The purification of human fibrinogen

Abstract

For investigations concerning the mode of action of the clearing factor, estimations of free fatty acids in mixtures of normal rat plasma and lipaemic dog plasma were performed using a modification of van de Kamer's (1948) method as described for faeces. Because the method developed may be of value for similar or other uses, it is given here. In a 100 ml. flask 2 ml. of the plasma-mixture, containing either heparin or citrate as an anticoagulant, are run into 6 ml. of phosphoric acid solution (metaphosphoric acid, 50 g., NaCl, 250 g. water to 1 1.). After a few minutes 16 ml. of ethanol, containing 0-4 % amyl alcohol, are added, followed by 20 ml. of distilled light petroleum (b.p. 40-60°). The flask is stoppered and the mixture shaken by hand for 1 min. After separation, the upper layer is filtered through dry paper and the funnel covered in order to minimize evaporation. Care should be taken to avoid contamination with traces of the other phase. Ten ml. of the light petroleum extract is evaporated on a boiling-water bath and the residue dried in vacuo. Neutralized ethanol (5 ml.) containing 0-6 mg. of thymol blue per 100 ml. are added and the residue dissolved by boiling gently under reflux for 3 min. Finally, the fatty acids are titrated under a stream of N2 with O-O0N-NaOH.The results are corrected for a blank (usually about 0060 ml. of 001N-NaOH), obtained by running 2 ml. of water through the same procedure.The reproducibility of the method suffices for many purposes. In twenty-four estimations giving results ranging from 021 to 210 0,-equiv. of fatty acids per 2 ml. of plasma tested, the differences between duplicate determinations were found to have a standard deviation of + 0-14 ,u-equiv.

Autologous Immune Complex Nephritis Induced With Renal Tubular Antigen

“…Preparation of Antisera.--Rabbit anti-rat RTE antigens, albumin, fibrinogen, and ~'-and ~zc-globulins as well as sheep anti-rabbit ~,-globulin were prepared as previously described (6,11,12). Rabbit antirat RTE contained antibodies to all three RTE-specific antigens, RTE-a~, RTE-oq, and RTE-a6 (6,11).…”

Pathogenesis of the Coagulation Defect Developing During Pathological Plasma Proteolytic (“Fibrinolytic”) States. I. The Significance of Fibrinogen Proteolysis and Circulating Fibrinogen Breakdown Products*

“…Purified human fibrinogen (91 per cent of the nitrogen being clottable by thrombin) was prepared by the method of Kekwick, Mackay, Nance and Record (26), dissolved in citrate buffer, and stored frozen in small aliquots. Fibrinogen was iodinated with 1"31 as previously described (20).…”

Fibrinogen mitogenic effect on hemopoietic cell lines: control via receptor modulation.

“…Human fibrinogen was purified from fresh human plasma as described by Kekwick et al (15). The fibronectin fraction was removed by using a gelatin-agarose column (16).…”