Abnormal antithrombin III with defective serine protease binding (antithrombin III "Denver").

A hereditary (three family members) deficiency of antithrombin III (AT-III) in which AT-III antigen (AT-III ag) is normal in spite of low heparin cofactor and antithrombin activity is described. Plasma levels were: AT-III ag, 0.92-0.96 U/ml; AT-III heparin cofactor activity, 0.54-0.62 U/ml; progressive antithrombin activity index, 0.13-0.18; anti-Xa activity, 0.50-0.56 U/ml. Plasma crossed immunoelectrophoresis (CIE) patterns performed with and without added heparin were normal, but serum CIE revealed a decreased complex peak. Purification of the patient's plasma AT-III by heparin-sepharose affinity chromatography showed a normal protein recovery and elution profile, but the purified AT-III fraction showed only 50% of the normal progressive thrombin neutralization and anti-Xa activity. When thrombin-antithrombin (TAT) complexes were formed by incubating with excess thrombin, SDS-polyacrylamide gel electrophoresis (PAGE) analysis revealed that half the patient AT-III formed TAT complexes while the remainder migrated as free AT-III. All the control AT-III formed TAT complexes. The patient's nonreacting AT-III (AT-III "Denver"), isolated by affinity chromatography, showed CIE and SDS-PAGE migration patterns characteristic of normal AT-III but failed to bind thrombin or Xa. Calculations from turnover studies in one patient and normal subjects with autologous 131I-AT-III suggested that AT-III "Denver" is removed from the plasma slightly more rapidly than normal. These studies indicate that the patients' variant AT-III molecule was characterized by normal heparin interaction but defective binding and inhibition of thrombin and Xa. These characteristics allow isolation of the nonreactive variant molecule by heparin-sepharose affinity chromatography.

Statistical identification of compartmental models with application to plasma protein kinetics.

A numerical method for fitting linear compartmental models to data is presented which is similar to the method proposed by Jennrich and Bright (R. I. Jennrich and P. B. Bright, Technometrics 18, 385 (1976] and Feldman (H. A. Feldman. Amer. J. Physiol. 233, R1 (1977] but avoids some of the numerical difficulties. The method is direct and does not use numerical integration thereby avoiding time consuming and expensive calculations. Emphasis is on statistical procedures for model selection. In addition to the usual F tests for comparing two models, Akaike's Information Criterion (AIC) is introduced for choosing from among several models. The combined use AIC with an F test, chi-square test, or confidence intervals on estimated parameters gives a practical method to obtain a balance between underfitting and overfitting. Application to models of plasma protein kinetics illustrates the method.

Further studies of the turnover of dog antithrombin III. Study of 131I-labelled antithrombin protease complexes.

Fresh plasma containing 131I-antithrombin III (*I-AT) was coagulated and incubated at 37 degrees C for 2 hr. A "complex peak," separated on heparin-agarose contained AT and *I-AT antigen but no heparin cofactor activity. Crossed immunoelectrophoresis showed only AT complexes. SDS PAGE showed 80% of the *I-AT in a major band (approximately 80,000 daltons), 15% in a minor band (approximately 100,000 daltons) and the rest in trace bands (approximately 60,000 and/or 115,000 daltons). Ammonia treatment of the complex peak released alpha-thrombin. After i.v. injection 80% of the complexed *I-AT, chiefly as the major band, left the plasma with t 1/2 approximately 15 min and was almost immediately catabolized to low molecular weight breakdown products. A major catabolic site was the liver. A simple kinetic model describes the findings approximately.

Antithrombin III deficiency: decreased synthesis of a biochemically normal molecule.

A 29-yr-old white female has suffered from recurrent venous thromboses over the last 12 yr. Plasma antithrombin III (AT-III) levels were 48% of normal by immunoelectrophoresis and 56% by chromogenic assay. Three of four siblings and the father had similar AT-III levels without associated venous thromboses. Heparin-Sepharose chromatography demonstrated normal behavior of the patient's AT-III. Her purified AT-III could not be distinguished from AT-III purified from a normal control either by SDS polyacrylamide gel electrophoresis or by crossed immunoelectrophoresis, and the heparin cofactor activity and the progressive antithrombin activity of both AT-III samples were identical. Turnover studies were made in the patient using her own purified AT-III labeled with 131I, (*I). The results did not differ significantly from studies made with autologous *I-AT-III in two normal control women. Her fractional breakdown rate of 0.54 total plasma AT-III per day compared with 0.45 and 0.52 in the controls. These studies indicate that the patient synthesizes a normal AT-III molecule at half normal rates.

Biochemical and functional study of antithrombin III in newborn infants.

Antithrombin III (AT-III) was isolated by heparin affinity chromatography from adult venous and newborn term and preterm umbilical cord blood. The purified proteins were compared by SDS-PAGE, rocket immuno-electrophoresis, protein concentration by microbiuret relative to optical density at 280 nm, heparin cofactor specific activity, progressive neutralization of thrombin and factor Xa at 37 degree C and pH related antithrombin kinetics. The structural evaluations revealed a fetal AT-III of molecular weight, charge and electrophoretic migration indistinguishable from adult AT-III. The functional studies showed that, on an equimolar basis, the rates of thrombin and Xa interactions with fetal AT-III were as rapid as those with adult AT-III. The catalytic rates of various concentrations of heparin were also equal. The newborn infant, therefore, displays a quantitative but not quantitative deficiency of AT-III.

Experimental studies of the fibrinogen response to hemorrhage.

Rabbits were bled 20 ml/kg body wt over approximately 1 h, and the changes in hematocrit, plasma total protein, and fibrinogen concentrations, red cell and plasma volumes, and masses of total protein and fibrinogen in the plasma during and for 4 days after the hemorrhage were monitored. Ten percent of plasma fibrinogen was transferred from the interstitial fluids during the hemorrhage, and total plasma fibrinogen was raised to 140% of initial levels by 1 day later. 131I-labeled fibrinogen was given intravenously 1 day before a 20 ml/kg hemorrhage, and simulation methods described in the APPENDIX were used to define changes in the posthemorrhage fractional rates: transcapillary transfer rate (j1), lymphatic return rate (j2), and catabolic rate of fibrinogen (j3): j1 was unchanged but j2 increased and j3 briefly declined. In the day after hemorrhage fibrinogen synthetic rate increased approximately threefold to restore plasma fibrinogen concentrations to initial levels, but then returned to normal or subnormal levels. The alterations in j2 and j3 save about 20% of the synthetic work that would otherwise be required to restore plasma fibrinogen levels to normal.

Atypical drusen in pseudoxanthoma elasticum.

Forty-three patients with pseudoxanthoma elasticum were evaluated ophthalmologically. The fundus appearance was characterized by angioid streaks and mottling of the retinal pigment epithelium. Rock-like atypical drusen were present in the posterior pole or peripheral retina in 75% of the patients examined and were an integral part of this fundus picture. These drusen changed little with time and were seen in early stages of pseudoxanthoma elasticum. The histopathology of these drusen is unknown.

Effects of prostaglandin E2, analogs, fatty acids, and indomethacin on fibrinogen level.

Dose-response relations in rabbits for 3-h intravenous infusion of prostaglandin E2 (PGE2) and (15S)-15-methyl-1-prostaglandin E2 methyl ester (MePGE2) on plasma fibrinogen and systolic blood pressure were determined and described by regression equations. MePGE2 was 20 times more active than PGE2. Fibrinogen synthetic rate responses to PGE2 and MePGE2 were estimated. Infusion of the PGE2 precursor, arachidonic acid, elevated plasma fibrinogen, but fibrinogen response to 0.5-9 mg/kg arachidonic acid was unrelated to dose and half that given by 3 mg/kg PGE2. Slow infusion of several other fatty acids raised plasma fibrinogen as effectively as arachidonic acid, but prostaglandins D2 and F2alpha had only a slight effect. Infusion of 30 times the indomethacin dose that blocks platelet prostaglandin synthetase did not alter the plasma fibrinogen response to arachidonic acid. Indomethacin did not inhibit plasma fibrinogen elevations following ACTH or endotoxin infusion, or subcutaneous turpentine injection. Intravenous infusion of two cyclic ether prostaglandin endoperoxide analogs, (15S)-hydroxy-9alpha, 11alpha-(epoxymethano) prosta-5Z, 13E-dienoic acid, and (15S)-hydroxy-11alpha, 9alpha(epoxymethano) prosta-5Z, 13E-dienoic acid, failed to increase plasma fibrinogen.

Laser treatment in maculopathy of pseudoxanthoma elasticum.

We report a case of Pseudoxanthoma Elasticum with exudative retinopathy which was treated with the Argon laser and in which the visual acuity was preserved and the subretinal neovascular membrane obliterated for ten months. Laser treatment seems to have helped this case although the same treatment has stimulated the development of subretinal neovascular membranes. These cases must be treated cautiously.

Resolution and repair of elastic tissue calcification in pseudoxanthoma elasticum.

Biopsy specimens of a skin lesion from a patient with pseudoxanthoma elasticum were obtained at the beginning and end of a 1 1/2-year period of clinically documented regression of the lesion. Light microscopic studies of these specimens showed that, during the period of regression, there was a decrease in the number of abnormal calcified elastic fibers in the dermis. Electron microscopic findings were interpreted as indicating breakdown and removal of mineralized elastic fibers at the beginning and formation of new elastic fibers at the end of the regression period. Cytoplasmic processes of active fibroblasts were intimately associated with fibers undergoing dissolution. The case thus represents a clinically and morphologically documented example of resolution and repair of elastic tissue calcification.

Neuropsychological findings with pseudoxanthoma elasticum.

It has been reported that the disease pseudoxanthoma elasticum (PXE) is associated with a high incidence of neurologic and psychiatric symptoms, which are possibly due to cerebrovascular ischemia. These reports are based mainly upon clinical observations made without reference to control group data. We have compared the results of 27 PXE patients with results of a control group on a battery of objective neuropsychological and personality tests. The PXE group showed only very mild deficits on extensive neuropsychological testing, a finding which argues against any marked involvement of cerebral vessels in these patients. Similarly, the personality test results did not reveal significant psychiatric disturbances which could be attributed to PXE. Possible reasons for the descrepancy with previous reports include the subjective data and much older PXE patients on which those reports are based. The need for systematic neuropsychological research with older PXE patients and control groups is suggested.

Fibrinogen synthesis stimulation by prostaglandin E1 and some other vasodilators.

Three-hour constant-rate intravenous infusion into rabbits of 1-3 mg prostaglandin E1 (PGE1) per kilogram markedly increased plasma fibrinogen 24 h later. 131I-labeled fibrinogen and model studies showed increased synthesis underlay this. Similar PGE1 doses lowered systolic blood pressure. Maintaining systolic blood pressure by infusing noradrenaline with the PGE1 did not alter plasma fibrinogen response to PGE1; plasma fibrinogen was unchanged by noradrenaline infusion. Regression equations relating plasma fibrinogen increment to PGE1 dose, plasma fibrinogen increment to dose and systolic blood pressure change, and systolic blood pressure change to dose are given as well as the constants relating plasma fibrinogen increment to dose using the Michaelis-Menten equation. Infusions of cyclic AMP, dibutyryl cyclic AMP, and cyclic GMP intravenously led to only small plasma fibrinogen increases. Daily intravenous infusions of PGE1 led to loss of both plasma fibrinogen and systolic blood pressure responses in two animals; a third animal showed only loss of the former and a fourth only loss of the latter response. PGE1 slightly enhanced the small plasma fibrinogen increase following intravenous bradykinin. Approximate arterial blood PGE1 concentrations resulting from the intravenous infusion of 1 mg mg PGE1 kg-1 3 h-1 are calculated. These are compared with measured values.

Some factors influencing metabolism and distribution of fibrinogen in man and rabbits.

To shed some light on the homeostatic regulation of plasma fibrinogen, metabolic studies were made in healthy females, and in normal, thyroidectomized, and thyroxine-treated rabbits. In females, compared with normal males, plasma fibrinogen concentration, plasma and interstitial fibrinogen decreased consequent to an increased fractional catabolic rate and a normal fibrinogen synthesis rate. The interstitial/plasma fibrinogen ratio remained unchanged. In normal rabbits, with increasing body weight fractional catabolic rate and catabolic rate decreased, while fibrinogen concentration and plasma fibrinogen remained constant owing to a simultaneous decrease in fibrinogen synthesis. In addition, fractional transcapillary transfer rate and transcapillary flux also decreased resulting in a shrinkage of interstitial fibrinogen. Thyroidectomy and thyroxine-injection markedly altered fibrinogen metabolism: thyroid hormone accelerated fibrinogen catabolism but also stimulated synthesis. The net result was an increase in plasma fibrinogen and fibrinogen concentration. The interstitial/plasma fibrinogen ratio decreased in thyroxine-treated, and increased in thyroidectomized animals. This study defines the variations of the fibrinogen system parameters in these physiologic and pathologic conditions, and illustrates some patterns of alterations in fibrinogen metabolism.

A micromethod for the quantitative analysis of calcium, magnesium, and phosphorus in human skin.

An inexpensive and simple micromethod is described for determining calcium, magnesium, and phosphorus in a 4 mm. punch biopsy of human skin. This small biopsy requires no sutures, and can be repeated frequently with minimal trauma to patients in long-term studies. Normal ranges of calcium (125 to 300 mg. per kilogram of dry defatted weight (DDW), phosphates (450 to 980 mg. per kilogram of DDW), and magnesium (75 to 112 mg. per kilogram of DDW) are similar to reported results on much larger biopsies. Skin calcium but not skin magnesium or phosphate was significantly higher in 40- to 76-year-old subjects when compared to the 20- to 39-year-old individuals. Comparison of an acid extraction method with the standard ashing procedure showed no significant differences in calcium and magnesium content, but the acid extractable phosphate was significantly lower than the total phosphorus found in the ash.