Antithrombin acts as a negative acute phase protein as established with studies on HepG2 cells and in baboons.

Patients with sepsis or after major surgery have decreased plasma levels of the anticoagulant protein antithrombin. In such patients elevated levels of interleukin-6 (IL-6) are present and this interleukin is known to induce positive and negative acute phase responses. To investigate the possibility that antithrombin acts as a negative acute phase response-protein we performed studies on the human hepatoma cell line HepG2 in vitro and baboons in vivo. HepG2 cells were treated with recombinant human IL-6, IL-1beta, or combinations of the latter two, and tested for production of antithrombin, fibrinogen and prealbumin (transthyretin). This treatment resulted in a dose dependent increase in fibrinogen concentration (with a maximum effect of 2.8-2.9-fold) and a dose dependent decrease in prealbumin (with a maximum effect of 0.6-0.7-fold) and antithrombin concentrations (with a maximum effect of 0.6-0.8-fold). Simultaneous treatment of the HepG2 cells with IL-6 (1,000 pg/ml or 2,500 pg/ml) and IL-1beta (25 pg/ml), provided more extensively decreased prealbumin (0.8 and 0.6-fold, respectively) and antithrombin concentration (0.7 and 0.6-fold, respectively) compared to the single interleukin treatment at these concentrations. Baboons treated with 2 microg IL-6 x kg body-weight(-1) x day(-1) showed increased plasma CRP levels (59-fold, p <0.05) and decreased prealbumin (0.9-fold, p <0.05) and antithrombin (0.8-fold, p <0.05) plasma levels, without evidence for coagulation activation. Our results indicate that antithrombin acts as a negative acute phase protein, which may contribute to the decreased antithrombin plasma levels observed after major surgery or in sepsis.

Fetal and neonatal development of antithrombin III plasma activity and liver messenger RNA levels in sheep.

In healthy term human newborns a unique hemostatic balance exists with reduced plasma concentrations of several coagulant and anticoagulant proteins, including antithrombin III (AT III). In preterm newborns even lower AT III concentrations are observed, with an associated thromboembolic risk. As part of our study program on the gene regulation of AT III, we investigated whether the increase in plasma AT III activity during fetal and neonatal development is particularly controlled at the transcriptional level. Plasma AT III activity and liver AT III mRNA content between the 8th wk of gestation and the 4th wk after birth were determined in sheep. AT III activity gradually increased from 34% of the mean adult level at 8-10 wk of gestation to 86% (2.5-fold) at term (21 wk), and remained in the adult range after birth. The mean body weight, and thus plasma volume, increased 57-fold. Therefore, the total plasma AT III activity increased 140-fold. The total liver AT III mRNA content increased only 14-fold between these fetal stages, mainly due to increased liver weight. Therefore, the total plasma AT III activity increased 10-fold more than the liver AT III mRNA content. In the neonatal period between d 1-3 and 28, the total plasma AT III activity increased only 2-fold more than the liver AT III mRNA content. We conclude that the increase in plasma AT III activity during the fetal period, and similarly the neonatal period, is not regulated at the transcriptional level. Furthermore, a unique fetal isoform of AT III was detected in sheep. This isoform had a 2500-D higher molecular mass compared with the other fetal, neonatal, and adult AT III isoform, and disappeared from the circulation between d 2 and 7 after birth. These AT III isoforms differ in their carbohydrate moiety.

Inflammatory bowel disease is associated with increased mucosal levels of bactericidal/permeability-increasing protein.

BACKGROUND & AIMS: Clinical sepsis seldom accompanies inflammatory bowel disease. The aim of this study was to measure colonic mucosal levels of the neutrophil product bactericidal/permeability-increasing protein (BPI), which kills gram-negative bacteria in addition to inactivating endotoxin. METHODS: Enzyme-linked immunosorbent assay and immunohistochemistry for BPI were performed on homogenates and tissue secretions of biopsy specimens from patients with ulcerative colitis (n=11) and Crohn's disease (n=5) and from normal controls (n=5). RESULTS: Mucosal neutrophil content (144 +/- 23 vs. 35 +/- 9 neutrophils/mg protein; P<0.007) and BPI content (2.07 +/- 0.75 vs. 0.12 +/- 0.02 ng/mg protein; P<0.002) were greater in the colitis groups and correlated closely (r=0.68; P<0.001). This relationship held for both ulcerative colitis (P<0.002) and Crohn's disease (P<0.01) with a trend towards greater levels in Crohn's disease. There was a trend towards higher BPI levels with an increasing endoscopic inflammation score (grade I, 1.32 +/- 0.6 ng/mg protein; grade II, 2.82 +/- 1.4 ng/mg protein). Immunohistochemistry and the biopsy culture showed BPI to be both intracellular and extracellular, to be present in the crypt lumen, and to be released into incubating medium. CONCLUSIONS: Mucosal levels of BPI are increased in colitis. Such localization may ameliorate mucosal responses to gram-negative bacteria and their products.

The influence of insulin, beta-estradiol, dexamethasone and thyroid hormone on the secretion of coagulant and anticoagulant proteins by HepG2 cells.

As a basis for regulatory studies on the influence of hormones on (anti)coagulant protein production by hepatocytes, we examined the amounts of the plasma proteins antithrombin III (AT III), protein C, protein S, factor II, factor X, fibrinogen, and prealbumin produced by the hepatoma cell line HepG2, into the culture medium, in the absence and presence of insulin, beta-estradiol, dexamethasone and thyroid hormone. Without hormones these cells produced large amounts of fibrinogen (2,452 +/- 501 ng/mg cell protein), AT III (447 +/- 16 ng/mg cell protein) and factor II (464 +/- 31 ng/mg cell protein) and only small amounts of protein C (50 +/- 7 ng/mg cell protein) and factor X (55 +/- 5 ng/mg cell protein). Thyroid hormone had a slight but significant effect on the enrichment in the culture medium of the anticoagulant protein AT III (1.34-fold) but not on protein C (0.96-fold) and protein S (0.91-fold). This hormone also significantly increased the amounts of the coagulant proteins factor II (1.28-fold), factor X (1.45-fold) and fibrinogen (2.17-fold). Insulin had an overall stimulating effect on the amounts of all the proteins that were investigated. Neither dexamethasone nor beta-estradiol administration did substantially change the amounts of these proteins. We conclude that the HepG2 cell is a useful tool to study the hormonal regulation of the production of (anti)coagulant proteins. We studied the overall process of protein production, i.e., the amounts of proteins produced into the culture medium. Detailed studies have to be performed to establish the specific hormonal effects on the underlying processes, e.g., transcription, translation, cellular processing and transport, and secretion.

South African founder mutations in the low-density lipoprotein receptor gene causing familial hypercholesterolemia in the Dutch population.

In South African Afrikaners, three point mutations in the gene coding for the low-density lipoprotein (LDL)-receptor are responsible for more than 95% of the cases of familial hypercholesterolemia (FH). To investigate whether one or more of these mutations originated in The Netherlands, a large group of Dutch heterozygous FH patients was screened for the presence of these three mutations. Of these, a missense mutation in exon 9 of the LDL-receptor gene, resulting in a substitution of Met for Val408, responsible for 15% of FH in Afrikaners, was found in 19 (1.5%) of 1268 FH patients of Dutch descent. Nine of the patients carrying the exon 9 mutation on one allele shared the LDL-receptor DNA haplotype with an FH patient from South Africa, who was homozygous for the same mutation. This would suggest that the mutation in these patients and in the South African patient have a common ancestral background. The remaining ten FH patients all shared a common haplotype, partly identical to the Afrikaner haplotype, which could have arisen from a single recombinational event. This mutation has not been described in persons other than of Dutch ancestry and supports the hypothesis that this mutation in exon 9 originated in The Netherlands and, in all likelihood, was introduced into South Africa by early Dutch settlers in the seventeenth century.

Analysis of the Afrikaner mutation in exon 9 of the low-density lipoprotein receptor gene in a large Dutch kindred suffering from familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is the most common genetic metabolic disorder, affecting about 1 in 500 persons in the general population. With novel techniques, it is possible to identify the molecular defects underlying FH in the gene coding for the low-density lipoprotein (LDL) receptor, thereby confirming the diagnosis of FH. In this study we present a large family with a specific mutation in exon 9 of the LDL-receptor gene (an Afrikaner mutation) and we demonstrate that by a large-scale case-finding study in this family, carriers of such a mutation can be detected. Of 63 family members, 13 were shown to be at risk for cardiovascular disease as judged by their lipoprotein profile or the presence of the Afrikaner mutation. Two persons were detected, affected with a dyslipidaemia other than FH. Medical management was initiated in order to reduce the high cardiovascular risk associated with this disorder.

Laboratory and clinical evaluation of an assay of thrombin-antithrombin III complexes in plasma.

We evaluated a recently developed commercial assay for quantifying thrombin-antithrombin III (TAT) complexes in human plasma. The assay is precise (within-assay CV less than 10%, between-assay CV less than 13%), and sensitive (detection limit 0.7 micrograms of TAT per liter of plasma). Measurements for healthy volunteers yielded a normal reference (95 percentile) interval of 0.8 to 5.0 micrograms/L (n = 50, mean 2.1 micrograms/L, range 1.1 to 7.5 micrograms/L). TAT concentrations were increased in 25 of the 41 patients who fulfilled the clinical criteria of disseminated intravascular coagulation (DIC, overall mean 15.8 micrograms/L) and in 30 of the 35 patients with deep-vein thrombosis of the leg (overall mean 9.4 micrograms/L). We assessed the accuracy of the TAT assay by comparison with established criteria for the laboratory diagnosis of DIC involving various cutoff values for antithrombin III, factor V, fibrinogen, platelet count, fibrin/fibrinogen degradation products, and activated partial thromboplastin time. The low specificity of the TAT assay with regard to some of these criteria indicates that the latter are probably insensitive.

Acquired alpha-2-antiplasmin deficiency in acute promyelocytic leukaemia.

We report a prospective study in nine consecutive adult patients with acute promyelocytic leukaemia (APL). The study objective was to assess the prevalence of activation of blood coagulation and/or activation of fibrinolysis in APL. Coagulation and fibrinolytic parameters relevant to the objective included antithrombin III, plasminogen, fibrin/fibrinogen degradation products and alpha-2 antiplasmin activity and antigen levels. The results of this study revealed consistently normal antithrombin III levels, both before and in the course of antileukaemic treatment. Plasminogen levels were slightly decreased or normal. However, a distinct alpha-2 antiplasmin activity deficiency in all patients was observed with levels even reaching zero in three patients, during chemotherapy. Alpha-2 antiplasmin activity levels were consistently lower than the alpha-2 antiplasmin antigen levels. The in vitro binding of alpha-2 antiplasmin activity to fibrin clots was severely reduced which appeared to be due to the reduced alpha-2 antiplasmin plasma levels. Upon crossed-immunoelectrophoresis against alpha-2 antiplasmin antiserum two alpha-2 antiplasmin antigen peaks were observed in the plasma of all nine patients. All abnormalities were reversible 4 d after completion of chemotherapy. In a second series of 12 consecutive APL patients we confirmed the consistency of the alpha-2 antiplasmin activity deficiency and normal antithrombin III plasma levels. In addition Protein C activity and antigen levels were normal or near normal in 10 and reduced in two patients. Thrombin-antithrombin III complexes were increased in 10 and normal in two patients. We conclude that some activation of blood coagulation is present in APL (increased thrombin-antithrombin III complex levels) but its contribution to the coagulopathy seems to be minor (normal antithrombin III and only slightly reduced protein C levels). The observed reduced alpha-2 antiplasmin content of the fibrin clot in vitro may result in vivo in a fibrin clot that is highly susceptible to fibrin degradation, thus aggravating the coagulopathy in APL.

Alpha 2-antiplasmin: functional characterization and metabolism in a heterozygote deficient patient.

An 81-year-old male with a mild life-long bleeding history and an alpha 2-antiplasmin (alpha 2-AP) plasma level of 55% biological activity and 41% antigen activity (normal range 80-140%) was studied. The ratio of plasminogen binding (PB):non-plasminogen binding (NPB) alpha 2-AP assayed by modified crossed immunoelectrophoresis (CIE) was 7.3/2.7 (controls 6.3 +/- 0.49 SD/3.7 +/- 0.49 SD). The patient's alpha 2-AP showed decreased affinity for fibrin, i.e. 8.3% versus 32.4% of normal control alpha 2-AP associated with fibrin during clotting of plasma. A metabolic study performed with human purified 125I-alpha 2-AP (PB/NPB 7.7/2.3) showed a plasma radioactivity disappearance half-life of 72.9 h (n 60.1 +/- 5.3 h) with a normal fractional catabolic rate and a reduced absolute catabolic (synthetic) rate of 0.70 mg/kg/day (n 2.10 +/- 0.60 mg/kg/day). The exchange between the central and third compartment was increased. The increased alpha 2-AP PB form and the increased plasma radioactivity disappearance half-life are suggestive of a slower conversion of the PB form into the NPB form and/or slower degradation of the PB form. The bleeding tendency in this patient could be explained by decreased synthesis of alpha 2-AP and decreased binding to fibrin.

Turnover of alpha-2-antiplasmin in patients with acute promyelocytic leukemia.

Patients with acute promyelocytic leukemia have a bleeding disorder which was thus far explained by a coagulopathy based on diffuse intravascular coagulation (DIC). We observed severe alpha-2-antiplasmin deficiency in a consecutive series of such patients. We postulate proteolysis rather than DIC, also because of increased turnover of 125I-alpha-2-antiplasmin, to be responsible for the coagulopathy. Alpha-2-antiplasmin deficiency may well contribute to the bleeding disorder in acute promyelocytic leukemia.

Automated amidolytic method for determining heparin, a heparinoid, and a low-Mr heparin fragment, based on their anti-Xa activity.

Using the chromogenic substrate S-2222, we have optimized and automated an amidolytic assay for heparin. The assay is based on the detection of anti-Xa activity generated by heparin in plasma. The method is reproducible (intra- and interassay CVs of 2.4 and 3.3%, respectively) and reliable in antithrombin III-deficient plasma. Results of this assay, obtained for plasma samples from patients and volunteers treated with heparin, correlate well (r = 0.899) with those of the test for activated partial thromboplastin time. Upon administration of a low-Mr heparinoid (Org 10172) and heparin fragment ( Kabi 2165), however, the activated partial thromboplastin time failed to detect anticoagulant activity, whereas the chromogenic heparin assay revealed anti-Xa activity. This automated amidolytic assay for heparin is therefore suitable not only for monitoring standard therapy with heparin but also for measuring the activity of recently developed heparin fractions.

Antithrombin III in oral contraceptive users and during normotensive pregnancy.

Plasma antithrombin III (AT III) was determined in four groups of subjects, by employing an automated chromogenic technique. In 25 women, discontinuing oral contraceptives led to a 9% elevation of AT III, while in 13 women AT III levels fell by 9% after starting with the pill. In 77 normotensive pregnant patients AT III levels were normal during the third trimester and did not differ from control values 6-8 weeks after delivery. Women taking the pill at that time did not have lower AT III levels than those who did not. Furthermore, AT III levels in 414 oral contraceptive users were the same as in 572 controls, when random samples were taken during pill cycle and menstrual cycle. It is concluded that although synthetic estrogens do cause a decrease in AT III levels, this decrease is probably the result of estrogen-induced hemodilution, which may also occur during the normal menstrual cycle. If low dose pills are thrombogenic, mass screening for AT III deficiency will not identify those at risk, with the exception of the rare cases of hereditary AT III deficiency.

PIP: Plasma antithrombin 3 (AT3) was determined in 4 groups of subjects, by employing an automated chromogenic technique. In 25 women, discontinuing oral contraceptives (OCs) led to a 9% elevation of AT3, while in 13 women, AT3 levels fell by 9% after introduction of OCs. In 77 normotensive pregnant patients, AT3 levels were normal during the 3rd trimester and did not differ from control values 6-8 weeks after delivery. Women taking the pill at that time did not have lower AT3 levels than those who did not. Furthermore, AT3 levels in 414 OC users were the same as in 527 controls, when random samples were taken during the pill cycle and menstrual cycle. It is concluded that although synthetic estrogens do cause a decrease in AT3 levels, this decrease is probably the result of estrogen-induced hemodilution, which may also occur during the normal menstrual cycle. If low dose pills are thrombogenic, mass screening for AT3 deficiency will not identify those at risk, with the exception of rare cases of hereditary AT3 deficiency.

"Morning-after pill" and antithrombin III.

Plasma antithrombin III (AT III) was studied longitudinally in 15 subjects (13 patients and 2 volunteers), who used the "morning-after pill" (5 mg ethinylestradiol daily for 5 days). The mean decrease in AT III level in the 13 patients was 17% of the pre-treatment value. From additional observations made in 2 of the patients and in the 2 volunteers it is concluded that this decrease is caused by hemodilution due to salt and water retention rather than by a decreased synthesis or increased consumption.

"Morning after pill" and antithrombin III.

PIP: The increased risk of thromboembolism in women using estrogen-containing (OCs) oral contraceptives has been related to decreased (AT3) antithrombin 3 levels of about 10%. A dose-dependent effect on AT3 has been suggested. Using an automated chromogenic technique, we have studied the effect on AT3 of a very high dose of ethinyl estradiol (5 mg daily for 5 days), popularly known as the "morning after pill," which in the Netherlands is prescribed to 45,000 women. The mean decrease in AT3 level in 13 patients of average age 23 was 17% of the pretreatment value (p=0.0013). Values in U/ml as mean + or - 50 were 1.03 + or - 0.12 on day 0, 0.86 + or - 0.12 on day 5, and 0.97 + or - 0.15 on day 12. The day 0 samples were taken immediately before the start of therapy and those on day 12 were taken 1 week after discontinuing therapy. The normal range is 0.80-1.40 U/ml. The effect of this dose was also studied in 2 volunteers. The 1st volunteer did not wish to continue after the first dose of 5 mg ethinyl estradiol because of vomiting. On day 2 AT3 had increased by 22% and on day 4 had decreased by 12% of the pretreatment level. The 2nd volunteer also vomited on day 1, but continued the medication. AT3 increased on day 2 and then fell to 18.5% of pretreatment level on day 4. Changes in AT3 ran parallel with changes in hematocrit (seen in figure). High doses of estrogen have been reported to cause an increase in blood volume of 18% and a decrease in hematocrit of 15%. Plasma volume increases by 11% during OC use. Estrogen induced retention of salt and water causing hemodilution rather than increased consumption or decreased synthesis, may explain the reported decreases in AT3 levels. This does not rule out the possibility that subnormal values contribute to a hypercoagulable state. In 1 of our patients on day 5 of treatment AT3 fell to 0.60 U/ml, which is within the range where thromboembolism may occur in certain settings, such as emergency surgery or a history of thrombosis.^ieng