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At least 19 recordsLinked to original sources
PubMed1976

Substitution of lanthanide ions for calcium ions in the activation of bovine prothrombin by activated factor X. High affinity metal-binding sites of prothrombin and the derivatives of prothrombin activation.

The substitution of lanthanide ions for Ca(II) in the Ca(II)-binding sites of prothrombin and the derivatives of prothrombin activation and in the metal-dependent conversion of prothrombin or prethrombin 1 to thrombin was studied at pH 6.8. Gd(III), Tb(III), La(III), Dy(III), Pr(III), Sm(III), and Ce(III) may be substituted for Ca(II) in the generation of thrombin from prothrombin or prethrombin 1 by activated factor X. The rates of thrombin generation in the presence of optimal concentrations of Gd(III) were about 25% for prothrombin and prethrombin 1 compared to the rate of thrombin generation with optimal concentrations of Ca(II). Maximal rates of thrombin generation were observed at 20 muM Gd(III) using prothrombin as substrate, compared to 10 muM Gd(III) when prethrombin 1 was employed. Using the steady state rate-dialysis method, the high affinity metal-binding sites of prothrombin and the products formed during prothrombin activation were characterized using 153Gd(III). Prothrombin has two high affinity binding sites for Gd(III) (Kd = 0.75 muM). Prethrombin 1 and prethrombin 2 each bind one Gd(III) tightly (Kd = 1.10 muM and 0.81 muM, respectively). Fragment 1, the phospholipid-binding portion of prothrombin, has two sites which bind Gd(III) tightly (Kd 0.16 muM). Fragment 2 has no high affinity metal-binding sites, but has intermediate affinity metal-binding sites (Kd greater than 1.6 muM). Thrombin has numerous high affinity binding sites (Kd less than 0.1 muM), suggesting that the conversion of prethrombin 2 to thrombin is associated with a significant change in tertiary structure. These results indicate that Gd(III) binds tightly to the metal-binding sites of these proteins and can substitute for Ca(II) in metal-dependent prothrombin activation. In the activation of prothrombin by activated factor X, these data suggest that Ca(II) is required for metal-dependent factor V and phospholipid binding and not as a cofactor in enzyme catalysis.

Animals↗
PubMed1975

Purification of an apparent rat liver prothrombin precursor: characterization and comparison to normal rat prothrombin.

Current evidence would suggest that prothrombin is synthesized from a liver precursor molecule in a vitamin K dependent step which involves the attachment of calcium binding groups to the precursor. A protein has now been isolated from the liver of warfarin-treated rats which has the properties predicted for this precursor. The purified precursor is a glycoprotein with a molecular weight indistinguishable from rat prothrombin. Both electrophoretic and isofocusing analyses indicate that the precursor is less negatively charged than prothrombin. Specific proteolysis of the precursor by thrombin, taipan snake venom, or clotting factor Xa yielded fragments indistinguishable from those formed by similar proteolysis of prothrombin. The rate of activation of the precursor to thrombin by factor Xa and Ca-2+ was not stimulated by the addition of phospholipid, while prothrombin activation is greatly stimulated under these conditions. All of the data obtained are consistent with the hypothesis that the protein isolated is a precursor to prothrombin, and that under the influence of vitamin K, this precursor is converted to prothrombin by the addition of a number of acidic calcium binding groups.

Animals↗
PubMed1975

The influence of cadmium ions on the adsorption of prothrombin onto A1(OH)3 as a means to purify prothrombin.

1. In the presence of CdSO4(1mM),Al(OH)3(1.3% w/v) completely adsorbs the coagulation factors VII, IX, and X from normal plasma, but factor II (prothrombin) is adsorbed for about 50% only.2. A purification procedure for factor II is developed, using Al(OH)3 adsorption in the presence of Cd2+ as a first step and using column chromatography only once. A 750-fold purification is obtained at a 24% yield. 3. Comparison of the prothrombin thus obtained, with prothrombin isolated by the method of Kisiel and Hanahan (Biochim. Biophys. Acta (1973) 304, 103-113) does not show significant differences in amino acid composition, N-terminal amino acid, molecular weight or immunological properties. 4. Comparison of the two prothrombin preparations in a thrombin-generating system shows that although the final yield of thrombin from a given amount of prothrombin in both preparations is the same, the initial velocity of thrombin formation from our preparation is comparable to that of native prothrombin, whereas the other preparation is converted significantly slower.

Adsorption↗
PubMed1976

Effect of polylysine on the activation of prothrombin. Polylysine substitutes for calcium ions and factor V in the factor Xa catalyzed activation of prothrombin.

Polylysine has been demonstrated to dramatically accelerate the rate of the factor Xa catalyzed activation of both prothrombin and prethrombin 1. Under the present experimental conditions (pH 8.0, 23 C), no detectable activation of prothrombin or prethrombin 1 occurs with either factor Xa or polylysine alone. The activation of prethrombin 2, the direct precursor of alpha-thrombin, by factor Xa is not stimulated by polylysine. The activation of either prothrombin or prethrombin 1 by factor Xa in the presence of polylysine is partially inhibited by the presence of 5 mM CaCl2. Electrophoretic analysis in sodium dodecyl sulfate showed that the products that were formed in the above activation system comigrated with the reaction products derived from prothrombin activated by factor Xa in the presence of calcium ions and phospholipid. It is suggested that polylysine stimulates the factor Xa-catalyzes activation of prothrombin by replacing the combination of calcium ions and factor V.

Animals↗
PubMed1975

Plasma abnormal prothrombin and microsomal prothrombin precursor in various species (38492).

Abnormal, biologically inactive forms of prothrombin have previously been shown to appear in the plasma of cows or humans given coumarin anticoagulants. We have previously shown that a protein with similar properties increases in the liver of rats given these vitamin K antagonists, and have postulated that this protein represents the liver precursor to plasma prothrombin. Eight species, rats, mice, guinea pigs, hamsters, rabbits, calves, dogs, and chickens, have now been surveyed for both plasma abnormal prothrombin and liver precursor activity. Large amounts of plasma abnormal prothrombin were found in the bovine, substantial amounts were seen in the chick, and small amounts in rat and mouse plasma. With the exception of the bovine, all anticoagulant treated animals showed elevated levels of liver precursor activity in microsomal preparations. The relationship of these observations to the mechanism of action of vitamin K is discussed.

Animals↗
PubMed1975

Vitamin K and the biosynthesis of prothrombin. V. Gamma-carboxyglutamic acids, the vitamin K-dependent structures in prothrombin.

Tryptic peptides obtained from normal prothrombin have been compared with those obtained from prothrombin synthesized by cattle given the vitamin K antagonist dicumarol. Two peptides were found which contain vitamin K-dependent structures. These peptides contain residues 4 through 10 and residues 12 through 44, respectively. One of these (residues 4 through 10) has previously been shown to contain gamma-carboxyglutamic acid residues. Digestion of this peptide with aminopeptidase M and carboxypeptidase B yielded a tetrapeptide (residues 6 through 9). Mass spectra of this peptide showed that it has the structure Leu-Glu(CO2)-Glu(CO2)-Val. The structure of the peptide containing residues 12 through 44 was determined by automated degradation in a peptide sequenator. The modified glutamic acid residues were identified by mass spectrometric comparison with the thiohydantoin derivatives of synthetic gamma-carboxyglutamic acid. This approach unequivocally demonstrated that all of the first 10 glutamic acid residues in prothrombin are carboxylated to form gamma-carboxyglutamic acid residues. Evidence is also presented that indicates that these gamma-carboxyglutamic acid residues constitute the entire vitamin K-dependent modification of prothrombin.

Amino Acid Sequence↗
PubMed1978

The role of blood clotting factor V in the conversion of prothrombin and a decarboxy prothrombin into thrombin.

Purified PIVKA-II exhibits some factor II (prothrombin) activity in the one-stage coagulation assay and this factor II activity does not come from residual amounts of factor II but originates from PIVKA-II itself. It is shown that PIVKA-II is converted by a normal prothrombinase complex (factor Va and factor Xa adsorbed onto a phospholipid interface) more readily than by phospholipids and factor Xa alone. This suggests that binding between PIVKA-II and factor Va is an essential feature in the formation of the enzyme . substrate complex and from this we infer that a direct interaction between factor Va and prothrombin plays a rôle in the prothrombinase . prothrombin complex.

Animals↗
PubMed1979

Decarboxylation of bovine prothrombin fragment 1 and prothrombin.

Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.

1-Carboxyglutamic Acid↗
PubMed1978

Metal ion induced conformational transitions of prothrombin and prothrombin fragment 1.

Circular dichroism experiments indicate that prothrombin fragment 1 undergoes essentially the same secondary structural change whether in the presence of Ca(2+), Mg(2+), or Mn(2+). Titration with any of these metal ions results in a sigmoidal titration curve indicative of cooperative binding. Mg(2+) and Ca(2+) have nearly identical transition midpoints, while that for Mn(2+) is an order of magnitude less. These results correlate well with the results of previous metal ion intrinsic fluorescence quenching experiments. Fragment 1 has previously been shown to undergo a second transition corresponding to dimerization at high calcium concentrations. The present circular dichroism experiments show that this transition does not result in a gross alteration of secondary structure in the fragment 1 molecule. Studies with prothrombin, similar to those with fragment 1, indicate a similar metal ion dependent conformational change but of smaller magnitude. As apparently only the fragment 1 portion of the molecule undergoes the transition, it would appear that the covalently linked fragment 1 is constrained from attaining the same conformation as the purified entity. This suggests that caution must be used in interpreting the results of metal ion binding studies using fragment 1 as an analogue for prothrombin.

Animals↗
PubMed1979

Physical studies on isolated human prothrombin fragment-2. Comparisons with human prothrombin fragment-1.

Variation of pH strongly affects the fluorescence intensity of human prothrombin fragment-1 in a manner suggesting contributions from a number of protropic equilibria including groups with apparent pKa values near 3.0. These results suggest a structural role for pK1a of gamma-carboxyglutamic acid noieties. Added calcium ions (9 mM calcium chloride) quench the fluorescence titration curve uniformly above pH 4. Below pH 4, however, the titration curve in the presence of calcium ions suggests that calcium-ion-dependent processes leading to fluorescence quenching are pH-dependent. Upon back titration of human fragment-1, from pH 9, hysteresis is observed. Human prothrombin fragment-2 fluorescence titration curves are relatively broad at low pH suggesting the titration of normal carboxyl groups. The titration curves of fragment-2 are not affected by the presence of calcium ions, and hysteresis occurs upon back titration from low pH values. Circular dichroism (CD) Cotton effects appear at 232 nm and 280 nm and a trough appears at 203 nm in the CD spectrum of human prothrombin fragment-2. The Cotton effects in the region from 230 nm to 300 nm are sensitive to pH, ellipticity values at 232 nm increasing from approximately 300 at pH 2.5 to 1300 (degree-cm/decimole) at neutral pH and finally become negative at high pH values. In contrast to fragment-1, at neutral pH the fragment-2 Cotton effect at 232 nm is insensitive to the presence of 8 mM calcium chloride.

Calcium↗