Genesio Murano

Preparation and properties of thrombin.

Abstract Purified bovine prothrombin preparations were converted to thrombin in 25% sodium citrate solution, and the thrombin was isolated by chromatography on Amberlite IRC-50 according to previously published procedures. In the ultracentrifuge So20 w = 3.7. Following a second chromatography on Amberlite IRC-50, 75 amino acid residues, predominately acidic, were deleted and 183 residues remained with the thrombin. The isoelectric point shifted from 5.75 to 6.2, and the specific activity increased from 4,200 to 8,230 U/mg protein. Acetylation also removed a postulated acidic moiety from the 3.7S thrombin. Ultracentrifugation of the twice chromatographed thrombin gave So20,w = 3.2. During centrifugation molecular association and aggregation occurred. The 3.2S thrombin contained 1.7% sialic acid, 5.02% orcinolreactive carbohydrate, 0.69% glucosamine, and 0.46% galactosamine. Molecular weight for only amino acids was 21,100, and with sialic acid and carbohydrate added it was 22,900. By thin-layer gel filtration we found 22,500, and with the same technique 33,000 for 3.7S thrombin and 40,000 for prethrombin. With TAMe as substrate Km for 3.7S thrombin = 2.97 × 10−4; 3.2S thrombin = 9.5 × 10−5, and for acetylated thrombin = 4.85 × 10−4. It was concluded that thrombin has two polypeptide chains. The N-termini are: threonine and isoleucine.

Human fibrinogen: Some characteristics of its S-carboxymethyl derivative chains☆

Abstract Human fibrinogen, a dimeric structure with a molecular weight of 340,000, is composed of three polypeptide chains: α (A), β (B), and γ, linked by S-S bridges. Following reduction with dithiothreitol and alkylation with tritiated iodoacetic acid, the three chains have been separated on CM-cellulose. Their molecular weights, determined by SDS-polyacrylamide gel electrophoresis were determined to be: α (A) = 64,000; β (B) = 57,000; and γ = 48,000. In preparation for amino acid sequence analysis, each purified chain derivative was cleaved with BrCN, and the fragments chromatographed on Sephadex G-100. Each chain had a characteristic radioactivity and optical density profile. This approach is helpful in isolating the carboxymethylated fragments of each chain. The N-terminal fragment of the α (A) chain, containing 51 amino acid residues, has been isolated and characterized by amino acid analysis, N-terminal analysis, and peptide mapping. Each chain derivative was digested with plasmin. Polyacrylamide gel electrophoresis and titration in the pH stat indicate that the α (A) chain is the preferred substrate for plasmin, a phenomenon previously observed in the intact fibrinogen molecule. This may explain the appearance of a heterogeneous population of α (A) chains upon chromatography of reduced alkylated fibrinogen (from pooled plasma) on CM-cellulose.

Primary structure of the amino terminal regions of chicken fibrin chains.

Abstract Fibrinogen and fibrin, isolated from chicken plasma, was studied in terms of a) its subunit polypeptide chain amino acid composition, b) their respective molecular size, and c) their partial NH2-terminal amino acid sequence. Based on the observation that intact fibrinogen yielded only tyrosine as an N-terminal residue, it was concluded that the N-terminal residues of both Aα and Bβ chains are probably blocked. Consistent with this observation was the finding that neither the isolated Aα and Bβ chain S-carboxymethyl derivatives could be sequenced. The γ-chain was sequenced 34 steps with 31 definite identifications. NH2-terminal amino acii sequence analysis was successfully carried out for 12 steps on all three (α, β, γ) S-carboxymethyl chains derived from fibrin. When compared to mammalian species (man and dog) the amino acid composition and partial NH2-terminal primary structure of the three fibrin chains did not differ dramatically. One major difference observed was the slightly reduced molecular size of the α-chain.

Further observations on the purification and properties of autoprothrombin III (Factor X)

Abstract Autoprothrombin III (Factor X) was separated from purified bovine prothrombin complex by chromatography on DEAE-cellulose and filtration through a Sephadex G-100 column. It had the same amino acid composition and approximately the same sedimentation characteristics in the ultracentrifuge as the first product obtained in this laboratory. The sedimentation constant was 3.58S. The product was studied by disc gel electrophoresis over a wide pH range. It was free of autoprothrombin C (Factor Xa), was stable, and had no tendency to activate spontaneously. On the basis of 27 Sephadex filtration experiments, the molecular weight was 81,900 ± 2,770. For autoprothrombin C, it was 53,500; by another method, it was 58,000. The N-terminal amino acids were alanine and serine, while those for autoprothrombin C were glycine and serine. Peptide maps for the zymogen and enzyme were similar except there were fewer peptides on the map of the latter. Based on a specific activity of 1,200 u/mg and a plasma concentration of 30 u/ml, the concentration of autoprothrombin III in bovine plasma most likely ranges from 2–3 mg%.

The coagulation of blood: Preliminary survey of thrombin and autoprothrombin zymogen structure☆

Abstract Numerous facts are consistent with the conclusion that bovine thrombin zymogen is a dimer of thrombin and is easily dissociated from autoprothrombin III (Stuart factor, F-X, autoprothrombin, prothrombokinase). The latter comprises about 4.4% of the total protein in our preparation. When thrombin forms, at least two peptides are removed. Prethrombin has one N-terminal amino acid in common with thrombin. Autoprothrombin C has an amino acid composition and specific activity similar to autoprothrombin III, but has only half the molecular weight. A likely possibility is that autoprothrombin III is a dimer of autoprothrombin C. Alternatively it could represent one mole of autoprothrombin C and one of F-VII.

An Improved Method for the Preparative Isolation of the S-Carboxymethyl Derivative Chains of Human Fibrinogen

Abstract An automated procedure for the preparative isolation of the S-carboxymethyl derivative chains of human fibrinogen on carboxymethyl cellulose is described. This involves the use of an apparatus which automatically controls the ion exchange chromatography elution gradient. This technique is advantageous in that it minimizes the need for rechromatography of the various fractions, and is more reproducible than previous techniques.

The “lysometer”: An automated device for the detection of fibrin dissolution, in vitro

Abstract The clot-lysis timing device consists of an electronic recording system and 24 controlled temperature sample wells. A 1.5–2.5 ml. clot is formed in a test tube by the addition of thrombin to plasma (or to a mixture of fibrinogen and plasminogen) containing varying quantities of urokinase, streptokinase or plasmin). Pre-cooling prevents enzymatic activity until the tube is placed in one of the 37 °C wells. A mechanical switch (at the base of each well) is a polled input to a computer. An opaque plastic sphere is placed on top of the clot. As the clot dissolves, (rate dependent on the quantity of lytic agent incorporated in the clot), the sphere travels to the bottom of the tube, interrupting an infrared light beam, which is another polled input to a computer. Each channel can be independently displayed on an L.E.D. readout. A four (or more) point standard curve (in duplicate) and four concentrations of an unknown (in duplicate) can be analyzed simultaneously. The instrument has been used successfully in the determination of the potency of urokinase, streptokinase and plasmin solutions.

Isolation and characterization of canine spleen alkaline phosphatase.

Alkaline phosphatase activity was extracted from the canine spleen using Triton X-100. The extract was partially purified employing Bio-Bead SM-2 and DEAE-cellulose column chromatography. Activity could be eluted off DEAE-cellulose, and two separate and distinct phosphatase fractions were demonstrated by polyacrylamide gel electrophoresis. Thermal inactivation studies indicated a lability similar to that of bone phosphatase, while chemical inhibition studies suggested activity similar to that found in liver. Metal ion activation was maximal with zinc and minimal with magnesium. Cathodic and anodic alkaline phosphatase could not be differentially characterized other than by electrophoresis.

Lack of Inhibition of Hog Renin by Heparin

These experiments were performed to test if heparin inhibits the production of angiotensin I from rat renin substrate acted upon by either rat or hog renin. Substrate was prepared from the plasma of nephrectomized rats; commercially prepared hog renin and heparin (0-50 units/ml, final concentration) were added and angiotensin I production was measured by radioimmunoassay following incubation of the mixture at 37-38 degrees C for 30 min. Heparin had no effect on the radioimmunoassay for angiotensin I, nor on the Vmax nor the Km of the renin-renin substrate reaction. Heparin (0 and 50 units/ml, final concentration) was added to rat plasma which contained renin and renin substrate. No effect of heparin on the rate of production of angiotensin I was observed in this system. In conclusion, rat and hog renin are not inhibited by heparin within the range of concentrations used.

Determination of plasminogen activity (fibrinolytic) with the lysometer.

In a recent report in this journal (1) an automated instrument (Lysometer) for the detection of fibrin dissolution in vitro, was described. Detailed procedures for the assay of purified urokinase, streptokinase, and plasmin, were also described (1). Studies have been extended to include the use of this instrument in the determination of the activity of purified plasminogen. The two stage procedure consists of incubating plasmfnogen (15 units) with urokinase (10 units) at 37 C for 30 min, and then assaying the generated plasmin by measuring the rate of clot dissolution (Fig. 1).