Hepatocyte-derived fibrinogen-related protein-1 is associated with the fibrin matrix of a plasma clot

doi:10.1016/j.bbrc.2006.09.018

Biochemical and Biophysical Research Communications

Volume 350, Issue 1, 10 November 2006, Pages 191-194

https://doi.org/10.1016/j.bbrc.2006.09.018 Get rights and content

Abstract

In order to study the multiple functions of fibrinogen and fibrin, we are investigating which proteins bind to the fibrin matrix of a plasma clot by using a proteomic approach. Extracts from washed plasma clots were analysed by 2-D gel electrophoresis. A relatively abundant spot was identified as hepatocyte-derived fibrinogen-related protein-1 (HFREP-1) by MALDI-TOF analysis, molecular mass (34 kDa), iso-electric point (pI 5.5) as well as by Western blot analysis. HFREP-1 in plasma almost completely bound to the fibrin matrix during clot formation. Several purified fibrinogen preparations proved to be contaminated with HFREP-1. It is concluded that HFREP-1 (also named hepassocin), a protein with liver cell growth regulatory properties, occurs in plasma and strongly associates with fibrin and possibly fibrinogen.

Section snippets

Materials and methods

Pooled platelet-poor citrated plasma was prepared from twelve healthy donors from the local blood bank. Clots of 500 μl plasma were prepared by supplementing plasma with calcium chloride (20 mM, f.c.), aprotinin (100 KIU/ml, f.c.) and thrombin (1 NIH U/ml, f.c). After an incubation period of 1 h at room temperature the clots were extensively washed by permeating them overnight at 4 °C with about 10 ml Tris-buffered saline, containing 100 KIU/ml aprotinin. The clots were compacted by centrifugation,

Results

Fig. 1A shows the protein mixture extracted from a washed plasma clot after 2-D gel electrophoresis using a pH range of 4–7 in the first dimension. The spot indicated by an arrow was excised, digested with trypsin and analysed by mass spectrometry. Mascot analysis identified the spot as HFREP-1 precursor, a protein of 312 amino acids including a predicted signal peptide of 17 [3] or 22 [5] amino acids (significant Mowse scores of 165, 172, and 201 in three separate experiments). The molecular

Discussion

HFREP-1 belongs to a fibrinogen superfamily that includes for instance fibrinogen, fibroleukin (fibrinogen-like protein-2), angiopoietin, angiopoetin-related protein, tenascin, and ficolin [6]. The members share a domain at their carboxy-termini, which is homologous to the carboxy-terminal two-thirds (about 270 amino acids) of the beta- and gamma chains of fibrinogen [7]. HFREP-1 has a domain with 41.1% and 41.3% homology to these parts of the beta- and gamma chains of fibrinogen, respectively

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Hepatocyte-derived fibrinogen-related protein-1 is associated with the fibrin matrix of a plasma clot

Abstract

Section snippets

Materials and methods

Results

Discussion

Adv. Protein Chem.

J. Thromb. Haemost.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.