Thrombin Generation Kinetics Under Constant and Pulsatile Shear Stress

Blood clot formation is a result of a coordinated process including platelet activation, adhesion, aggregation, as well as coagulation. The generation of thrombin is a major step in the coagulation cascade. Thrombin is a strong agonist on platelet activation and is responsible for cleaving soluble plasma fibrinogen into insoluble fibrin. Fibrin network can trap red blood cells, white blood cells and platelets, forming a clot or a thrombus. Clots can effectively stop bleeding while thrombi formed on the blood vessel wall can disturb/restrict blood flow. The formation of thrombin is governed by the formation of the prothrombinase complex, which is formed with factor Xa and factor Va in the presence of membrane phospholipids on activated platelets. Platelets are known to activate in response to the presence of pro‐thrombotic proteins as well as pathological shear stress induced by disturbed blood flow. This study examines how altered blood shear stress affects the kinetics of thrombin generation in the presence of pro‐coagulant proteins or vascular endothelial cells (ECs). Using a custom‐designed cone and plate shearing device, fresh human platelets obtained from random healthy donors were exposed to physiological or pathological shear stress (at 3, 10 and 30 dynes/cm 2 , constant or pulsatile) for 30 minutes in the presence of one of three pro‐coagulant substrates, i.e., von Willebrand Factor (vWF), tissue factor, and collagen, or a confluent monolayer of human coronary artery ECs (quiescent or activated with liposaccharide). Thrombin generation rate was measured using a prothrombinase assay; and platelet adhesion to the substrate protein or endothelial cells was measured using immunofluorescence microscopy. Platelets were identified using Alexa‐Fluor 488‐conjugated phalloidin (to cytoskeleton F‐actin) and their size (approximately 4 μm). Adherent platelets were counted by two or more individuals independently. The results demonstrated that shear stress (3, 10, 30 dynes/cm 2 , pulsatile or constant) did not seem to have a significant effect on thrombin generation rate when vWF or collagen was present. Elevated shear stress (at 30 dynes/cm 2 , pulsatile and constant) caused significant increases ( P <0.05) in thrombin generation rate when tissue factor was present. When quiescent ECs were present, thrombin generation rate was much lower ( P <0.05) under low (3 dynes/cm 2 ) or normal (10 dynes/cm 2 ) pulsatile shear stress. However, when activated ECs were present, thrombin generation rate was high overall (compared to quiescent ECs), regardless of shear stress magnitude of pulsatility. Results from platelet adhesion studies indicated that immobilized vWF, tissue factor, and collagen, as well as activated ECs, all support and promote platelet adhesion. However, platelet adhesion to quiescent ECs was lower compared to all the other substrates, especially under pulsatile shear stress. These results suggest that, in the presence of pro‐coagulant proteins/surfaces, platelet thrombin generation rates and adhesion is not only dependent on shear stress (magnitude and pulsatility). It is also likely to be a diffusion‐limited rather than concentration‐limited reaction. Support or Funding Information This work is in part supported by American Heart Association (Grant‐In‐Aid to WY). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .