Regulated Phosphatidylserine Exposure on Platelets Mediates Fibrin Formation in Hemostasis and Thrombosis.
Authors: Shi Jialan , Pipe Steven W. , Rasmussen Jan T. , Heegaard Christian W. , Gilbert Gary E. 
Published: April 2020

Abstract
Thrombin-stimulated platelets support activity of phosphatidylserine (PS)-dependent blood coagulation reactions. However, only 2–6% of stimulated platelets expose sufficient PS to bind annexin V, leading to the supposition that procoagulant reactions are localized to the annexin V positive platelets and to microparticles shed by platelets. We hypothesized that thrombin-stimulated platelets expose sufficient PS to support the prothrombinase and factor Xase complexes but insufficient to meet the threshold for annexin V binding. We evaluated lactadherin, a PS-binding milk protein, as a reagent to detect platelet PS exposure. Thrombin or TRAP-treated platelets bound lactadherin with 3200 ± 700 sites/plt, but did not bind annexin V, as detected by flow cytometry. To confirm that lactadherin binding truly reported PS exposure, we performed activation experiments upon platelets loaded with 1-Palmitoyl-2- [6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-Glycero-3-Phospho-L-Serine (NBD-PS). Stimulation of platelets with 10 μM TRAP led to 8 ± 2 % PS exposure vs &gt; 90% PS exposure for 10 μM A23187. PS exposure was maximal within 1 min of exposure to TRAP and was reversible with &gt;70% of exposed PS re-internalized within 30 min. In situ platelet fibrin deposition was monitored utilizing a novel flow cytometry assay. Platelet rich plasma was recalcified and supplemented with 50 pM factor Xa, 100 μM GPRP. Platelet-bound fibrin, lactadherin and annexin V binding sites were measured at time intervals. Fibrin accumulated on lactadherin + platelets at a rate greater than or equal to the rate on platelet microparticles or annexin V + platelets. Lactadherin inhibited &gt; 98% of prothrombinase and factor Xase activity on platelets while annexin V inhibition reached a plateau of ~ 80%. The localization and importance of regulated PS exposure in vivo was evaluated in mouse models. Anesthetized mice were injected intravenously with 1 μg of lactadherin and 1 μg annexin V. Three minutes after FeCl3 injury of exposed mesentery, mice were perfused with fixative. Immunohistochemistry showed veins edged with fibrin and decorated with platelets. Lactadherin co-localized with CD41+ platelets along the vascular wall but was less intense on platelets lodged within fibrin aggregates that extended into the vessel lumen and did not stain platelets in sequestered blood. Annexin V stained only scattered platelets and endothelial cells and did not correlate well to sites of fibrin deposition. To evaluate the hemostatic importance of platelet PS, 10 μg of lactadherin was injected intravenously prior to tail snip; bleeding volumes increased from 33 ± 29 μl to 128 ± 39 μl, n experimental = 8. Rose bengal/laser-induced carotid thrombosis delayed from 34 ± 9 min to 80 ± 20 min, n = 10 after 8–21 μg lactadherin. Four treated animals did not develop a thrombosis. In summary, use of lactadherin as a PS probe has revealed that the capacity for regulated PS exposure, at levels below the annexin V binding threshold, is a general platelet property and that low level PS exposure is sufficient to support thrombin and fibrin generation in vitro and in vivo. These results have implications about the mechanism through which PS exposure is regulated as well as for the potential value of PS exposure as a diagnostic or therapeutic target.