title: Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse.
abstract: BACKGROUND
  Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics.
  
  
  RESULTS
  An N-terminal-azido thrombin-sensitive fluorescent peptide (ThS-P) with a thrombin-releasable quencher was linked to anti-CD41 using click chemistry to generate a thrombin-sensitive platelet binding sensor (ThS-Ab). Rapid thrombin cleavage of ThS-P (K(m) = 40.3 μm, k(cat) = 1.5 s(-1) ) allowed thrombin monitoring by ThS-P or ThS-Ab in blood treated with 2-25 pm tissue factor (TF). Individual platelets had > 20-fold more ThS-Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s(-1) ), ThS-Ab fluorescence increased between 90 and 450 s for 0.1-1 molecule-TF μm(-2) and co-localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s(-1) ), thrombin and fibrin were detected at the thrombus-collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti-mouse CD41 ThS-Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co-localized and where the thrombus was most mechanically stable.
  
  
  CONCLUSIONS
  ThS-Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients.
authors.affiliation: Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering Department of Biochemistry and Molecular Biophysics Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Welsh
authors.forename: T V
authors.initials: TV
authors.lastname: Colace
authors.forename: R W
authors.initials: RW
authors.lastname: Muthard
authors.forename: T J
authors.initials: TJ
authors.lastname: Stalker
authors.forename: L F
authors.initials: LF
authors.lastname: Brass
authors.forename: S L
authors.initials: SL
authors.lastname: Diamond
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001777:Blood Coagulation; D001792:Blood Platelets / Q000166:cytology*; D003094:Collagen / Q000737:chemistry; D005337:Fibrin / Q000737:chemistry; D006487:Hemostasis; D006801:Humans; D007700:Kinetics; D007834:Lasers; D051379:Mice; D046210:Microfluidic Analytical Techniques; D044085:Microfluidics; D010455:Peptides / Q000737:chemistry; D040201:Platelet Membrane Glycoprotein IIb / Q000737:chemistry; D011312:Pressure; D021381:Protein Transport; D013917:Thrombin / Q000737:chemistry*; D013925:Thromboplastin / Q000737:chemistry; D013927:Thrombosis / Q000378:metabolism; D013997:Time Factors
chemical_list: D000906:Antibodies; D010455:Peptides; D040201:Platelet Membrane Glycoprotein IIb; D005337:Fibrin; D003094:Collagen; D013925:Thromboplastin; D013917:Thrombin