AFM investigation of APAC (antiplatelet and anticoagulant heparin proteoglycan)

Maximilian Winzely,Riitta Lassila,Laurin Lux,Karina Barreiro,Annukka Jouppila,Gernot Friedbacher,Georg Ramer,Bernhard Lendl

doi:10.1007/s00216-021-03765-y

Abstract

Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm3, and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method.Graphical abstract

Highlights

Vascular damage is induced by disease-triggered inflammation or plaque rupture and vessel intervention
The results for different dilutions of APAC with and without M gCl2 clearly show that surface coverage was increased with the addition of M gCl2 (Fig. 1d–f)
Addition of M gCl2 to the adsorption solution leads to a change of the surface charge from negative to positive, thereby enhancing the adsorption of APAC [21, 22]

Summary

Introduction

Vascular damage is induced by disease-triggered inflammation or plaque rupture and vessel intervention. The exposed subendothelial matrix components such as collagen, laminin, and fibronectin activate the hemostasis [1]. Platelets respond first by adhering on collagen to seal the injury site. Under arterial shear force conditions, platelet interaction with von Willebrand factor (VWF) is essential for binding and retaining on collagen [2]. VWF is derived from Weibel-Palade bodies of endothelial cells and alpha granules of megakaryocytes or platelets [3,4,5]. VWF monomers (~ 250 kDa) assemble as coiled bundles up to 20,000 kDa, but at hemostatic challenge, they elongate to long structures with multiple binding sites to endothelial matrix proteins, platelets, and fibrin. VWF integrates with coagulation system by carrying

Methods

Results

Conclusion