Expression of hirudin fusion proteins in mammalian cells: a strategy for prevention of intravascular thrombosis

Circulation. 1998 Dec 15;98(24):2744-52. doi: 10.1161/01.cir.98.24.2744.

Abstract

Background: Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation.

Methods and results: An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti-hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4-P-selectin fusion proteins accumulated in adrenocorticotropic hormone-containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane.

Conclusions: Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology
  • Animals
  • Antithrombins / genetics*
  • Antithrombins / metabolism
  • Antithrombins / pharmacology
  • Blood Coagulation / drug effects
  • CD4 Antigens / chemistry
  • CD4 Antigens / genetics
  • CD4 Antigens / metabolism
  • Catalytic Domain / drug effects
  • Cell Line / chemistry
  • Cell Line / metabolism
  • Cell Line / ultrastructure
  • Cytoplasmic Granules / chemistry
  • Cytoplasmic Granules / metabolism
  • Hirudins / genetics*
  • Hirudins / metabolism
  • Hirudins / pharmacology
  • Histocompatibility Antigens Class I / genetics
  • Humans
  • Membrane Proteins / genetics
  • P-Selectin / genetics
  • P-Selectin / metabolism
  • Protein Sorting Signals / genetics
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / genetics*
  • Thrombin / drug effects
  • Thrombin / metabolism
  • Thrombosis / therapy
  • Transfection / methods

Substances

  • Amino Acid Chloromethyl Ketones
  • Antithrombins
  • CD4 Antigens
  • Hirudins
  • Histocompatibility Antigens Class I
  • Membrane Proteins
  • P-Selectin
  • Protein Sorting Signals
  • Recombinant Fusion Proteins
  • Thrombin
  • phenylalanyl-prolyl-arginine-chloromethyl ketone