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A simple reproducible procedure has been developed for the large scale isolation of bovine factor X and prothrombin in highly purified states. Both proteins isolated by this method show a single component in four different electrophoretic systems. The specific activity of prothrombin obtained by this procedure is 1300 ± 100 NIH thrombin units per mg of prothrombin, whereas the specific activity of factor X is 90 ± 10 units of activated factor X (factor Xa) per mg of factor X, when assayed by the standard technique (Bachmann, F., Duckert, F., andKoller, F. (1958) Thromb. Diath. Haemorrh. 2, 24). The over-all yields of bovine prothrombin and factor X are 70 mg and 4.5 mg, respectively, per liter of starting plasma. The isolation procedure involves adsorption of factor X and prothrombin on to barium citrate, their elution from the barium citrate complex with EDTA, extensive dialysis against 0.02 mtrisodium citrate and 0.9% sodium chloride, ammonium sulfate fractionation, diisopropylfluorophosphate treatment, and DEAE-cellulose chromatography. DEAE-cellulose chromatography in (sodium) citrate buffer at pH 6.0 effectively and completely separates prothrombin from factor X. Factor X is then further purified by DEAE-Sephadex chromatography. Both proteins are completely stable in 50% glycerol-H2O (v/v) at -20° for at least 18 months.

Bovine prothrombin with tritium incorporated into its sialic acid residues has been prepared by the method of Van Lenten and Ashwell ((1970) J. Biol. Chem.246, 1889–1894). The resulting radiolabeled protein ([sialyl-3H]prothrombin) was electrophoretically indistinguishable from the untreated protein. The thrombin activity generated by factor Xa activation of [sialyl-3H]prothrombin was about 90 % of that obtained from nonlabeled prothrombin.

The effect of the Arg506→ Gln mutation in factor VLEIDENon thrombin generation was evaluated in a reconstituted system using the purified components of the tissue factor (TF) pathway to thrombin and the components of the protein C pathway. Recombinant full-length tissue factor pathway inhibitor (RTFPI) was included in the system because of a previously observed synergistic inhibitory effect of TFPI and the protein C pathway on TF-initiated thrombin generation. Thrombin generation initiated by 1.25 pmfactor VIIa·TF in the absence of the protein C pathway components occurs following an initiation phase, after which prothrombin is quantitatively converted to 1.4 μmthrombin. The factor VLEIDENmutation did not influence thrombin generation in the reconstituted model in the absence of the protein C pathway. In the presence of 2.5 nmTFPI, 65 nmprotein C, and 10 nmrecombinant soluble thrombomodulin (Tm), thrombin generation catalyzed by normal factor V was abolished after the initial formation of 25 nmthrombin. In contrast, persistent thrombin generation was observed in the presence of factor VLEIDENin the same system, although the rate of thrombin generation was slower compared with the reaction without protein C and Tm. The rate of thrombin generation with factor VLEIDENincreased with time and ultimately resulted in quantitative prothrombin activation. When the TFPI concentration was reduced to 1.25 nm, thrombin generation is still curtailed in the presence of normal factor V. In contrast, under similar conditions using factor VLEIDEN, the protein C pathway totally failed to down-regulate thrombin generation. The dramatic effect of a 50% reduction in TFPI concentration on the inhibitory potential of the protein C pathway on thrombin generation catalyzed by factor VLEIDENsuggests that the observed synergy between TFPI and the protein C pathway is directly governed by the TFPI concentration and by cleavage of the factor Va heavy chain at Arg506. This cleavage appears to have a dramatic regulatory effect in the presence of low concentrations of TFPI. Markedly increased thrombin generation in the presence of both 1.25 nmTFPI and factor VLEIDENwas also observed when antithrombin-III was added to the system to complete the natural set of coagulation inhibitors. Protein S (300 nm) had a minimal effect in the model on the inhibition of thrombin generation by protein C, Tm, and TFPI, with either normal factor V or factor VLEIDEN. Protein S also failed to significantly potentiate the action of the protein C pathway in the presence of antithrombin-III in reactions employing normal factor V or factor VLEIDEN. The absence of an effect of protein S in the model, which employs saturating concentrations of phospholipid, suggests that the reported interactions of protein S with coagulation factors are not decisive in the reaction. Altogether the data predict that TFPI levels in the lower range of normal values are a risk factor for thrombosis when combined with the Arg506→ Gln mutation in factor VLEIDEN.

Bovine lactoferrin has a molecular weight of 77,100 ± 1500 in dilute aqueous solution as determined by the method of sedimentation equilibrium in the ultracentrifuge. In 6 mguanidine hydrochloride in the presence or absence of mercaptoethanol, bovine lactoferrin has a molecular weight of 72,500 to 77,200 ±1300 as measured by sedimentation equilibrium in the ultracentrifuge or by gel filtration on agarose columns. The molecular weight obtained by gel electrophoresis in the presence of sodium dodecyl sulfate is 76,000 ± 2400. Tryptic peptide maps reveal a number of peptides approximately equal to the content of lysine plus arginine in the molecule. Quantitative end group analysis shows 1 residue of alanine at the amino terminus of the molecule. These data demonstrate that bovine lactoferrin is composed of a single polypeptide chain. Analysis of the carbohydrate content revealed 1 residue of terminal sialic acid, 10 to 11 residues of N-acetyl glucosamine, 5 to 6 residues of galactose, and 15 to 16 residues of mannose per molecule of lactoferrin. Another protein, isolated as a major contaminant in commercial preparations of bovine α-lactalbumin showed identical properties to bovine lactoferrin except that it appeared, based upon molecular weight measurements, to be an aggregating system.

Bovine prothrombin can be labeled with fluorescein isothiocyanate to produce a fluorescent proenzyme which maintains up to 85% of native prothrombin-specific activity. Other proteins labeled similarly were analyzed by sodium dodecyl sulfate gel electrophoresis, and it was found that the chromophores did not affect the molecular weights as determined by the electrophoretic system. Sodium citrate activation of both labeled and unlabeled prothrombin produced molecular weight species consistent with our previously proposed model (Mann, K. G., Heldebrant, C. M., andFass, D. N. (1971) J. Biol. Chem.246, 6106–6114): prothrombin (72,000), Intermediate 1 (65,000), Intermediate 2 (39,000), and Intermediate 3 (25,000). The thrombins are produced from Intermediate 2 and consist of multichain disulfide-linked structures; α-thrombin (33,000 + 6,000), β-thrombin (18,000 + 10,000), and γ-thrombin (14,000 + 10,000 + 4,000). An analog of the physiological coagulation system was constructed using serum and a phospholipid source. Labeled prothrombin when activated in this mixture yielded products which, by virtue of their fluorescence, could be distinguished from the serum proteins. All precursors of thrombin which are produced during citrate activation are also produced during serum activation. The large thrombin, α-thrombin, is composed of 33,000- and 6,000-dalton chains in both activating systems. These results indicate that prothrombin activation in biological systems produces intermediates and products identical with those formed in highly purified synthetic activation systems. Our studies also indicate that the kinetics of the activation process in serum is demonstrably different from that previously described in highly purified systems.

The intermediates formed during the activation of prothrombin in 25% sodium citrate solutions have been isolated and partially characterized. Their properties are in agreement with those predicted by a previously proposed model for prothrombin activation in 25% sodium citrate: prothrombin (72,000 daltons) → intermediate 1 (65,000 daltons) → Intermediate 2 (39,000 daltons) and Intermediate 3 (25,000 daltons); Intermediate 2 (39,000 daltons) → thrombin (α-thrombin, 39,000 daltons) (Mann, K. G., Heldebrant, C. M., andFass, D. N. (1971) J. Biol Chem.246, 6106–6114).