Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear

Abstract

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26–34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.

Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear

“…This would imply that external shear forces play a role in the concept of intracellular actomyosin-dependent mechanosensitive forces for αIIbβ3 activation [ 21 ]. As we have recently shown [ 15 ], the GPIbα-VWF axis contributes to the formation of platelet aggregates under shear.…”

“…For flow assays, the two synthetized CIB1-binding peptides were used at an affordable high dose for platelet-inhibiting peptides of 50 μg/mL in whole blood [ 15 ]. Preincubation of blood with the pCIB or pCIB m resulted in no more than small visual effects on thrombus formation at microspots of collagen I ( Figure 6 Ai–iii).…”

“…Knowing that multiple shear-dependent platelet adhesion mechanisms [ 15 ] and platelet-platelet interactions [ 11 ] can determine the thrombus-forming process, we used our experience in the targeted design and synthesis of anti-platelet peptides [ 13 , 15 ] to reveal the importance of less understood signaling pathways. In this context, a less well-studied protein acting downstream of αIIbβ3 in platelets is protein tyrosine kinase 2 (PTK2, also known as focal adhesion kinase) [ 16 , 17 , 18 ].…”

Molecular Mechanisms of Hemostasis, Thrombosis and Thrombo-Inflammation

“…Another important player in thrombus formation is von Willebrand factor (vWF), which is converted to the open conformation by surface immobilization in combination with shear stress, then allowing platelet adhesion to the vessel wall via the GPIbα receptor. The study of Hrdinova et al [ 9 ] provides further understanding of the multiple contributions of the vWF A1 domain to the thrombotic process. Stable cyclic peptides were designed in silico and chemically synthesized to specifically interfere with the opened conformation of the vWF A1 domain and the platelet GPIbα receptor.…”

“…Stable cyclic peptides were designed in silico and chemically synthesized to specifically interfere with the opened conformation of the vWF A1 domain and the platelet GPIbα receptor. Three peptides inhibited vWF-dependent platelet adhesion and thrombus formation on collagen in whole blood under flow, although they were not as effective as a blocking anti-VWF A1 domain antibody [ 9 ]. This shows that the design of peptides based on structure results in physiologically active peptide-based inhibitors, even for intricate complexes such as GPIα-vWF A1.…”