Preparation of thrombokinase from bovine plasma

Thrombokinase is prepared from bovine plasma by a procedure involving: treatment with diatomaceous silica, adsorption on barium sulfate, flowing elution with two successive phosphate buffers, ammonium sulfate fractionation, "spontaneous" activation in concentrated solution, and isoelectric precipitation. The yield of nitrogen is 0.002 per cent, corresponding to 1.2 mg. protein per liter of plasma. When diluted back to the volume of parent plasma, and complemented by calcium plus cephalin, the product causes appreciable activation of prothrombin in 1 minute. Thus, the quantity of thrombokinase obtainable is compatible with a physiologic role. In the more complex system used for routine assay, thrombokinase can be supplied by crude plasma at a dilution of 1/500. In parallel tests, the product appears to be more active than its parent plasma, although it contains only 0.002 per cent of the nitrogen. However, the thrombokinase of the product has been activated, whereas the thrombokinase of the plasma is probably in an inactive precursor state. When diluted back to the volume of parent plasma, to a concentration of 0.2 microgram nitrogen per ml., thrombokinase can slowly activate prothrombin in the presence of oxalate, and without the addition of accessory factors. Activation of prothrombin in the presence of oxalate is faster with higher concentrations of thrombokinase.     

Thrombokinase of the Blood as Trypsin-Like Enzyme

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity.     

Outstanding Characteristics of Thrombokinase Isolated from Bovine Plasma

Thrombokinase has been isolated from bovine plasma by a procedure which begins with the highly purified product of a previously described method, chromatographs it on DEAE-cellulose, and then fractionates it by continuous flow electrophoresis, yielding 0.2 mg per liter of oxalated plasma. The electrophoretic fraction has shown a single boundary in the ultracentrifuge; and its esterase activity on toluenesulfonylarginine methyl ester has been about the same as that of thrombokinase previously isolated by repeated electrophoretic fractionations. Thrombokinase is a euglobulin with minimum solubility near pH 5.0. It is most stable within the pH range 7.5 to 9.5; but there is also a peak in the stability curve near pH 1.8. A few micrograms of thrombokinase per milliliter can activate prothrombin in the presence of EDTA. A few thousandths of a microgram causes rapid production of thrombin in the system: prothrombin, thrombokinase, calcium chloride, phosphatide, "accelerator." But, thrombokinase has less than 1/175 the proteolytic activity of crystallized trypsin.     

Fractionation of plasma globulin for prothrombin,thrombokinase, and accessory thromboplastin

1. Crude globulin from more than 1,000 liters of citrated bovine plasma has been used in developing a procedure for moderately large scale separation of clotting factors. Fraction A, prothrombin, kinase, and thrombin fractions were prepared. Fraction A contained both kinase and accessory thromboplastin, the latter predominating when fraction A was diluted. 2. When prothrombin was activated by kinase, the rate of thrombin production was enhanced by the addition of platelets, or brain lipid, or dilute fraction A. These accessory thromboplastins caused this acceleration only when calcium chloride was added. Even with calcium, they were not effective unless kinase was present. 3. In contrast, the action of kinase was not entirely dependent on either ionic calcium or accessory thromboplastin. The concentrated kinase fraction activated prothrombin in the presence of excess oxalate. Although kinase often contaminates highly purified thrombins, it is probably distinct from thrombin. The ratio of kinase to thrombin was 100 times as great in the kinase fraction as in the thrombin fraction. 4. The kinase fraction, diluted 45,000-fold, to protein-nitrogen concentrations as low as 0.02 microgram per ml., accelerated the conversion of crude prokinase in three-stage tests. 5. The findings are consistent with the following concept of the basic enzymatic mechanism: See PDF for Structure It is now added that calcium and accessory thromboplastin exert their effects by impinging on the basic mechanism, in a chemically secondary or indirect manner.     

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.     

Three-stage analysis of blood coagulation

1. Blood-clotting mechanism has been analyzed by a procedure which devotes a separate experimental step to each of the three primary reactions: 1. Prothrombokinase --> thrombokinase 2. Prothrombin --> thrombin 3. Fibrinogen --> fibrin 2. Activation of prothrombin by thrombokinase followed the course of a unimolecular reaction, and the concentration of thrombokinase determined the initial rate. By this relation thrombokinase was measured, and the activation of its precursor was charted. 3. When the activation of prothrombokinase was plotted against time, the experimental points fell close to the theoretical curve for a simple autocatalytic reaction. Moreover, the process was accelerated by seeding with a small amount of crude thrombokinase. It was concluded that the activation of prothrombokinase involves an autocatalytic or chain reaction. 4. The three-stage procedure made possible the separate estimation of the power to activate prothrombin, on one hand, and the capacity to accelerate the transformation of prothrombokinase on the other. Drastic losses of both activities occurred when crude thrombokinase solutions were heated at 60 degrees C., or adsorbed with barium sulfate. 5. The concentration of calcium was important for the normal progress of prothrombin activation, and also for the transformation of prothrombokinase.     

A note on the mathematical theory of the effect of nervous stress on coronary thrombosis

The “classical” view of formation of blood clots is that the disintegration of platelets releases thrombokinase which activates prothrombin into thrombin. The latter combining with fibrinogen produces fibrin, the substance of the clot. There is, however, ample evidence of hormonal and other biochemical factors as playing a role in the process of clotting. Through the necessity of a breakdown of platelets has been challenged, yet theirpresence is considered as necessary for clotting. Thefirst part of this paper is based on the assumption that either a breakdown or even an injury of a platelet produces thrombokinase. An injury is likely to occur due to collisions between platelets, between platelets and erythrocytes or between platelets and leukocytes. A probability is ascribed to the production of thrombokinase by a collision. Thus not every collision is effective. The frequency of those collisions is calculated according to classical physics. Inasmuch as mental stress usually causes vasoconstriction and therefore an increase of concentration of the formed elements of blood, the frequency of collisions and therefore the production of small amounts of thrombokinase increases with stress. If thrombokinase formed by the collision remains preserved and therefore accumulates in the blood, we arrive at an expression which gives us the necessary total duration of stress to produce thrombosis. According to that equation, even in the absence of stress, any individual if he lives long enough should eventually develop a thrombosis. If, however the thrombokinase is destroyed, then it is necessary that during a sufficiently short time enough effective collisions occur to increase the amount of thrombokinase to a danger level. With this picture we find only theprobability of a formation of a thrombus as a function of the intensity of stress and its duration. The probability remains finite event for zero stress, showing that even individuals who are always relaxed may develop thrombi. In the second part of the paper an outline of a biochemical approach is given. Inasmuch as we know very little about the biochemical mechanism involved, it is simply assumed that during a stress of intensityS and duration Δ there is a probability of a thrombosis occurring. Then by applying the theory of probabilities, an expression is derived giving the probability of occurrence of a thrombus after a given number of stress incidences. The expressions are different from those obtained from the first approach but lead basically to the same results. A comparison of both or of their combination, with available data may give indications as to which of the factors plays a preponderant part.     

Rat prothrombin: Purification,characterization, and activation

Prothrombin has been purified from rat plasma and its properties compared to prothrombin isolated from other species. The molecular weight, amino acid composition, and amino-terminal sequence of rat prothrombin are similar to human and bovine prothrombin. Rat prothrombin binds to phospholipid in the presence of calcium ions, and calcium-binding measurements indicate that it may bind somewhat more calcium than does bovine prothrombin. The proteolytic cleavage of purified rat prothrombin by Factor X_a or thrombin yields the same peptides that are formed from similar proteolysis of bovine prothrombin. Factor V and phospholipid were shown to enhance the rate of Factor X_a and calcium ion generation of thrombin from rat prothrombin.     

STUDIES ON BLOOD COAGULATION : I. THE R?LE OF PROTHROMBIN AND OF PLATELETS IN THE FORMATION OF THROMBIN

1. A method is described for the preparation and titration of prothrombin and thrombin. 2. Confirming the views of Morawitz, Howell (1916–17, 1925), and Bordet, thrombin cannot be regarded as an artificial by-product of coagulation (Wooldridge, Nolf (both quoted from Morawitz)). Calcium, a platelet factor, and a plasma factor (prothrombin) interact to form thrombin, and this then acts upon fibrinogen to form fibrin. The amount and rate of thrombin formation in the first reaction are independent of the presence or absence of fibrinogen. After a variable latent period, thrombin suddenly appears in large quantities, coincident with or immediately preceding the deposition of fibrin if fibrinogen is present. 3. The amount of thrombin formed in a mixture of prothrombin, Ca and platelets is independent of the platelet or Ca concentration, and depends primarily upon the amount of prothrombin used. The platelets (or cephalin) enormously accelerate the transformation of prothrombin to thrombin, and this acceleration seems to be their physiological rôle in the coagulation process. 4. Contrary to previous reports, platelets have not been demonstrated to contain significant quantities of prothrombin. 5. The available data do not allow any definite decision as to whether the platelet factor actually combines with prothrombin to form thrombin, or merely catalyzes the transformation. The very slow formation of thrombin in the complete absence of platelets may be due to dissolved traces of platelet material released during the physical manipulation of the plasma (centrifuging, Berkefeld filtration). 6. There was no evidence for a species-specific activity of platelets in the transformation of prothrombin to thrombin.     

Chromatography of blood-clotting factors and serum proteins on columns of diatomaceous earth

1. In batch adsorptions with prothrombin solutions, hyflo was the weakest adsorbent, standard super-cel intermediate, and filter-cel strongest. Of these three grades of diatomaceous earth, hyflo has the smallest surface area per gram and filter-cel the largest. In parallel breakthrough experiments, a column of standard super-cel had a capacity almost six times that of a hyflo column. 2. After partial removal of impurities by diatomaceous earth, prothrombin preparations contained less thrombokinase, were more stable, and displayed less tendency to form thrombin "spontaneously." Thrombokinase (or its precursor) was removed from a preparation of prothrombin by passage through a filter cake of standard super-cel. The specific activity of the prothrombin was increased; and 62 per cent of the activity was recovered. 3. Prothrombin was adsorbed from an ammonium sulfate solution at pH 5.26 by columns of hyflo or standard super-cel. When eluted by phosphate solutions, the protein moved down the columns more readily at higher pH and higher concentration of phosphate salts, within the pH range 5.0 to 6.6, and within the phosphate range 0.1 to 1.0 M. 4. Thrombin was adsorbed on a column of standard super-cel at pH 5.11. As successive eluents passed through the column, the thrombin emerged between two bands of impurities. The specific activity of the thrombin was raised; and 83 per cent of the activity was recovered. 5. With a column of standard super-cel, and with a series of eluents within the pH range 5.1 to 6.3, total serum proteins were separated into four major bands. About 94 per cent of the protein was recovered.     

HD1, a thrombin-directed aptamer, binds exosite 1 on prothrombin with high affinity and inhibits its activation by prothrombinase

Incorporation of prothrombin into the prothrombinase complex is essential for rapid thrombin generation at sites of vascular injury. Prothrombin binds directly to anionic phospholipid membrane surfaces where it interacts with the enzyme, factor Xa, and its cofactor, factor Va. We demonstrate that HD1, a thrombin-directed aptamer, binds prothrombin and thrombin with similar affinities (K(d) values of 86 and 34 nm, respectively) and attenuates prothrombin activation by prothrombinase by over 90% without altering the activation pathway. HD1-mediated inhibition of prothrombin activation by prothrombinase is factor Va-dependent because (a) the inhibitory activity of HD1 is lost if factor Va is omitted from the prothrombinase complex and (b) prothrombin binding to immobilized HD1 is reduced by factor Va. These data suggest that HD1 competes with factor Va for prothrombin binding. Kinetic analyses reveal that HD1 produces a 2-fold reduction in the k(cat) for prothrombin activation by prothrombinase and a 6-fold increase in the K(m), highlighting the contribution of the factor Va-prothrombin interaction to prothrombin activation. As a high affinity, prothrombin exosite 1-directed ligand, HD1 inhibits prothrombin activation more efficiently than Hir(54-65)(SO(3)(-)). These findings suggest that exosite 1 on prothrombin exists as a proexosite only for ligands whose primary target is thrombin rather than prothrombin.     

Human placental anticoagulant protein: isolation and characterization

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.     

Improved Procedures for the Purification of Selected Vitamin K-Dependent Proteins

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4, 340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8, 200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XlVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIItm. Auto-III throtnbin Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin and, furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-11 Im was also not converted to the active enzymewhen the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The “activation peptide” released by RVV-X from the NH₂-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same “activation peptide” was isolated, but Auto-C was obtained instead of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.     

Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity

fgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-L-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent V(max) value of 6 mol/min/mol fgl2 and an apparent K(m) value for prothrombin of 8.3 microM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent V(max) value to 3670 mol/min/mol fgl2 and decreasing the apparent K(m) value for prothrombin to 7.2 microM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser(89) was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.     

Expression, isolation, and characterization of an active site (serine 528----alanine) mutant of recombinant bovine prothrombin

An active site mutant bovine prothrombin cDNA (Ser528----Ala) has been constructed, subcloned, and expressed in Chinese hamster ovary cells. The recombinant mutant prothrombin, expressed at the level of 1.5-2.0 micrograms/ml of cell medium, was fully carboxylated (9.9 +/- 0.4 mol of gamma-carboxyglutamic acid/mol of prothrombin). The mutant prothrombin could be activated to thrombin by Taipan snake venom and activated to meizothrombin by ecarin in a manner comparable to native bovine prothrombin or recombinant wild-type bovine prothrombin. The mutant meizothrombin thus formed was stable and did not autolyze. The initial rate of cleavage of mutant prothrombin catalyzed by the full prothrombinase was only 28% of the rate of cleavage of native prothrombin, while recombinant wild-type prothrombin was cleaved at the same rate as the native molecule. The mutant thrombin, obtained from the mutant prothrombin in situ by prothrombinase or Taipan snake venom activation, showed no enzymatic activity toward either fibrinogen or a synthetic chromogenic substrate, D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride (S2238). The mutant thrombin also bound dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, a specific fluorescent inhibitor of the thrombin active site, with a weaker binding affinity (kd = 5.4 x 10(-8) M) than did native thrombin (kd = 1.7 x 10(-8) M). These results indicate that the mutant recombinant prothrombin described here is a useful tool for the study of meizothrombin or thrombin without the complications arising from the proteolytic activities of these molecules. Study of the activation of this mutant has already revealed a functional link between the site of initial cleavage by the prothrombinase and the conformation at the nascent active site of prothrombin.     

Blood coagulation induced by the venom of Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator

In this paper, we show that the procoagulant action of Bothrops atrox venom is due in part to a protein component that activates prothrombin. The venom prothrombin activator was purified by ion-exchange chromatography and gel filtration. It was separated from a protease by affinity chromatography in a p-aminobenzamidine-CH-Sepharose column. It is a protein of about Mr 70,000, consisting of a single polypeptide chain. We have studied the kinetics of activation of prothrombin under different experimental conditions. The prothrombin activator from B. atrox venom is insensitive to reagents of serine and thiol proteases but is inactivated by ion chelators and by various divalent ions. These results suggest that it is a metalloenzyme. The prothrombin activator from B. atrox venom is inactive on the chromogenic substrates S-2337 and S-2238, and it is selective for prothrombin since it does not act on other blood coagulation factors such as fibrinogen and factor X. We have also studied the pattern of peptide cleavages produced in the human prothrombin molecule during the activation by the activator from B. atrox venom and compared it to that obtained with ecarin, a prothrombin activator from Echis carinatus venom. In the presence of thrombin inhibitors, e.g., hirudin, we found that the activators from B. atrox venom and ecarin act in a similar, or identical, manner by producing a thrombin intermediate, meizothrombin. In the absence of thrombin inhibitors, several peptides are generated, and alpha-thrombin is produced as a consequence of meizothrombin action.     

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.     

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.     

Interaction of short chain and long chain fatty acid phosphoglycerides and bile salts with prothrombin

An equimolar mixture of phosphatidylserine and (dioleoyl)phosphatidylethanolamine could substitute for brain cephalin preparations in the single stage prothrombin assay. However, no clot promoting activity was observed on the addition of any of the individual long chain fatty acid-containing phospholipids. Short chain fatty acid-containing phospholipids, such as diheptanoylphosphatidylcholine, diheptanoylphosphatidylethanolamine, diheptanoylphosphatidic acid, and dihexanoylphosphatidylcholine, or dihexanoylphosphatidylethanolamine were inhibitory under all conditions studied. Similar effects of these two general classes of phospholipids were observed in a two-stage thrombin generation system, in which a mixture of bovine Factor Xa, Factor Va, and Ca²⁺ were interacted with prothrombin.In the presence of 25 mM Ca²⁺, dioleoylphosphatidic acid or brain phosphatidylserine alone, and with other long chain phospholipids, formed complexes with bovine plasma prothrombin. On the other hand, dioleoyl-, diheptanoyl- or dihexanoylphosphatidylcholine under comparable conditions showed no binding to prothrombin. There appeared to be a small degree of binding of diheptanoylphosphatidic acid to prothrombin, but it was insufficient to cause any significant change in apparent molecular weight of prothrombin. A mixture of prothrombin, Factor V, diheptanoylphosphatidic acid/diheptanoylphosphatidylcholine and Ca²⁺ eluted in the void volume of Sephadex G-200, but showed a much reduced coagulant activity. Though a net negative charge on the phospholipid surface is required for phospholipid-protein interactions, this does not necessarily promote coagulant activity.Bile acids and bile salts, such as cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, lithocholic acid and dehydrocholic acid, exerted varying levels of stimulation on the prothrombin assay and thrombin generation system, but were not as effective as the phospholipids. Interestingly, no interaction of these bile acids or salts with prothrombin was noted in the presence of Ca²⁺. The results of these experiments suggest that negatively charged micelles per se are not sufficient for binding alone and that other chemical and physical characteristics of phospholipids are of prime importance.