Cellular aggregation, platelet activation andvon Willebrand factor self-association under hydrodynamic flow.

摘要

Blood flow plays an important role in regulating cellular activation and aggregation rates in circulation. In this thesis, the mechanisms of cellular aggregation and shear-induced platelet activation (SIPAct) are studied by applying fluid shear to cell suspensions in a cone-plate viscometer.; Firstly, we performed a theoretical analysis of fluid flow and particle interactions in the cone-plate viscometer. At high shear rates, centrifugal forces at the cone surface induce non-linear secondary flow in this device. Our analysis indicates that secondary flow causes positional variations in intercellular collision frequency and adhesion efficiency in the viscometer. In addition, the adhesion efficiency is dependent not only on the shear rate, but also the sample volume and the cone angle. Experiments performed with isolated neutrophils confirmed these predictions. The results suggest that secondary flow may significantly influence biological experiments in the viscometer.; Next, we quantitatively examined the aspects of fluid flow that regulate SIPAct using the viscometer. We observed that a threshold shear stress of ∼80dyn/cm 2 is necessary to induce platelet activation. Results also reveal a two-step mechanism for SIPAct: Fluid shear and von Willebrand factor (vWF) are required in the first step where binding between the platelet GpIb and vWF likely occurs. Subsequently, high shear forces alone in the absence of vWF in suspension can induce platelet activation.; In other experiments, purified vWF was subjected to shear in the viscometer and vWF morphology was assessed using light scattering. These studies demonstrate the ability of hydrodynamic forces to induce vWF aggregation in suspension. vWF self-association may be an additional feature involved in controlling thrombosis in circulation.; Finally, a mathematical model was developed to compute hydrodynamic forces applied on intercellular aggregates, cell-surface receptors and soluble molecules. Calculations reveal that: (i) The force applied on neutrophil-platelet doublets is ∼3-fold lower than that on neutrophil-neutrophil doublets. Thus, alterations in cell size may dramatically alter adhesion molecule requirement for efficient cell binding. (ii) Forces on platelet GpIb and vWF are of comparable magnitude, but are orders of magnitude lower than those applied on cell doublets. The calculation scheme may find application in studies of vascular biology and receptor biophysics.