(676g) Fluorogenic Peptide Linked to Anti-CD41 Detects Platelet-Localized Thrombin Activity within Blood Clots Formed Under Flow in Microfluidic Assays or in Mouse

Colace, T., University of Pennsylvania
Stalker, T. J., University of Pennsylvania
Brass, L. F., University of Pennsylvania
Muthard, R., Penn State University, University Park

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 (Km = 22 µM, kcat
= 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.