alpha 2-Plasmin inhibitor metabolism in patients with liver cirrhosis.

We describe the metabolism of purified human alpha 2-plasmin inhibitor in patients with liver cirrhosis to determine whether low plasma concentrations of alpha 2-plasmin inhibitor are the result of impaired synthesis or increased catabolism or both. A kinetic study was performed with 131I-alpha 2-plasmin inhibitor as a sensitive parameter of fibrinolysis in 14 patients with histologically proved liver cirrhosis compared with six healthy control subjects. Eight patients had macronodular cirrhosis (with positive hepatitis B surface antigen), and six had micronodular cirrhosis as a result of alcohol abuse. None of the patients had clinical signs of ascites, and in all the disease was stabilized. alpha 2-Plasmin inhibitor levels biologically and immunologically measured were decreased in all patients. Ten microCi 131I-alpha 2PI was injected intravenously, the disappearance of plasma radioactivity was measured, and turnover data were calculated according to the function x(t) = A1e-alpha 1t + A2e-alpha 2t + Be-beta t. Mean (+/- SD) turnover data in the control subjects were plasma radioactivity half-life 60.1 +/- 5.3 hours, fractional catabolic rate constant of the plasma pool 0.0318 +/- 0.0106 hr-1, and absolute catabolic (synthetic) rate constant 2.10 +/- 0.60 mg/kg/day. The alpha 1-phase was 1.26 +/- 0.23, and the transcapillary influx constant (k2,1) was 0.974 +/- 0.109 hr-1. In the patients, plasma radioactivity half-life was 58.7 +/- 12.09 hr, and fractional catabolic rate constant of the plasma pool 0.0283 +/- 0.0043 hr-1. The alpha 1-phase 4.74 +/- 6.48 and the transcapillary influx (k2,1) 3.08 +/- 3.9 hr-1 were both significantly increased compared with control values (p less than 0.05 and p less than 0.05, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Neonatal antithrombin III.

Antithrombin III (AT-III) heparin cofactor activity and its antigen levels have been determined in 106 plasma samples from 42 term and preterm neonates. In contrast to healthy adult controls, a reduced activity/antigen (act/ag) ratio (ranging from 0.26 to 0.86) was observed in 90% of the samples and was independent of the state of health of the infant. By modifying the routine assay techniques, laboratory artefacts were excluded as the cause of the observed discrepancy. The relative increase in antigenic AT-III could not be accounted for by circulating AT-III-thrombin complexes, or by increased heparin cofactor II plasma levels in neonates.

Antithrombin III metabolism in patients with liver disease.

Liver diseases are associated with complex haemostasis defects, in which platelets, coagulation, and fibrinolysis may all be affected. The low plasma concentrations of clotting factors often found can be the result of many changes such as impaired synthesis, increased catabolism due to intravascular coagulation, or alternate distribution. In this study, we investigated the metabolism of purified human antithrombin III(AT III) labelled with 125I in 25 patients with histologically established liver disease and in nine control subjects. The results showed that, in general, low plasma concentrations of AT III in liver cirrhosis are not due to consumption in the central compartment but rather to altered transcapillary flux ratio. Such altered transcapillary flux ratios may already exist even with normal plasma AT III concentrations. Altered ratios are not only found for coagulation proteins but also for albumin and thus may be a general phenomenon of liver disease. In micronodular cirrhosis the alpha phase, the transcapillary efflux (k1, 2) and influx (k2, 1) were significantly increased compared with the normal subjects.

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.

Rapid microanalysis of coagulation parameters by automated chromogenic substrated methods - application in neonatal patients.

Daily monitoring of coagulation parameters in critically ill premature born neonates is only possible on small amounts of blood obtained by heelpuncture. Therefore, automated spectrophotometric micro-assays for antithrombin III (AT III), factors II and X, plasminogen and alpha 2 antiplasmin were applied on capillary and venous blood samples concurrently obtained in adults and healthy neonates. No statistically significant difference for any of the parameters was revealed. High levels of platelet factor 4 present in serial capillary samples of adults, did not interfere with the heparin dependent AT III assay. There was no evidence of thrombin or thromboplastin generation in these capillary samples, when examined for Va or VII activities. The levels of AT III, factors II and X and of plasminogen in neonates were 35-45% of the adult levels, in contrast to alpha 2 antiplasmin which was in the adult range.

Further evaluation of an automated amidolytic factor X assay in monitoring anti-vitamin K treatment.

A mechanized factor X assay was tested in 2222 plasma samples of patients being treated with oral anticoagulants. The correlation coefficient between this assay and Thrombotest was 0.78. The therapeutic range for factor X amidolytic activity was 150 to 300 units/l. Amidolytic factor X activity and Thrombotest provide similar information about the state of anticoagulation within the same patient, including patients that are not well balanced. The ranges for factors II, VII, IX and X clotting activity in 57 patients on long-term therapy (Thrombotest within the therapeutic range between 190 and 95 s) were 80-300, 70-600, 40-420 and 50-330 units/l, respectively. The range for factor X amidolytic activity in this group of patients was 150-470 units/l.

Antithrombin III in normal pregnancy.

Plasma antithrombin III (AT III) was determined in 94 women during and after normal pregnancy employing an automated amidolytic technique. The patients were selected on the following criteria: no toxaemia, spontaneous delivery at term, birth-weight above the 10th percentile and discharged with a healthy baby. AT III levels during pregnancy and early puerperium were not lower than own control values obtained 6-8 weeks after delivery.

Postoperative hemostatic profile in relation to gram-negative septicemia.

Gram-negative septicemia presents a particular problem in the ICU. Septicemia is usually diagnosed by fever, chills, and shock. Results of blood cultures become available a few days later. Major surgery induced a marked decline in antithrombin III (AT III) and plasminogen (PLG) to a mean level of 0.60 U/ml (normal value: 0.80-1.40 U/ml) on the 2nd and 3rd postoperative days. Around the 5th postop day, these values again attained mean preoperative levels. Seventy-six surgical ICU patients were investigated preoperatively and for 10 days postoperatively to relate postop septicemia to changes in the hemostatic profile. In 15 patients with gram-negative septicemia verified by positive culture, AT III and PLG barely recovered from the postop decrease and remained significantly lower (p less than 0.05) after the 3rd postop day, compared to 61 surgical ICU patients without septicemia. The behavior of alpha 2antiplasmin (alpha 2AP) values was equal in both groups. This difference in hemostatic profile preceded the clinical manifestations of septicemia and the results of blood culture by several days. Leucocyte or platelet counts provided no reliable information on the early development of septicemia in these surgical patients. It is concluded that persistent low plasma AT III and PLG levels in the postop phase are early indicators of a developing gram-negative septicemia.

Quantitative endotoxin determination in blood with a chromogenic substrate.

A method is described for the quantitative determination of endotoxins in blood. The method is based upon the endotoxin-dependent activation of a proenzyme present in Limulus amebocyte lysate. This activated enzyme is measured by using the chromogenic substrate S 2422. Inhibitors and activated coagulation factors possibly interfering in the assay are removed by dilution and boiling. The method has been proven to be fast (2.5 h), sensitive and reproducible with a detection limit of 10 ng/l. Preliminary results comparing the results of blood cultures with the endotoxin assay indicate a good correlation.

Optimization of a mechanized amidolytic factor-X assay; influence of reaction conditions and reagents.

We describe the effect of pH and NaCl concentration on the activation of factor X in plasma by Russell's viper venom and on the amidolytic activity of the activated enzyme towards factor Xa-sensitive chromogenic substrates. An increasing NaCl concentration results in a decrease in the activation rate of factor X by Russell's viper venom, whereas in this step no pH effect is observed. Increasing NaCl concentrations decrease the Km and Vmax for both the factor Xa-sensitive chromogenic substrates, S 2222 and S 2337. Km values were lowest between pH 7.8 and 8.6; Vmax increased with increasing pH. Comparison of NaCl and KCl in the activation step as well as in the amidolytic step shows that the observed effect is specific for Na+, not just an effect of ionic strength. No difference was detected between activation by the crude venom or its purified factor X-activating enzyme. Also, values with the substrates S 2222 or S 2337 were the same in the mechanized amidolytic factor X assay.

"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

Severe antithrombin III deficiency in a patient with pre-eclampsia. Observations on the effect of human AT III concentrate transfusion.

Severe acquired antithrombin III (AT III) deficiency was observed in a patient with severe pre-eclamptic toxaemia. Plasma AT III concentration of 0.25 U/ml was found in both functional and immunological assays. The patient was treated with human AT III concentrate as a result of the development of progressive disseminated intravascular coagulation (DIC), the further deterioration of renal function, the risk for thromboembolic complications and the possible adverse effects of heparin therapy. The selective correction of AT III activity resulted in a rapid disappearance of coagulation abnormalities. The patient underwent uncomplicated caesarian section. This observation indicates that acquired severe AT III deficiency may occur as an early feature of DIC in severe pre-eclamptic toxaemia.

Evaluation of a new chromogenic assay for factor VII and its application in patients on oral anticoagulant treatment.

A new chromogenic method has been developed and rigorously standardized for the estimation of factor VII in defibrinated diluted plasma. This method employs a mixture of CaCl2-rabbit brain thromboplastin as activator, diluted factor VII deficient plasma as source of factor X and the chromogenic substrate S2222 for the measurement of factor Xa. The chromogenic method was insensitive to cold- and kaolin-induced activation of factor VII, this in contrast to the one-stage clotting assay. Results obtained with the chromogenic method revealed good correlation with the clotting method in 33 normal subjects, in 42 patients on oral anticoagulant therapy and in five patients with severe congenital factor VII deficiency. A good correlation was also obtained with 'Thrombotest'. Comparative estimation of factor VII and of factor VII cross-reacting material in supernatants of BaCl2 adsorbed plasma of coumarin treated patients revealed that the chromogenic method does not measure decarboxy factor VII. Detailed investigations revealed a half life for decarboxy factor VII of 2.1 +/- 0.6 h.

Mechanized amidolytic technique for determination of factor X and factor-X antigen, and its application to patients being treated with oral anticoagulants.

We describe a mechanized chromogenic assay for factor X, the results of which correlate well with those for the one-stage clotting assays for factor X in which it is activated either via the extrinsic pathway by thromboplastin or directly by Russell's viper venom. We purified human factor X and raised monospecific antibodies to it in rabbits. We used our chromogenic assay for factor X to develop a factor-X-inhibitor neutralization assay for determination of factor-X antigen. Patients receiving oral anticoagulant treatment had significantly different factor-X activities after activation via thromboplastin or with Russell's viper venom. The concentration of factor-X antigen, although decreased, significantly exceeded factor-X clotting activity or chromogenic activity in this group of patients. Results of the chromogenic assay for factor X correlated well with results of "Thrombotest," a clotting test introduced by Owren (Lancet ii: 754, 1959) to control anticoagulant therapy. For patients taking oral anticoagulant drugs, the therapeutic range by our assay is 180 to 300 units/L.

Studies on the mechanism of ristocetin-induced platelet aggregation: binding of factor VIII to platelets.

The effect of ristocetin on the binding of [125I]factor VIII to platelets was studied. High and low affinity F.VIII binding sites exist on platelets. The high affinity sites bind 13 times more F.VIII than the low affinity sites. Ristocetin increased the binding of F.VIII to both types of binding sites by increasing the affinity of F.VIII for the platelet and increasing the total number of platelet binding sites. Chymotrypsin-treated platelets were not aggregated by ristocetin and F.VIII: these platelets have less of the major platelet membrane glycoproteins and bind much less [125I]F.VIII than do buffer-treated platelets with and without ristocetin.