(148f) Platelet Margination, Adhesion, and Activation in Secondary Flows Are Necessary for Thrombus Propagation in an in vitro Model of Venous Thrombosis

Background: The histology of venous thrombi implies a role for blood flow in thrombus propagation. The alternating layered structure of red, fibrin-rich regions that begin at the vessel wall in the valve sinus followed by white, platelet-rich regions, called the lines of Zahn, suggests a mechanism of platelet accumulation to the initial fibrin-rich regions. The geometry of the valve sinus defined by a large cavity distal to the expansion created by the valve leaflets yields unique flow patterns. Flow through fixed venous valves of dogs shows flow separation that results in a large primary vortex adjacent to the valve cusps, and a secondary vortex in the deepest recess of the valve pocket. The fluid velocity in this secondary vortex is extremely slow and corresponds to the most hypoxic area of the valve sinus. Vortical flows in the valve sinus have also been observed by ultrasound in human venous valves. Platelets are essential for thrombus formation under flow and thrombus growth in murine models of venous thrombosis (VT). The murine models used in VT however do not capture the vortical flows and geometry that are characteristic of human valves where VT initiates. The objective of this study was to determine the importance of platelets on thrombus propagation an in vitro model of VT that mimics the hemodynamics of human venous valves. Methods: The flow chamber consists of a 150 µm x 150 µm channel that undergoes a 1:3 expansion, which is similar to the stenosis ratio of venous valves, with a varying undercut angle (90, 120, 135 or 150°) to mimic different positions of a valve leaflet. In order to match flow vortices in our system with human venous valves, we used the Reynolds number (Re) to scale our flow rates. The viscosity of blood is a as function of hematocrit and the channels size. Here, we use the semi-empirical equation from Pries et al.[1] to calculate the viscosity in order to appropriately match Re between flow conditions. Velocity fields and streamlines for the device were calculated using commercially available computational fluid dynamics software (COMSOL). The streaklines of platelet sized fluorescent beads (2 µm) mixed with red blood cells (RBC) in buffer were used to characterize the flow field for Re of 1-100. For experiments where blood clots are formed, tissue factor (TF) was adsorbed in the valve sinus in order to initiate the coagulation cascade. Human whole blood was separated into platelet rich plasma (PRP) and packed RBC and reconstituted to hematocrits of 0, 0.2, 0.4 or 0.6. Platelets were labeled with DiOC6 prior to and/or with annexin V (phosphatidylserine stain) after an experiment and exogenous Alexa 555-fibrinogen was added to visualize fibrin. Thrombus formation was measured by confocal microscopy as plasma, plasma with RBC, PRP, or reconstituted blood (PRP and RBC) was perfused over 30 minutes at flow rates that yield primary and secondary vortices similar to those measured in humans. Results: CFD simulations and streakline imaging showed flow separation for Re > 1 and the presence of a primary vortex for all expansion angles near the valve cusp. A secondary vortex was observed for expansion angles of 135° and 150° in the valve pocket. The size of the secondary vortex was more strongly dependent on expansion angle than Re. RBC were required for significant quantities of platelets and fluorescent beads to enter into the primary vortex, suggesting the need for platelet margination upstream of the expansion. Plasma or RBC suspensions without platelets resulted in a fibrin gel that was confined to the deepest part of the pocket that coincided with the secondary vortex. Both platelet and RBC were required for the thrombus to grow out of the valve sinus over and into the main flow channel. It appears that there is a critical platelet flux necessary to support additional coagulation, and that flux is dependent on RBC-platelet collisions. The thrombus that forms with reconstituted blood shows a layered structure whereby a dense layer of platelets in the primary vortex adhered to the initial fibrin gel formed in the secondary vortex. This dense platelet layer is followed by another fibrin rich layer. These data suggest that fibrin adhered platelets support coagulation beyond the near-wall region coated with TF. Indeed, platelet adhered at this interface are positive for the procoagulant lipid phosphatidylserine as measured by annexin V staining.

 Conclusions: Immobilized TF can initiate coagulation in a model venous valve sinus within deep regions defined by a secondary vortex, but platelets and RBC are necessary to spatially propagate a thrombus. Platelets entrained in the primary vortex in the valve sinus, a process that is enhanced by RBC, adhere in a dense layer to fibrin. This platelet layer supports additional coagulation, and the formation of a fibrin rich thrombus that grows beyond the valve sinus.

 [1] A. R. Pries, D. Neuhaus, and P. Gaehtgens, “Blood viscosity in tube flow: dependence on diameter and hematocrit,” American Journal of Physiology - Heart and Circulatory Physiology, vol. 263, no. 6, pp. H1770–H1778, Dec. 1992.