Shear-induced unfolding triggers adhesion of von Willebrand factor fibers

Abstract

von Willebrand factor (VWF), a protein present in our circulatory system, is necessary to stop bleeding under high shear-stress conditions as found in small blood vessels. The results presented here help unravel how an increase in hydrodynamic shear stress activates VWF's adhesion potential, leading to the counterintuitive phenomena of enhanced adsorption rate under strong shear conditions. Using a microfluidic device, we were able to mimic a wide range of bloodflow conditions and directly visualize the conformational dynamics of this protein under shear flow. In particular, we find that VWF displays a reversible globule-stretch transition at a critical shear rate ␥ crit in the absence of any adsorbing surface. Computer simulations reproduce this sharp transition and identify the large size of VWF's repeating units as one of the keys for this unique hydrodynamic activation. In the presence of an adsorbing collagen substrate, we find a large increase in the protein adsorption at the same critical shear rate, suggesting that the globule unfolding in bulk triggers the surface adsorption in the case of a collagen substrate, which provides a sufficient density of binding sites. Monitoring the adsorption process of multiple VWF fibers, we were able to follow the formation of an immobilized network that constitutes a ''sticky'' grid necessary for blood platelet adhesion under high shear flow. Because areas of high shear stress coincide with a higher chance for vessel wall damage by mechanical forces, we identified the shear-induced increase in the binding probability of VWF as an effective self-regulating repair mechanism of our microvascular system. blood flow ͉ mechanical activated proteins ͉ polymer physics O ur circulatory system is exposed to an amazingly wide span of shear rates, ranging from 1 to 10 5 s Ϫ1 (1). This range calls for adhesion mechanisms during blood clotting that are different for different regimes of shear rates. At small shear rates, which are found in rather wide vessels, objects such as vesicles or cells bind to vessel walls once the contact area and the adhesion strength is large enough (2). At high shear rates, which are found in small arteries, hydrodynamic lift forces inhibit formation of a sufficiently large contact area, which makes adsorption of soft objects from the blood onto vessel walls increasingly difficult. In fact, theoretical analyses predict that adhering compact and soft objects (such as vesicles or platelets) will roll and detach from a surface at a particular shear stress and, more importantly, will remain unbound if the shear rate is increased further (3, 4). Experiments on leukocyte adhesion confirmed these theoretical predictions quite nicely (2). However, the experimental finding for blood platelet adhesion in small arteries contradicts this scenario: von Willibrand factor (VWF)-mediated platelet adhesion, which is necessary to stop bleeding in small vessels, is strongly enhanced under high shear-flow conditions (5). Therefore, this example of shear-induced adsorption m...

The effects of shear flow on protein structure and function

“…61 Recently, fluorescence microscopy has been used to directly visualize individual fluorescently labeled vWF multimers under hydrodynamic stress in a microfluidic device. 42 vWF was fluorescently labeled with Alexa fluor 488, which was attached to the primary amines of the glycoprotein using tetrafluorophenyl ester. It was found that shear rates greater than 1000 s 21 triggered a reversible stretching of vWF in solution.…”

“…45 More importantly, the results 41 contradict the observed abrupt stretching of vWF ([20,000 kDa) in a microfluidic device at a threshold shear rate of 10 3 s 21 . 42 Clearly, the larger, more complex vWF would be expected to be more susceptible to shear stress relative to cytochrome c. However, it is noteworthy that specific conformational characteristics such as a-helix and b-sheet composition, and intra-molecular interactions (e.g., hydrophobic, electrostatic) and covalent bonds (e.g., disulfide), would be an important determinant of protein stability.…”

“…109 Interestingly, it has been shown that molecular confinement enhances the deformation of entangled polymers under squeeze flow, 110 a condition which models pulsatile blood flow. Similarly, there are a considerably number of reports 27,34,35,42,59,60,[111][112][113] citing haemodynamic shear stress as the primary facilitator of the proteolytic cleavage of large vWF multimers freshly released from endothelial cells as well as vWF-mediated platelet adhesion during haemostasis. The accumulation of large vWF multimers in the plasma may lead to disease states including thrombotic thrombocytopenic purpura, microvascular occlusion and atherosclerosis, due to the development of microvascular thrombi resulting from the complexation of the hyperactive vWF multimers and platelets at regions of low physiological shear.…”

Blood-clotting-inspired reversible polymer–colloid composite assembly in flow

“…Note that this behaviour cannot be sustained indefinitely, and for extremely high shear rates one expects flow-induced dissolution of the aggregates. The enhancement in thrombus formation is particularly true in the presence of vWF, and it has been shown that the formation of the plug can be driven purely by flow in a reversible fashion [16][17][18] . Such behaviour is completely contrary to our intuition based on the fact that we typically use strong flows to disintegrate or dissolve matter, and thus the clotting scenario in which flow controls the reversible assembly of complex composites (with presumably tailored properties) represents a completely new aggregation paradigm that cannot be understood in terms of purely diffusion-limited and/or reaction-limited aggregation concepts.…”

Kinetic study of von Willebrand factor self-aggregation induced by ristocetin

“…Beside inducing the binding to GpIb or to collagen surface, "activated" VWF was demonstrated to self-aggregate [13,31]. However, the data on molecular dimensions of VWF were obtained on the protein studied as a function of shear rate (up to >5000 sec -1 ) in a viscometer and successively transferred in a light…”