Fluorogenic Peptide Linked to Anti-CD41 Detects Platelet-Localized Thrombin Activity within Blood Clots Formed Under Flow in Microfluidic Assays or in Mouse

Background:  Thrombin is subject to advection and diffusion upon its production during coagulation.  In order to monitor thrombin activity on platelets we developed a thrombin-sensing molecule capable of localizing to growing thrombi.   Methods and Results:  A thrombin sensitive peptide (ThS-P), with a fluorescent reporter, was chemically linked to an anti-CD61 antibody using click chemistry to generate a thrombin sensitive platelet specific sensor (ThS-Ab) (Figure 1A).  The kinetics of thrombin cleavage of ThS-P by thrombin was determined (K_m = 22 µM, k_cat = 1 s^-1).  Flow cytometry determined that platelets specifically showed a ~20 fold increase in fluorescence signal in response to thrombin generation in whole blood (Figure 1B).  ThS-Ab signal showed a dose response to various thrombin environments created in a microfluidic model of thrombosis, and was capable of identifying spatially distinct regions of thrombin generation in a thrombus (Figure 1C).  A separate microfluidic model capable of generating varied pressure gradients was utilized to study thrombin localization within intraluminal thrombi.  Altered pressure gradients influenced the localization of thrombin and were visualized using ThS-Ab signal. Thrombin generation in vivo was monitored using a mouse laser injury model and infused ThS-Ab whose signal temporally co-localized with fibrin deposition.  Conclusion:  We generated a platelet surface specific thrombin sensor, which provides real time information on the localization of thrombin within a thrombus.