Characterization of GPVI- or GPVI-CD39-Coated Nanoparticles and Their Impact on In Vitro Thrombus Formation.

Jeremy A Nestele,Netanel Korin,Valerie Dicenta,Marcel Kremser,Oliver Borst,Tilman E Schäffer,Konstantin Krutzke,Anne-Katrin Rohlfing,Daniela Eißler,Frederic Emschermann,Moran Levi,Alexander Bild,Meinrad Paul Gawaz

doi:10.3390/ijms23010011

Abstract

Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.

Highlights

We demonstrated that NP-GPVI and NP-GPVI-cluster of differentiation 39 (CD39) effectively adhered to collagen fibers under both static and dynamic conditions, using NP-bovine serum albumin (BSA) as an inert control
adenosine diphosphate (ADP)-dependent aggregation in vitro significantly reduced thrombus coverage by 15% (6.89 ± 1.16%) and NP-GPVI-CD39 reduced thrombus coverage by 30% (5.67 ± 1.23%) at LS, compared to NP-BSA (8.1 ± 0.83%)
Nanoparticles been successfully used in cancer genicity and toxicity

Summary

Introduction

According to the World Health Organization, approximately 30% of all deaths are linked to cardiovascular diseases (CVD), including, e.g., coronary heart disease and cerebrovascular disease, and, thereby, represent the predominant cause of death worldwide [1]. Atherothrombosis and vascular ischemia form core pathophysiological features of CVD with platelets playing a key role in thrombogenesis [2,3]. Antithrombotic agents have become a fundamental pillar in the prevention of CVD [4]. Classical platelet inhibiting agents are cyclooxygenase inhibitors and adenosine diphosphate (ADP) receptor blockers. Due to their systemic mode of action and the inhibition of platelet function, they

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Results

Conclusion