Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy

Biochemistry. 1999 Apr 6;38(14):4572-85. doi: 10.1021/bi9824792.


It has been hypothesized that protease-activated receptors may be activated and attenuated by more than one protease. Here, we explore a desensitization mechanism of the PAR1 thrombin receptor by anticoagulant proteases and provide an explanation to the enigma of why plasmin/tissue plasminogen activator (t-PA) can both activate and deactivate platelets prior to thrombin treatment. By using a soluble N-terminal exodomain (TR78) as a model for the full-length receptor, we were able to unambiguously compare cleavage rates and specificities among the serum proteases. Thrombin cleaves TR78 at the R41-S42 peptide bond with a kcat of 120 s-1 and a KM of 16 microM to produce TR62 (residues 42-103). We found that, of the anticoagulant proteases, only plasmin can rapidly truncate the soluble exodomain at the R70/K76/K82 sites located on a linker region that tethers the ligand to the body of the receptor. Plasmin cleavage of the TR78 exodomain is nearly equivalent to that of thrombin cleavage at R41 with similar rates (kcat = 30 s-1) and affinity (KM = 18 microM). Specificity was demonstrated since there is no observed cleavage at the five other potential plasmin-cleavage sites. Plasmin also cleaves the TR78 exodomain at the R41 thrombin-cleavage site generating transiently activated exodomain. We directly demonstrated that plasmin cleaves these same sites in full-length membrane-embedded receptor expressed in yeast and COS7 fibroblasts. The rate of plasmin truncation is similar between the extensively glycosylated COS7-expressed receptor and the nonglycosylated yeast-produced receptor. Mutation of the R70/K76/K82 sites to A70/A76/A82 eliminates plasmin truncation and desensitization of thrombin-dependent Ca2+ signaling and converts PAR1 into a plasmin-activated receptor with full agonist activity for plasmin. Plasmin does not desensitize the Ca2+ response of platelets or COS7 cells to SFLLRN consistent with intermolecular ligand-binding sites being located to the C-terminal side of K82. Truncation of the wild-type receptor at the C-terminal plasmin-cleavage sites removes the N-terminal tethered ligand or preligand, thereby providing an effective pathway for PAR1 desensitization in vivo.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blood Platelets / drug effects
  • Blood Platelets / metabolism
  • COS Cells
  • Calcium Signaling / drug effects
  • Fibrinolysin / pharmacology*
  • Fibroblasts
  • Glycosylation
  • Humans
  • Hydrolysis
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Peptide Fragments / biosynthesis
  • Peptide Fragments / genetics*
  • Peptide Fragments / metabolism*
  • Platelet Activation / drug effects
  • Platelet Aggregation Inhibitors / pharmacology
  • Protein Structure, Tertiary
  • Rabbits
  • Receptor, PAR-1
  • Receptors, Thrombin / biosynthesis
  • Receptors, Thrombin / genetics*
  • Receptors, Thrombin / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Saccharomyces cerevisiae / genetics
  • Solubility
  • Thrombolytic Therapy*
  • Tissue Plasminogen Activator / pharmacology


  • Peptide Fragments
  • Platelet Aggregation Inhibitors
  • Receptor, PAR-1
  • Receptors, Thrombin
  • Recombinant Proteins
  • Tissue Plasminogen Activator
  • Fibrinolysin