Thrombus Structure Regulates Plasma Loss after Vascular Injury in Vivo
Authors: Welsh John Douglas , Taliaferro Joshua P , Stalker Timothy J. , Diamond Scott L. , Brass Lawrence F. 
Published: October 2019

Abstract
Introduction. Hemostatic thrombi are comprised of platelets, fibrin and, to a variable extent, trapped red cells. In contrast to pathological thrombus formation, the hemostatic response serves to limit bleeding. We and others have shown that hemostatic thrombi formed after local injury develop a characteristic structure in which a core of closely-packed, fully-activated platelets and fibrin is overlaid by a shell of less-activated platelets. Intravital microscopy in mouse models shows that red cell extravasation ceases after the accumulation of relatively small numbers of platelets, but plasma loss continues considerably longer, delivering potentially-bioactive proteins into extravascular tissue planes. Here we examined the impact of thrombus structure and platelet packing density on plasma extravasation, using laser-induced injuries in cremaster muscle venules as a model system.
Methods. Albumin conjugated to photoactivatable caged fluorescein molecules (cAlb) was used to track protein loss in real time. Real time intravital confocal microscopy and light pulses administered every 15 seconds after injury allowed the kinetics of protein loss and metrics of thrombus structure to be compared.
Results. The results show that the rapid loss of plasma proteins begins to slow immediately following the injury, but persists well beyond the end of red cell extravasation. cAlb leakage plateaued at about 40% of its initial value approximately 100 sec after injury. Infusion of the integrin aIIbb3 antagonist, eptifibatide (Integrilin) had no effect on fibrin accumulation, but reduced peak platelet deposition and thrombus height by approximately 72% and 51% respectively. It also completely prevented the normal time-dependent reduction in plasma protein leakage, which continued at near-initial rates at the end of the observation period. Under the same experimental conditions, the direct-acting P2Y12 antagonist, Cangrelor, which largely eliminates the thrombus shell in this injury model while having little impact on core size, decreased peak platelet deposition and thrombus height by 42% and 37%. In contrast to eptifibatide, Cangrelor only slowed the rate of decline of plasma extravasation without introducing a lag period. Finally, we performed comparative studies with otherwise-identical mice in which platelet-dependent clot retraction was reduced by replacing two critical Tyr residues in the cytoplasmic domain of the b subunit of aIIbb3with Phe (diYF). We have shown that the diYF substitution affects packing density in the thrombus core in this model. We now show that this has no effect on the initial decline in plasma extravasation following injury, but reduces the extent of decline starting approximately 75 sec after injury.
Conclusions. Taken together, these results show that both thrombus size and retraction impact the hemostatic potential of a thrombus, and provide in vivo measurements of how antiplatelet agents impact thrombus integrity. In the mouse cremaster model the thrombus structural defects only impacted plasma leakage, but we hypothesize that in different vascular beds where blood flow and injury type vary, these structural defects may lead to increased bleeding. Our results demonstrate the impact of platelet accumulation and retraction in forming a fully-competent hemostatic plug, provide a potential technique for predicting clinical bleeding induced by anti-thrombotic pharmaceuticals, and open the door to observing the impact of the sustained loss of plasma-borne molecules even when red cell loss has ceased.

Disclosures
Brass: Medicines Company: Research Funding.