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.     

Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation

The conversion of prothrombin into thrombin by Factor Xa requires the cleavage of two peptide bonds in prothrombin. Dependent on the order of cleavage, prethrombin 2 or meizothrombin occurs as intermediate. Since prethrombin 2 has as yet been the only observed intermediate, prothrombin activation is generally considered to proceed via prethrombin 2. In this paper we present new methods that allow differentiation between meizothrombin and thrombin formed during the initial phase of prothrombin activation. These methods, which make use of the different reactivities of meizothrombin and thrombin toward fibrinogen and antithrombin III plus heparin, enabled us to show the generation of considerable amounts of meizothrombin during Factor Xa-catalyzed prothrombin activation. Both meizothrombin and thrombin incorporated the active site-directed fluorescent chloromethyl ketone 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-CH2Cl. Gel electrophoretic analysis of chloromethyl ketone-treated aliquots of prothrombin activation mixtures confirmed meizothrombin formation. These observations demonstrate that prothrombin may also be converted into thrombin via meizothrombin.     

Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.     

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.     

Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan snake)

The prothrombin activator from the venom of Oxyuranus scutellatus (Taipan snake) was purified by gel filtration on Sephadex G-200 and ion-exchange chromatography on QAE-Sephadex. The activator is a large protein with a molecular weight of approximately 300,000, which is composed of subunits of Mr 110,000 and 80,000 and two disulfide-linked polypeptides of Mr 30,000. One or both of these Mr 30,000 subunits contain the active site. The venom activator readily converts Factor Xa-specific chromogenic substrates and is also able to activate prothrombin (Km = 166 microM, Vmax = 2.5 mumol of prothrombin activated per min/mg of venom). Gel electrophoretic analysis of prothrombin activation indicates that the venom activator randomly cleaves the Arg274-Thr275 and Arg323-Ile324 bonds of prothrombin since both thrombin and meizothrombin are formed as reaction products. Venom-catalyzed prothrombin activation is not affected by bovine Factor Va but is greatly stimulated by phospholipids plus Ca2+ ions. This stimulatory effect is explained by a decrease of the Km for prothrombin. In the presence of 50 microM phospholipid vesicles (25% phosphatidylserine/75% phosphatidylcholine; mole/mole), the Km is 0.34 microM and the Vmax is 7.1 mumol of prothrombin activated per min/mg of venom. The purified venom activator contains gamma-carboxyglutamic acid residues which presumably function in the interaction between the venom activator and phospholipids. Treatment of the activator with 0.8 M NaSCN strongly reduces its ability to activate prothrombin but has no effect on its amidolytic activity. The prothrombin-converting activity of the NaSCN-treated activator can be restored with bovine Factor Va. During prolonged gradient gel electrophoresis, the Mr 300,000 activator dissociates into smaller subunits. This causes a loss of the prothrombin-converting activity, while the amidolytic activity is recovered in a protein with an apparent molecular weight of 57,000. This protein can, however, rapidly activate prothrombin in the presence of Factor Va or in the presence of a protein component of Mr 220,000 that also migrates on the gel. These results suggest that the prothrombin activator from the O. scutellatus venom is a multimeric protein complex consisting of a Factor Xa-like enzyme and a Factor Va-like cofactor.     

Prothrombin activation by immobilized thrombin]

The ability of thrombin, immobilized on BrCN-activated Sepharose 4B, to split prothrombin, was studied. Immobilized thrombin retained up to 70% of its esterase activity and about 5% of its coagulating activity; it was also found to induce partial proteolysis of prothrombin. Two products of prothrombin degradation isolated, i.e. P1 (m. w. 50.000-52.000) and P2 (m. w. 22.000-24.000), did not show either the thrombin or the prothrombin activities. P1 was converted into thrombin under the action of tripsin or Factor Xa. The rate of conversion was considerably increased after addition of Factor V, thromboplastin and Ca2+ ions. Intravenous administration of P1 to rats resulted in changes in the coagulating system of blood, which may be probably indicative of the stimulation of the anticoagulating system. P2 possessed no thrombogenic activity.     

Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation

Regulatory exosite I of thrombin is present on prothrombin in a precursor state (proexosite I) that specifically binds the Tyr(63)-sulfated peptide, hirudin(54-65) (Hir(54-65)(SO(3)(-))) and the nonsulfated analog. The role of proexosite I in the mechanism of factor Va acceleration of prothrombin activation was investigated in kinetic studies of the effects of peptide binding. The initial rate of human prothrombin activation by factor Xa was inhibited by the peptides in the presence of factor Va but not in the absence of the cofactor. Factor Xa and factor Va did not bind the peptide with significant affinity compared with prothrombin. Maximum inhibition reduced the factor Va-accelerated rate to a level indistinguishable from the rate in the absence of the cofactor. The effect of Hir(54-65)(SO(3)(-)) on the kinetics of prothrombin activation obeyed a model in which binding of the peptide to proexosite I prevented productive prothrombin interactions with the factor Xa-factor Va complex. Comparison of human and bovine prothrombin as substrates demonstrated a similar correlation between peptide binding and inhibition of factor Va acceleration. Inhibition of prothrombin activation by hirudin peptides was opposed by assembly on phospholipid vesicles of the membrane-bound factor Xa-factor-Va-prothrombin complex. Factor Va interactions of human and bovine prothrombin activation are concluded to share a common mechanism in which proexosite I participates in productive interactions of prothrombin as the substrate of the factor Xa-factor Va complex, possibly by directly mediating productive prothrombin-factor Va binding.     

The action of factor Xa, thrombin and trypsin on human factor II

The proteolytic action of human and bovine Factor Xa, bovine thrombin and bovine pancreatic trypsin Factor II at pH 7.5 and 25°C was monitored by sodium dodecylsulfate gel electrophoresis and thrombin assays. Purified human and bovine Factor Xa, and trypsin, were found to activate Factor II to thrombin. The conversion of Factor II to thrombin by either Factor Xa or trypsin was found to proceed through two thrombogenic intermediates. The reaction pathway appears to be sequential in that the Factor II (75 000 daltons) is first cleaved to a 55 000-dalton thrombogenic product (Intermediate 1) and a 25 000-dalton non-thrombogenic product (Fragment 1). Intermediate 1 is subsequently converted to an inactive 37 000-dalton thrombogenic protein (Intermediate 2) and a 16 000-dalton protein (Fragment 2). Intermediate 2 is finally converted to an active 37 000-dalton thrombin (α-thrombin). Purified bovine thrombin readily converted Factor II to Intermediate 1 and Fragment 1, but possessed little capacity to catalyze subsequent cleavages to produce active thrombin. The ability of thrombin to cleave Factor II was entirely obviated in the presence of hirudin. Under the conditions of the incubation, the maximum thrombin yield obtainable by Factor Xa or trypsin activation was 50% when compared to the two-stage potential thrombin.     

Human thrombins. Production, evaluation, and properties of alpha-thrombin.

Human alpha-thrombin, the thromboplastin activation product of prothrombin with high clotting and esterase activity, was produced from Cohn Fraction III paste. The procedure started with 0.4 to 3.2 kg of frozen paste and was completed in 2 or 3 days. Some 23 g of thrombin were recorded for 65 quantitated preparations made from 11 lots of Fraction III paste. These preparations were obtained at protein concentrations of 3.9 +/- 1.3 mg/ml with a yield of 340 +/- 110 mg/kg of paste, which represented 48 +/- 14% of the clotting potential extracted as prothrombin. They had specific clotting activities of 2.8 +/- 0.4 U.S. (NIH) units/microng of protein and titrated to 88 +/- 8% active with p-nitrophenyl-p'-guanidinobenzoate (NPGB). Those (N - 29) examined by labeling with [14C]diisopropyl phosphorofluoridate (iPr2P-F) and electrophoresing in sodium dodecyl sulfate (SDS)-polyacrylamide gels were found to contain only (N = 4) or predominantly alpha-thrombin (97 +/- 3%) and corresponding amounts of ists degradation product, beta-thrombin (2.6 +/- 3.1%). No plasmin(ogen), prothrombin complex factors (II, VII, IX, IXalpha, X, Xalpha), or prothrombin fragments were detected in representative preparations. As produced in 0.75 M NaCl, pH approximately 6, thrombin was stable for approximately 1 week at 4 degrees and for greater than 1 year at less than or equal to 50 degrees; freeze-dried thrombin stored at 4 degrees for greater than 1 year displayed stable clotting activity and no vial to vial variation, permitting its use for reference purposes. Human thrombin generated by Taipan snake venom activation was compared with that produced by rapid thromboplastin activation: after treatment with [14C]iPr2P-F, greater than 95% of the label in both thrombins migrated at the same rate during electrophoresis in SDS; identical pairs of NH2-terminal residues were released in three consecutive Edman degradation cycles.     

Effect of phosphatidylserine in hemocoagulating action of factors V, VII and autoprothrombin C

It was found that phosphatidylserine inhibits the activating effect of factors V, VII and autoprothrombin C on the thromboplastin time of plasma. This is in agreement with the previously observed phosphatidylserine ability to form an inactive complex with the substrate of thrombinogenesis, i.e. prothrombin. The excess of autoprothrombin C results in changes in the kinetic parameters of the inhibiting effect of phosphatidylserine on the thrombinogenesis, which is indicative of the interaction between autoprothrombin C and phosphatidylserine. Data from gel-filtration on Sephadex G-100 and differential spectrophotometry support the evidence for the formation of the autoprothrombin C-phosphatidylserine complex. During ion-exchange chromatography and gel-filtration the autoprothrombin C preparations are eluted as an acute symmetrical peak; using electrophoresis in agarose gel, two fractions were revealed in the preparations. The first fraction migrates with gamma-globulins, whereas the second one--with alpha-globulins. The presence of phosphatidylserine in the autoprothrombin C solution does not change its migration rate during electrophoresis.     

The preparation of activated Factor X and its action on prothrombin

The preparation of activated Factor X from reaction mixtures of bovine Factor X and Russell's-viper venom is described. The molecular weight of purified protein varies about a mean value of 40000; this variation is the result of at least two forms of Factor Xa. The action of activated Factor X, together with purified Factor V, was studied on purified prothrombin and the reaction products were isolated. In addition to thrombin, two other polypeptides with molecular weights of 16000 and 19500 were recovered.     

On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides.

A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII by thrombin must be accompanied by a limited proteolysis of the arginyl-glycyl bond located in the N-terminal region of the a chain, liberating the "Activation peptide." The possibility of activating Factor XII with other porteinases was examined using Factor Xa [EC 3.4.21.6], Factor XIIa, kallikreins [EC 3.4.21.8], urokinase [EC 3.4.99.26], trypsin [EC 3.4.21.4], ficin [EC 3.4.22.3], papain [EC 3.4.22.2], and bromelain [EC 3.4.22.4]. Among these enzymes, only bromelain and trypsin showed clear activating effects.     

Characterization of the interaction between factor Xa and bovine aortic endothelial cells

Cultured bovine aortic endothelial cells incubated with Factor Xa activate prothrombin. Factor V, synthesized by the endothelial cells, or plasma Factor V and calcium are required for the reaction. In the present study, it has been demonstrated that 125I-Factor Xa binds specifically to endothelial cells. In addition, the activation of prothrombin by Factor Xa and aortic endothelial cells has been further characterized. The binding of 125I-Factor Xa to endothelial cells was saturable and reversible. The equilibrium dissociation constant (Kd) for 125I-Factor Xa binding was 3.6 X 10(-9) M, with 39000 molecules bound per cell. 125I-Factor Xa, inactivated by diisopropylfluorophosphate did not bind specifically to endothelial cells, indicating that the active site of Factor Xa was required for binding. Factor Xa, but not activated protein C, competed with 125I-Factor Xa for binding. Autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of cell lysates indicated that the radiolabeled material that bound to the cells had electrophoretic mobility identical to Factors Xa alpha and Xa beta. Although Factor X partially inhibited the binding of 125I-Factor Xa, Factor Xa did not inhibit the binding of 125I-Factor X, indicating that the zymogen and enzyme bound to different receptors. The relationship of the 125I-Factor Xa binding which was measured in these studies to aortic endothelial cell prothrombin activation is unclear since an anti-Factor V IgG blocked prothrombin activation but not Factor Xa binding. Additionally, 125I-Factor Xa binds to nonvascular cells; these cells do not activate prothrombin in the presence of Factor Xa. Moreover, the calcium requirements for each reaction and the saturation curves of 125I-Factor Xa binding and prothrombin activation differ. Although these data do not exclude a relationship between Factor Xa binding and prothrombin activation, the binding of 125I-Factor Xa to aortic endothelium measured in these studies may be related to a separate cellular function. To further characterize prothrombin activation by Factor Xa and endothelial cells, the rates of thrombin generation by intact bovine aorta or endothelial cells derived from this tissue were compared and were found to be equivalent. These data indicate that vascular endothelium may serve as a physiologic surface for hemostasis.     

Human prothrombin activation.

Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl sulfate electrophoretic analysis of activation products indicated that human prothrombin activation is similar to bovine prothrombin activation. Molecular weight analysis of human prothrombin and intermediated by sodium dodecyl sulfate co-electrophoresis with bovine prothrombin and its intermediates resulted in molecular weights of 70,000 for prothrombin, 51,000 for intermediate 1, 41,000 for intermediate 2, 23,000 for intermediate 3, and 13,000 for intermediate 4. Amino acid compositions of human prothrombin and intermediates are similar to those for bovine prothrombin and intermediates. NH2-terminal sequence studies of human prothrombin, intermediates, and alpha-thrombin A and B chains placed the intermediates in the parent human prothrombin molecule as described for the bovine system. Intermediate 3 is the NH2-terminal of prothrombin, and intermediate 1 is the COOH-terminal segment of the zymogen. Intermediate 4 is the NH2-terminal of intermediate 1. Intermediate 2', the immediate precursor of alpha-thrombin, is the COOH-terminal segment of intermediate 1. In general, a high degree of homology in the primary structure of prothrombin and intermediates was observed between the human and bovine system. The NH2-terminal sequences of human intermediate 2' and alpha-thrombin A chain are identical. However, human intermediate 2' isolated in a manner identical with that used for the isolation of bovine intermediate 2 is homologous with bovine intermediate 2, beginning with residue 14.     

The contribution of bovine Factor V and Factor Va to the activity of prothrombinase.

The rates of prothrombin activation under initial conditions of invariant concentrations of prothrombin and Factor Xa were studied in the presence of various combinations of Ca2+, homogeneous bovine Factor V, Factor Va, phosphatidylcholine-phosphatidylserine vesicles, and activated bovine platelets. Reactions were monitored continuously through the enhanced fluorescence accompanying the interaction of newly formed thrombin with dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide. The complete prothrombinase (Factor Xa, Ca2+, phospholipid, and Factor Va) behaved as a "typical" enzyme and catalyzed the activation of prothrombin with an apparent Vmax of 2100 mol of thrombin/min/mol of Factor Va or Factor Xa, whichever was the rate-limiting component. Regardless of whether the enzymatic complex was composed of Factor Xa, Ca2+, and plasma Factor Va plus phospholipid vesicles, or activated platelets in the place of the latter components, similar specific activity values were observed. The combination of Factor Va, Ca2+, and phospholipid enhanced the rate of the Factor Xa-catalyzed activation of prothrombin by a factor of 278,000. Factor Va itself when added to Factor Xa, Ca2+, and phospholipid, enhanced the rate of prothrombin activation by a factor of 13,000. Unactivated Factor V appears to possess 0.27% of the procoagulant activity of thrombin-activated Factor Va. From the kinetics of prothrombinase activity, an interaction between Factor Xa and both Factor V and Factor Va was observed, with apparent 1:1 stoichiometries and dissociation constants of 7.3 x 10(-10) M for Factor Va and 2.7 x 10(-9) M for Factor V. The present data, combined with data on the equilibrium binding of prothrombinase components to phospholipid, indicate that the model prothrombinase described in this paper consists of a phospholipid-bound, stoichiometric complex of Factor Va and Factor Xa, with bound Factor Va serving as the "binding site" for Factor Xa, in concert with its proposed role in platelets.     

Functional properties of an endothelial cell cofactor for thrombin-catalyzed activation of protein C

Thrombin-catalyzed activation of Protein C is accelerated by a human endothelial cell surface cofactor. The cofactor occurs also on mouse hemangioma cells (a transformed endothelial cell line), but not on cultured human smooth muscle cells or fibroblasts. The cofactor remains bound to the cell surface during Protein C activation. The cofactor is saturable with respect to both Protein C (Km = 0.72 +/- 0.07 microM) and thrombin (Km = 0.48 +/- 0.05 nM). Diisopropylphosphoryl-thrombin is a competitive inhibitor of the cofactor-dependent reaction with Ki = 0.56 +/- 0.1 nM. Prothrombin Fragment 1, the peptide derived from prothrombin that retains phospholipid binding capacity, does not inhibit activation of Protein C when present in a 7:1 molar excess over Protein C. Platelet Factor 4 (20 microgram/ml) also fails to inhibit Protein C activation. It is concluded that the endothelial cell provides a surface on which Protein C can be activated under physiological conditions.     

Effects of anticoagulant from Spirodela polyrhiza in rats

A fibrinolytic protease was purified from an Oriental medicinal herb, Spirodela polyrhiza (Choi, H. S., et al., Biosci. Biotechnol. Biochem., 65, 781-786 (2001)). The protease hydrolyzed not only fibrin but also fibrinogen. The enzyme had an anticoagulant activity measured with activated partial thromboplastin time, thrombin time, and prothrombin time in rat plasma. It doubled all three at 69, 29, and 221 nM, respectively. The protein had anticoagulant activity when given intravenously and orally. The maximum delay in the activated partial thromboplastin time was at the dose of 0.52 and 4.2 mg/kg for intravenous and oral administration, respectively. This protein may be useful in clinical applications for anticoagulation.     

A method for preparation of dry thrombin for topical application

Thrombin for topical hemostasis can be prepared from bovine or human blood plasma. The prothrombin is isolated by means of adsorption on DEAE-Sephadex A-50 and consecutively activated by CaCl₂ and thromboplastin. Thrombin is precipitated and purified by acetone. The specific activity of the thrombin preparation is 122 + 23 IU/mg protein while the yield is 36,360 ± 6623 IU/liter plasma.     

Molecular characteristics of bovine factor X activated in concentrated sodium citrate solution

Preparations of the zymogen form of bovine factor X were incubated in 25% $\text{w}\text{v}$ sodium citrate at room temperature. The rate of activation of factor X was dependent on the extent of contamination with factor VII, prothrombin, and thrombin. The activated factor X was isolated by DEAE-cellulose chromatography. Analysis of the final product by sedimentation velocity centrifugation coupled with measurements of the rate of boundary spreading, high-speed sedimentation equilibrium, and gel filtration chromatography provided evidence for a single molecular species undergoing reversible association-dissociation with a monomeric molecular weight of 48,000. In the absence of mercaptoethanol a single band was seen by disc electrophoresis and by SDS-acrylamide electrophoresis but after disulfide reduction two components of molecular weights 30,000 and 17,000 were visible. The protein contained large amounts of acidic amino acids but no carbohydrate. The N-terminal amino acids were alanine and isoleucine and 1 mole C-terminal arginine per mole protein was found. These characteristics are very similar to those of factor X activated with Russell's viper venom.When a BaSO₄ eluate of bovine plasma rich in prothrombin was allowed to stand in 25% sodium citrate both thrombin and activated factor X were generated. Chromatography of the isolated activated factor X on Sephadex G-200 as well as disc electrophoresis showed that it behaved identically with the enzyme obtained from purified zymogen and was clearly distinguishable from autoprothrombin c, a glycoprotein possessing qualitatively similar biological activity (Seegers, W. H., Cole, E. R., Harmison, C. R., and Marciniak, E. (1963) Can. J. Biochem. Physiol.41, 1047).     

Blood coagulation studies in normothermic,hibernating, and aroused Spermophilus franklini

The blood coagulation system of Spermophilus franklini was evaluated from normothermic, hibernating, and aroused individuals. Clotting time, thrombin time, prothrombin time, and partial thromboplastin time were measured to test the state of coagulability. The concentrations of the formed elements and the titers of five plasma factors were also determined.During hibernation, clotting time significantly increased above normothermic levels. Arousal resulted in clotting time returning toward normothermic values. Both thrombin time and partial thromboplastin time significantly increased above normothermic levels in blood from hibernators. The two tests exhibited normothermic levels in arousing individuals. Prothrombin time did not increase in blood from hibernating animals.Erythrocytes, leukocytes, and platelets were found to be significantly reduced in number in hibernating animals. Leukocyte and platelet numbers returned to normothermic levels during arousal.Factor VII, Factor X, prothrombin, and heparin concentrations did not significantly change from normothermic levels in hibernating individuals. Factor V, however, displayed a 45% decrease in concentration in hibernating individuals, with arousal resulting in near-normothermic levels. Aroused individuals displayed a doubling of prothrombin concentrations relative to normothermic individuals.