Quantification of fibrin deposition in flowing blood with peroxidase-labeled fibrinogen. High shear rates induce decreased fibrin deposition and appearance of fibrin monomers.
To study fibrin incorporation into thrombi at different wall shear rates, a new method to study fibrin deposition on extracellular matrixes underlying stimulated endothelial cells under flow conditions was developed. For this method, we used fibrinogen labeled with peroxidase (Fg-PO). Fg-PO was fully exchangeable for Fg in the clotting assays tested, and PO activity was bound to fibrin-specific fragments. Fg-PO containing fibrin could be stained for microscopic studies with 3,3'-diaminobenzidine and could be quantified by oxidation of phenylenediamine. The absorbance values at 492 nm were converted to fibrin quantities via a standard curve. To study fibrin deposition, Fg-PO was added in trace amounts to whole blood anticoagulated with low-molecular-weight heparin, and perfusion studies were performed over endothelial cell matrixes containing tissue factor. In parallel perfusion studies, 125I-labeled Fg was added in trace amounts to whole blood instead of Fg-PO. Both quantitative methods demonstrated decreased fibrin deposition after perfusions at 1,300 sec-1 compared with fibrin deposition after perfusions at 300 sec-1, while fibrinopeptide A generation was independent of the wall shear rate. The decrease in fibrin deposition at 1,300 sec-1 was accompanied by the appearance of fibrin monomers in the perfusate. This suggested that the decrease in fibrin incorporation at 1,300 sec-1 was due to the impaired polymerization of fibrin monomers. This impairment was probably due to a decrease in local fibrin monomer concentration as a result of the increased removal of monomers from the surface at 1,300 sec-1.
Tijburg
P N
PN
Department of Haematology, University Hospital Utrecht, The Netherlands.
Ijsseldijk
M J
MJ
Sixma
J J
JJ
de Groot
P G
PG
D000911:Antibodies, Monoclonal; D000925:Anticoagulants / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005337:Fibrin / Q000032:analysis* / Q000187:drug effects; D005340:Fibrinogen / Q000502:physiology*; D006801:Humans; D007124:Immunoenzyme Techniques; D008955:Models, Cardiovascular; D012212:Rheology; D013927:Thrombosis / Q000503:physiopathology*
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D005337:Fibrin; D005340:Fibrinogen