title: A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature.
abstract: Previous studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbβ3antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5'-diphosphate (ADP) P2Y12receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue.
authors.affiliation: Department of Medicine and Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA.
authors.forename: John D
authors.initials: JD
authors.lastname: Welsh
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA.
authors.forename: Ryan W
authors.initials: RW
authors.lastname: Muthard
authors.affiliation: Department of Medicine and.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stalker
authors.affiliation: Department of Medicine and.
authors.forename: Joshua P
authors.initials: JP
authors.lastname: Taliaferro
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA.
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
authors.affiliation: Department of Medicine and.
authors.forename: Lawrence F
authors.initials: LF
authors.lastname: Brass
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000032:analysis / Q000378:metabolism*; D005337:Fibrin / Q000032:analysis / Q000378:metabolism; D006487:Hemostasis; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D055806:Microvessels / Q000293:injuries* / Q000378:metabolism / Q000473:pathology*; D015539:Platelet Activation; D010976:Platelet Count; D013927:Thrombosis / Q000097:blood / Q000378:metabolism / Q000473:pathology*
chemical_list: D001798:Blood Proteins; D000244:Adenosine Diphosphate; D005337:Fibrin