The interaction of fibulin-1 with fibrinogen. A potential role in hemostasis and thrombosis.
The fibulins are an emerging family of extracellular matrix and blood proteins presently having two members designated fibulin-1 and -2. Fibulin-1 is the predominant fibulin in blood, present at a concentration of 30-40 micrograms/ml (approximately 1000-fold higher than fibulin-2). During the course of isolating fibulin-1 from plasma by immunoaffinity chromatography, a 340-kDa polypeptide was consistently found to co-purify. This protein was identified as fibrinogen (Fg) based on its electrophoretic behavior and reactivity with Fg monoclonal antibodies. Radioiodinated fibulin-1 was shown to bind to Fg transferred onto nitrocellulose filters after SDS-polyacrylamide gel electrophoresis. In enzyme-linked immunosorbent assay, fibulin-1 bound to Fg (and fibrin) adsorbed onto microtiter well plastic, and conversely, Fg bound to fibulin-1-coated wells. The binding of Fg to fibulin-1 was also observed in surface plasmon resonance assays, and a dissociation constant (Kd) of 2.9 +/- 1.6 microM was derived. In addition, fluorescence anisotropy experiments demonstrated that the interaction was also able to occur in fluid phase, which suggests that complexes of fibulin-1 and Fg could exist in the blood. To localize the portion of Fg that is responsible for interacting with fibulin-1, proteolytic fragments of Fg were evaluated for their ability to promote fibulin-1 binding. Fragments containing the carboxyl-terminal region of the Bbeta chain (residues 216-468) were able to bind to fibulin-1. In addition, it was found that fibulin-1 was able to incorporate into fibrin clots formed in vitro and was immunologically detected within newly formed fibrin-containing thrombi associated with human atherectomy specimens. The interaction between fibulin-1 and Fg highlights potential new roles for fibulin-1 in hemostasis as well as thrombosis.
Tran
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Department of Biochemistry, J. H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
Tanaka
A
A
Litvinovich
S V
SV
Medved
L V
LV
Haudenschild
C C
CC
Argraves
W S
WS
D001665:Binding Sites; D001704:Biopolymers; D002135:Calcium-Binding Proteins / Q000097:blood* / Q000302:isolation & purification / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D005454:Fluorescence Polarization; D006487:Hemostasis; D006801:Humans; D011485:Protein Binding; D013927:Thrombosis / Q000097:blood*
D001704:Biopolymers; D002135:Calcium-Binding Proteins; C060820:fibulin; D005340:Fibrinogen