Activation-Independent Platelet Adhesion and Aggregation under Elevated Shear Stress.
Authors: Ruggeri Zaverio M. , Orje Jennifer N. , Habermann Rolf , Federici Augusto B. , Reininger Armin J. 
Published: April 2020

Abstract
The response of platelets to vascular injury involves adhesion onto reactive substrates followed by activation and aggregation, the latter mediated by the integrin αIIbβ3 with bound adhesive proteins such as fibrinogen and von Willebrand factor (VWF). Hemostasis and pathological arterial occlusion, however, occur in distinct hydrodynamic environments. A 90% lumen reduction in a coronary artery may cause shear rates of 20,000–40,000 s−1, values that are 10-fold higher than in arterioles where platelets participate in hemostasis after trauma. How hemodynamic forces influence platelet function is not fully understood. We have now found that platelets adherent to an immobilized VWF surface in a flow field can promote aggregation without activation, provided that soluble VWF be present and the shear rate exceed 10,000 s−1. In these studies blood was treated with 10 μM prostaglandin (PG) E1, thus the definition of aggregation is extended to a process of platelet cohesion mediated by physiologic blood components even in the absence of activation. Upon blood perfusion, individual rolling platelets covered the VWF surface at the shear rate of 3,000 s−1. In contrast, at 20,000 s−1 rolling platelets formed aggregates within 100–200 μm from the point of initial contact with immobilized VWF, equivalent to a few seconds. These aggregates became larger with varying shape and velocity while translocating in the direction of flow, but were predominantly elongated during periods of prolonged arrest. We then used plasma-free blood cells suspended in buffer to evaluate whether addition of soluble VWF was sufficient to mediate activation-independent platelet aggregation. Single platelet adhesion to immobilized VWF was the same with or without soluble VWF at the shear rate of 3,000 s−1. At 24,000 s−1, in contrast, adhesion was minimal without soluble VWF, but in its presence activation-independent aggregates formed as in whole blood. To elucidate the nature of the links that support activation-independent platelet aggregation, we used reflection interference contrast microscopy (RICM). This showed that, from a discrete point of tight adhesion and over a period of several seconds, the platelet body was stretched by the fluid drag into a structure reaching lengths of 20 μm or more but less than 1 μm thick at the narrowest point. Additional platelets then adhered to these elongated ones, and some that were stretched by the fluid drag contributed to the increasing length of string-like formations, while many retained their discoid morphology. In the end, stretched platelets joined to one another formed continuous structures that reached lengths of 100–200 μm and remained attached to immobilized VWF for minutes, while hundreds of discoid platelets adhered to them and to one another with arrest times of variable duration until they eventually detached as rolling aggregates. Thus, stretching of platelets by fluid drag and binding of soluble VWF are required to form elastic links that connect platelets into activation independent aggregates.