Cross-linked alpha s gamma t-chain hybrids in plasma clots studied by 1D- and 2D electrophoresis and western blotting.

Fibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the alpha-chains or the gamma-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute alpha gamma-chain hybrids or superimposed alpha- and gamma-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia's Multiphor II system.

Characterization of fibrinogen/fibrin derivatives isolated from normal and fibrinaemic plasma and serum using paramagnetic particles coated with a monoclonal antibody (mAb) to D-dimer.

Paramagnetic particles coated with a monoclonal antibody to D-dimer (mAb S4) were used to isolate and concentrate D-dimer and D-dimer-containing complexes in plasma and serum. Antibody-captured material was eluted with SDS-urea buffer and examined by either SDS-polyacrylamide or submerged SDS-agarose gel electrophoresis followed by Western blotting. The protein pattern was visualized by either polyclonal antibodies to human fibrinogen or monoclonal antibodies specific for fibrinogen derivatives containing fibrinopeptide A (FpA; mAb Y18), the N-terminus of the beta-chain in fibrin (mAb 59D8) or the gamma-chains (mAb J88B).

Early cross-linked fibrin in human plasma contains alpha-polymers with intact fibrinopeptide A.

alpha-polymer formation, as opposed to gamma-chain dimerization has been considered a relatively late event in factor XIII-induced fibrin stabilization. Recently it has been shown, however, that plasma from healthy individuals and from patients with fibrinaemia contains small amounts of soluble fibrin/fibrinogen oligomers interlinked through dimerized gamma-chains as well as cross-linked alpha-chains. The present work was carried out to see if these early alpha-chain polymers also arise during coagulation of plasma in vitro.

Soluble, cross-linked fibrin(ogen) hybrid oligomers do not stimulate t-PA conversion of plasminogen.

Cross-linked hybrid oligomers of fibrinogen and fibrin are found in plasma from fibrinaemic patients and normal individuals as well as in preparations of purified human fibrinogen. The present study was undertaken to see if such hybrid oligomers have the same stimulatory effect on tissue plasminogen activator (t-PA) conversion of plasminogen as do polymeric and monomeric fibrin. Hybrid oligomeric fibrin(ogen) material was provided by subjecting purified human fibrinogen to gel filtration in urea-containing buffer at pH 5.

Observations on the cell biology of tissue factor in endothelial cells.

Human umbilical vein endothelial cells (HUVEC) are inducible for tissue factor (TF) activity in culture. Based on experiments using ECGF (4-20 micrograms/ml) with heparin (90 micrograms/ml), we obtained the following results: 1) In confluent HUVEC cultures, ECGF had essentially no influence on the levels of inducible TF. 2) In growing HUVEC cultures, ECGF reduced the TF response shortly after seeding but full response was regained when cells were kept confluent for 2-3 days.