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Biomedical subjects

R L Lundblad

Publications and source records attributed to R L Lundblad.

At least 73 records · Page 4Linked to original sources
PubMed1979

Correlation of in vivo and in vitro inhibition of thrombin by plasma inhibitors.

Rabbit antithrombin III and thrombin were purified to homogeneity to determine the in vivo relationship of these proteins in an autologous system. These proteins, radiolabeled with Na[125I], were injected into rabbits to determine the circulatory half-life. The mean half-life values were 125I-antithrombin III, 54.75 +/- 3.10 hr; 125I-thrombin, 7.25 +/- 1.49 hr; 125I-thrombin-antithrombin III, 7.25 +/- 1.09 hr; 125I[thrombin-antithrombin III], 11.13 +/- 0.88 hr; and Tos-Lys-CH2Cl-125I-thrombin, 27.75 +/- 3.18 hr. All the mean half-life values were statistically different from that of thrombin alone except for the two forms of thrombin-antithrombin III complex. Following injection of the radiolabeled proteins, plasma samples were obtained and gel-filtered to analyze the molecular weight distribution of the radiolabel. An identical elution position on gel filtration of 125I-antithrombin III with native antithrombin III was observed. The 125I-thrombin distributed into two peaks of radioactivity, with a molecular weight of 100,000 (79%) and a molecular weight greater than 200,000 (21%). The 100,000 dalton peak is consistent with a thrombin--antithrombin III complex, and the greater than 200,000 dalton peak is consistent with a thrombin-alpha 2-macroglobulin complex as confirmed by in vitro immunochemical studies. Thrombin inactivated with Tos-Lys-CH2Cl also showed two peaks of radioactivity on gel filtration, one peak which was excluded from the column and the other peak with an elution volume that was consistent with the position of native thrombin.

Antithrombin III↗
PubMed1978

On the preparation of bovine alpha-thrombin.

An improved method for the preparation of bovine alpha-thrombin is described. The procedure involves that activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2.200-2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strength and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of alpha-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to "aged" thrombin. In addition, the catalytic properties of alpha-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

Animals↗
PubMed1978

Thrombin binding to thrombasthenic platelets.

Platelets from two patients with Glanzmann's thrombasthenia showed decreased iodination of surface glycoproteins GPIIb and GPIII. Despite these changes, the binding of [125I] alpha-thrombin to the thrombasthenic platelets was normal. Binding was linear up to a thrombin concentration of 0.1 to 0.2 U/ml, at which point a change in the slope of the binding curve was observed. At lower concentrations of thrombin, 1,000 to 2,000 molecules of thrombin were bound per platelet, with an apparent Kdiss of 0.1 to 0.3 U/ml. With high concentrations of thrombin, thrombasthenic platelets bound 30,000 to 65,000 molecules of thrombin per platelet at saturation, with an apparent Kdiss of 5 to 10 U/ml. The release of [14C]serotonin by thrombasthenic platelets as a function of thrombin concentration was also similar to release by normal platelets. These studies indicate that the receptor(s) for thrombin on the plasma membrane of platelets from patients with Glanzmann's disease are intact and that membrane glycoproteins GPIIb and GPIII play little or no role in either the initial binding of thrombin to platelets or the transmission of this surface stimulus to release-inducing mechanisms.

Adult↗
PubMed1977

Structure-function relationships in the interaction of alpha-thrombin with blood platelets.

Highly purified alpha-thrombin has been chemically modified in an attempt to determine which features of the molecule are important for normal platelet-thrombin interactions. Modifying agents included diisopropylphosphorofluoridate and 1-chloro-3-tosylamido-7-amino-L-2-heptanone, which modify serine and histidine, respectively, at the catalytic site, as well as N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, which modify a single tryptophan at or near the fibrinogen-binding site. Active site-directed modification did not appreciably affect the binding characteristics, but prevented platelet activation. In contrast, modification of tryptophan at the macromolecular substrate-binding site resulted in the loss of high affinity binding of thrombin to platelets, while low affinity binding was apparently unaffected. This modification altered but did not abolish the ability of thrombin to effect platelet aggregation and release of [14C]serotonin. These results suggest that residues at the catalytic site are not involved in binding and that the macromolecular substrate-binding site of alpha-thrombin participates in high affinity binding to platelets. These data are also consistent with the existence of at least two types of binding sites for thrombin on the platelet surface as well as more than one platelet-binding region on the thrombin molecule.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1977

The modification of tryptophan in bovine thrombin.

2-Hydroxy-5-nitrobenzyl bromide, at a 100-fold molar excess, was observed to react withthrombin at pH 4.0 to give a modified enzyme which possessed 20% of the fibrinogen clotting activity and 80% of the esterase activity compared to a control preparation. Spectrophotometric analysis of the modified protein indicated that this effect on catalytic activity was associated with the incorporation of 1 mol of reagent per mol of thrombin. Amino acid analysis showed no loss of amino acids other than tryptophan. The reaction of N-bromosuccinimide with thrombin at 2-fold molar excess resulted in the modification of one tryptophan per mol of enzyme with the loss of 80% of the fibrinogen clotting activity with, as above, a considerably smaller loss of esterase activity. Oxidation of thrombin with N-bromosuccinimide decreased the extent of subsequent tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Thrombin modified with 2-hydroxy-5-nitrobenzyl bromide showed a 3-4 fold increase in Km and a decrease in V for the ester substrate. The reaction of thrombin with 2-acetoxy-5-nitrobenzyl bromide, a substrate analogue, also resulted in the inactivation of the enzyme. The data are interpreted to show the presence of a tryptophan residue at or near the enzyme's substrate binding site.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1977

Purification and partial characterization of deoxyribonuclease I from bovine parotid gland.

Deoxyribonuclease I has been purified from bovine parotid gland. The purification procedure utilizes an acid extraction of minced parotid gland, salt fractionation, gel filtration, and ion-exchange chromatography. The last step, chromatography on Sulfopropyl-Sephadex, resolves the enzymatic activity into several fractions. The major fraction, designated DNase A, was subjected to further investigation. This enzyme has, as expected, an alkaline pH optimum and an obligate requirement for divalent cations. The presence of calcium chloride protects DNase A from inactivation by proteolytic enzymes. Despite the previously described immunologic dissimilarity, there appears to be a large amount of homology between the parotid and pancreatic DNase's.

Amino Acids↗
PubMed1977

Prothrombin complex concentrates: potentially thrombogenic materials and clues to the mechanism of thrombosis in vivo.

Factors affecting the coagulant activity of two different prothrombin complex concentrates have been investigated using a sensitive in vitro assay developed in this laboratory. One concentrate contained substantial amounts of potentially thrombogenic material, while the other, which was deliberately fortified with antithrombin III and heparin during production, was judged to be relatively nonthrombogenic. The coagulant activity of the thrombogenic concentrate has been partially identified and was due largely to the presence of coagulation factos IXa and Xa. Neither concentrate contained detectable thrombin. However, after incubation with calcium or various polyamines, large amounts of additional coagulant material, including thrombin, appeared. Heparin and antithrombin III not only neutralized the thrombogenic materials present in the thrombogenic concentrate, but also inhibited the de novo generation of coagulant enzymes during incubation with calcium. The implication of these studies on the preparation of prothrombin complex concentrates and on host susceptibility to thrombosis during the clinical use of these concentrates is discussed.

Antithrombins↗
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↗
PubMed1976

The complete amino-acid sequence of rat thyrocalcitonin.

The complete amino acid sequence of rat thyrocalcitonin has been determined by automated Edman degradations of the intact molecule, a cyanogen bromide fragment, and by degradations of mixtures of peptides produced by hydrolysis of the hormone with trypsin and chymotrypsin. The sequence determined was H2N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Leu-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ser-Ile-Gly-Val-Gly-Ala-Pro-NH2. This sequence differs in only two positions from that found in the human hormone, i.e. leucine-16 in the rat vs phenylalanine-16 in the human, and serine-26 in the rat vs alanine-26 in the human. These similarities and differences are consistent with the previously reported immunological properties of the hormones isolated from these two species.

Amino Acid Sequence↗
PubMed1976

Characteristics of the association between prothrombin fragment 2 and alpha-thrombin.

The esterolytic activity of bovine alpha-thrombin on the synthetic substrate N-alpha-p-tosyl-L-arninine methyl ester (TosArgOMe) is stimulated when the prothrombin activation fragment, prothrombin fragment 2, is added as previously reported by this laboratory (Heldebrant, C. M., and Mann, K. G. (1973), J. Biol. Chem. 248, 3642). A similar stimulation of beta-thrombin is observed upon addition of prothrombin fragment 2. The binding constant of prothrombin fragment 2 to alpha-thrombin has been determined by the method of Gutfreund ((1972), Enzymes, Physical Principles, Wiley, New York, N.Y., pp 67-71). The dissociation constant is 7.7 X 10(-10)M, and there is one molecule of prothrombin fragment 2 bound per molecule of alpha-thrombin. Prethrombin-2 competes for prothrombin fragment 2, so the enhancement of the esterolytic activity of alpha-thrombin by prothrombin fragment 2 was used as a probe to determine the dissociation constant for the binding of prothrombin fragment 2 to prethrombin 2. The dissociation constant for this association is 1.3 X 10(-10)M. The kinetic parameters for the reaction of alpha-thrombin on TosArgOMe were determined in the absence and presence of prothrombin fragment 2 and are as follows: (a) in the absence of prothrombin fragment 2, Km(app) = 1.92 X 10(-4)M, and k3(app) = 35.8 mol of TosArgOMe/mol of alpha-thrombin s(-1); (b) in the presence of prothrombin fragment 2,Km(app = 1.76 X 10(-4)M, and k3(app) = 60.5 mol of TosArgOMe/mol of alpha-thrombin s(-1). Thus, the stimulatory effect of bovine prothrombin fragment 2 on bovine alpha-thrombin is reflected in k3(app) and not in Km(app). In contrast to the stimulatory effect of prothrombin fragment 2 on the thrombin-catalyzed hydrolysis of TosArgOMe, it inhibits the activity of alpha-thrombin toward N-alpha-benzoyl-L-arginine ethyl ester and N-alpha-benzoyl-L-arginine p-nitroanilide. The inhibition of activity toward these substrates by prothrombin fragment 2 is also reflected in k3(app). Activity toward the nonspecific substrate p-nitrophenyl butyrate was completely inhibited by the addition of prothrombin fragment 2. Prothrombin fragment 2 has no effect on the inhibition of alpha-thrombin activity by the active-site serine inhibitors diisopropyl phosphofluoridate, phenylmethanesulfonyl fluoride, or p-nitrophenyl guanidinobenzoate. Inhibition by the active-site-histidine-modifying inhibitor, N-alpha-p-tosyl-L-arginine chloromethyl ketone, was enhanced by the addition of prothrombin fragment 2. Soybean trypsin inhibitor reduces the stimulation by prothrombin fragment 2, but only at high molar ratios. Prothrombin fragment 2 has no effect on the clotting activity of alpha-thrombin, nor inhibition of this activity by heparin, hirudin, or diisopropyl phosphafluoridate. Bovine prothrombin fragment 2 enhances the esterolytic activity of both human and bovine alpha-thrombin, but human prothrombin fragment 2 does not enhance the esterolytic activity of either human or bovine alpha-thrombin.

Animals↗