Purified human fibrinogen

Purified human fibrinogen (Hyphen BioMed, France) was bound covalently to 5-µm spherical silica pedestals anchored to the bottom of a chamber as previously described^{11 (link),13 (link),15 (link)}. Briefly, pedestals coated with a thin layer of polyacrylamide were activated with 10% glutaraldehyde (30 min, 37 °C), after which fibrinogen (1 mg/ml in the activation mixture) was immobilized for 2 hr at 4 °C. The chamber was then washed with 20 volumes of the same buffer to remove non-covalently adsorbed protein, blocked with 2 mg/ml bovine serum albumin (BSA) in 0.055 M borate buffer, pH 8.5, 150 mM NaCl, 3 mM CaCl₂ for 30 min at 4 °C, and equilibrated with 20 mM HEPES buffer, pH 7.4, containing 150 mM NaCl. To generate surfaces coated with monomeric fibrin, the fibrinogen-coated pedestals were treated with human α-thrombin (1 U/ml, 37 °C, 1 hr) followed by thorough washing (20 volumes of the flow chamber) and equilibration in 0.1 M HEPES buffer, pH 7.4, containing 150 mM NaCl, 3 mM CaCl₂, 1 mM MnCl₂, 2 mg/ml BSA, and 0.1% (v/v) Triton X-100. Treatment with thrombin has been previously shown to generate biologically active fibrin molecules on the bead surface^{10 (link),22 (link)}. Because the monomeric fibrin was covalently attached to the bead surface, substantial fibrin oligomerization was not possible.

Immuno-depleted lyophilized TFPI (tissue factor pathway inhibitor, def-TFPI) and PS (protein S, def-PS) plasmas were obtained from Diagnostica Stago (Asnières, France); their fibrinogen concentrations were 2.2 and 2.4 g/L, respectively. Lyophilized heparinized plasma (calciparin 0.2 UI/mL, Diagnostica Stago) had a final fibrinogen concentration of 2.8 g/L. A frozen normal plasma pool (NP) was obtained from normal donors; its final fibrinogen concentration was 2.5 g/L (Diagnostica Stago). Therefore, there were 3 kinds of plasmas studied: 2 hypercoagulants plasmas (def-TFPI and def-PS), 1 hypocoagulant plasma (heparinized), and 1 normocoagulant plasma (NP). Purified human fibrinogen was from Hyphen Biomed (Neuville-sur-Oise, France).
Plasma fibrinogen concentrations were determined with the Clauss method using Fib5 reagent on a STAR coagulometer (Diagnostica Stago).