Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation <i>in vitro</i>

Abstract

Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of SRGs but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create shear rate gradients during blood perfusion. VWF-GPIbα interactions were increased at sites of shear rate gradients compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRGs but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFV-A1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRGs on VWF dependent platelet aggregation and developed the shear-gradient sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of shear rate gradients. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal haemostasis.

Atherothrombosis‐on‐Chip: A Site‐Specific Microfluidic Model for Thrombus Formation and Drug Discovery

“…Previous studies supported this observation, as blood was recommended to use immediately or within 2 h of collection. [27,[32][33][34][35] The effect of blood age was also checked in the device with 15% stenosis, and the results showed a similar pattern (Figure S2, Supporting Information).…”

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

“…In support of the current data, we and others have previously shown that the role of GPIbα in flow-dependent platelet aggregation was most prominent at high pathological wall-shear rate gradients, i.e., as present at stenotic sites in the arterial circulation [46,47]. A recently published single-chain antibody, scFV-A1, designed to target VWF activated by shear, was shown to play an inhibiting effect in stenotic chambers, i.e., at sites of sharp shear rate changes [48]. Therefore, stenotic flow chambers may be a useful tool to further study the effects of the present cyclic peptides under conditions of pathological VWF-dependent platelet activation.…”

Design of artificial vascular devices: Hemodynamic evaluation of shear-induced thrombogenicity

“…When blood interacts with a foreign material, a rapid absorption of plasma protein occurs. This is believed to be the precursor of thrombosis, as it involved an aggregation of platelets and fibrin on the surface, trapping RBCs (Hoefer et al, 2020). In laminar conditions, velocity and concentration gradients, together with collisions, spontaneously transport the platelets towards the walls, enhancing these interactions.…”

“…It is understood that immobilized VWF is likely to remain in its unfolded conformation at shear rates around 3,000 s -1 whereas an elongation of soluble VWF needs higher shear up to 5,000 s -1 (Siedlecki et al, 1996;Kuwahara et al, 2002;Schneider et al, 2007). However, recent observations have questioned the existence of such a threshold, showing in particular that VWF could be involved at lower shear magnitudes of, around 1,500 s -1 (Receveur et al, 2020) and in the context of shear rate gradients (Hoefer et al, 2020;Van Rooij et al, 2021). In a shear gradient context, it was observed that VWF unfolding increased at two-fold lower flow rates when compared to constant shear flows (Zhang, 2009;Sing and Alexander-katz, 2010).…”