Substrate Properties Affect Thrombin Generation Kinetics

Platelet activation and coagulation play critical roles in hemostasis. It is well established that altered flow and shear stress can cause platelet activation. We recently reported that flow and shear stress can also affect platelet thrombin generation kinetics. In the present study, we aimed to investigate how different substrates, i.e., tissue factor, von Willebrand factor, and vascular wall endothelial cells, affect thrombin generation kinetics under shear flow. Fresh human platelets were exposed to physiological (0.1–1.2 Pa pulsatile) or pathological (0.05 – 0.3 Pa pulsatile) shear stress in the presence of prothrombin, factor Xa, and calcium in a cone and plate shearing device. The wells were precoated with tissue factor or von willebrand factor, or covered with a monolayer of confluent human coronary artery endothelial cells (untreated or activated with liposaccharide ‐ LPS). Thrombin generation was quantified using Chromozym‐TH absorbance at 405 nm. The results demonstrated that with the presence of tissue factor, shear stress magnitude hardly affected thrombin generation rate. When von Willebrand factor was available, thrombin generation rate increased by approximately 21% under normal pulsatile shear stress (compared to no shear conditions), which was further increased by low pathological shear stress to 34% (compared to no shear condition). When platelets were exposed to shear stress in the presence of healthy (untreated) coronary artery endothelial cells, thrombin generation rate decreased by about 1/3 under normal shear stress. However, when endothelial cells were pre‐activated by LPS, thrombin generation rate increased under all conditions (no shear, normal shear and low pathological shear). But pathological low shear stress did not cause significant changes in thrombin generation rate compared to normal pulsatile shear stress. These results are different from what we reported earlier, that with the presence of coagulation factors (i.e., prothrombin, factor Xa, and calcium), low pulsatile shear stress induced faster thrombin generation compared to normal shear stress. It was also observed that under normal pulsatile shear stress, thrombin generation rate almost doubled when the plate was coated with von Willebrand factor, compared to tissue factor. When endothelial cells were activated, thrombin generation rate increased to about 2.5 folds of that seen with tissue factor. Similar trend was observed under low pathological shear stress. These results suggest that substrate properties could affect thrombin generation rate greatly. Therefore, to quantify thrombin generation kinetics, not only shear stress (magnitude and exposure time) and the presence/concentration of coagulation factors, but also substrate properties need to be considered. Support or Funding Information This study is supported by an American Heart Association Grant‐in‐Aid Award (16GRNT30440002).