free tPA samples demonstrated equivalent in vitro fibrinolytic activity against human plasma clots, as determined by ELISA D-dimer assays. The FXIIIa targeted fibrinolytic nanoagent efficiently bound the margin of intravascular thrombi as detected by IVFM. In in vivo fibrinolysis studies normalized for activity, the FXIIIa-targeted agent lysed pulmonary emboli with similar efficacy as free tPA (p>0.05). Conclusions The applicability of a FXIIIa-targeted thrombolytic nanoagent in the treatment of thromboembolism was demonstrated in vitro and in vivo. Future studies are planned to investigate the safety profile and overall efficacy of this class of nanoagents, and to further optimize their thrombus-targeting profile and lytic action.

IntroductionStudies have shown reduced antiviral responses in kidney transplant recipients (KTRs) following SARS-CoV-2 mRNA vaccination, but data on post-vaccination alloimmune responses and antiviral responses against the Delta (B.1.617.2) variant are limited.Materials and methodsTo address this issue, we conducted a prospective, multi-center study of 58 adult KTRs receiving mRNA-BNT162b2 or mRNA-1273 vaccines. We used multiple complementary non-invasive biomarkers for rejection monitoring including serum creatinine, proteinuria, donor-derived cell-free DNA, peripheral blood gene expression profile (PBGEP), urinary CXCL9 mRNA and de novo donor-specific antibodies (DSA). Secondary outcomes included development of anti-viral immune responses against the wild-type and Delta variant of SARS-CoV-2.ResultsAt a median of 85 days, no KTRs developed de novo DSAs and only one patient developed acute rejection following recent conversion to belatacept, which was associated with increased creatinine and urinary CXCL9 levels. During follow-up, there were no significant changes in proteinuria, donor-derived cell-free DNA levels or PBGEP. 36% of KTRs in our cohort developed anti-wild-type spike antibodies, 75% and 55% of whom had neutralizing responses against wild-type and Delta variants respectively. A cellular response against wild-type S1, measured by interferon-γ-ELISpot assay, developed in 38% of KTRs. Cellular responses did not differ in KTRs with or without antibody responses.ConclusionsSARS-CoV-2 mRNA vaccination in KTRs did not elicit a significant alloimmune response. About half of KTRs who develop anti-wild-type spike antibodies after two mRNA vaccine doses have neutralizing responses against the Delta variant. There was no association between anti-viral humoral and cellular responses.