Flow Initiation of Thrombosis

The dynamics of thrombus buildup depend on prevailing hemodynamics in conjunction with biochemical determinants at the injury site (i.e., tissue factor, vWF, and collagen) along with prevailing biological attributes of the flowing blood. Fluid flow affects: platelet fluxes to the wall, the time available to form bonds, force loading of bonds, cellular deformation, endothelial metabolism, and removal of reactive species created during thrombotic events. The functional role of blood derived TF at near zero or sub-picomolar levels is particularly difficult to study but important to fundamental issues about the dynamical stability of blood to clot only when necessary. Using flow chambers and microarrays with spotted features presenting lipidated tissue factor and fibrillar collagen, we determined the critical levels of surface TF needed to trigger fibrin formation and thrombus buildup under flow with human whole blood (with added corn typsin inhibitor). At a venous level of flow (wall shear rate of 100 s^-1), a surface concentration of 3.6 molecules-TF/μm² was needed to trigger fibrin formation within 5 minutes of perfusion. In arterial flows (500 or 1000 s^-1), levels of 8 to 10 molecules-TF/μm² were needed to trigger fibrin formation. While the addition of 100 fM TF to blood under no flow conditions had negligible effect, this small perturbation increased fibrin formation 2.5-fold under flow conditions. In addition to patterned microarrays/parallel-plate flow chambers, we have also developed membrane flow chambers to control the flux of a solute into flowing blood. This allowed us to determine a critical threshold of ADP required to trigger clotting under flow. At the lowest ADP flux (1.5 x 10^-18 mol/μm²-sec), we observed little to no aggregation. At the higher fluxes, we observed monolayer (2.4 x 10^-18 mol/μm²-sec) and multilayer (4.4 x 10^-18 mol/μm²-sec) aggregates of platelets and found that the platelet density within an aggregate increased with increasing ADP flux. Finally, in recognition of the growing need for in vitro tools to study small volumes (~100 μL) of whole blood from genetically-modified mice, we developed microfluidic chambers that allow study of a focal (100 μm x 100 μm collagen patch) thrombotic event in real time. Wild type murine platelets adhered and aggregated on the collagen patch in a biphasic shear dependent manner with increasing deposition from 100 to 400 sec^-1 but decreasing deposition at 1000 sec^-1. Platelets lacking a functional α₂β₁ receptor were unable to adhere to the collagen.  These approaches have allowed a determination of critical levels of surface tissue factor and ADP flux needed to trigger clotting as well as the role of sub-pM TF levels. These flow approaches seek to reconcile an understanding of blood function as measured in static assays and flow assays.