Metabolic control may alter antithrombin III activity but not its plasma concentration in diabetes: a possible role for nonenzymatic glycosylation.

The effects of metabolic control on both antithrombin III (AT III) activity and AT III plasma concentration in 20 insulin-treated diabetic subjects have been evaluated. Basal AT III activity was significantly lower in diabetic subjects versus healthy controls (P less than 0.001), whereas no difference was found in AT III concentration. A good correlation was found between AT III activity and AT III concentration (r = 0.81; P less than 0.001) in healthy controls, but this correlation was not significant in diabetic subjects (r = 0.12; P = NS). In those subjects a linear inverse correlation was found to exist between AT III activity and level of glycosylated proteins (r = -0.43; P less than 0.05). Diabetic subjects were also examined after 1 and 2 mo of restored metabolic control, obtained by human insulin (DNA-recombinant) therapy. Improved metabolic control was characterized by an increase of AT III activity (P less than 0.05), a decrease of mean daily blood glucose, and stable HbA1 and glycosylated proteins (P less than 0.05), while AT III concentration did not vary. On the other hand, a significant inverse correlation between AT III activity and glycosylated proteins was found during both the first and second months (r = -0.54 and r = -0.53, respectively; P less than 0.01). Moreover, no correlation between AT III activity and AT III concentration was found. These data suggest that impaired metabolic control may alter the biologic activity of AT III in diabetes, but not its plasma concentration.

Abnormal properties and behaviors of antithrombin III found in a thrombophilic patient: defective biological functions and dissimilar antigenic determinants.

Abnormal properties of antithrombin III have been found in a 55-year-old male who has been thrombophilic over the last seven years. They are characterized by defective inhibition of thrombin and activated blood coagulation factor X, reduced affinity to heparin and partial immunological identity with the normal molecule. The antithrombin III molecule, however, preserves a single-chain structure and an apparently identical molecular weight with that of the normal molecule. It is, thus, very unlikely that the impaired functions of antithrombin III in the patient's plasma are induced by possible proteolytic modifications of the molecule by thrombin or other related activated blood coagulation factors. Since no other members of his immediate family have been found to be affected, the abnormality may be acquired rather than genetically determined, although further investigation is necessary for the elucidation of the abnormality of the molecule.

Antithrombin III and heparin cofactor II in patients with chronic renal failure undergoing regular hemodialysis.

Heparin enhances the inhibition rate of thrombin by both antithrombin III (AT III) and heparin cofactor II (HC II). We studied the activity of these two plasma proteins in patients with chronic renal failure (CRF) undergoing regular hemodialysis as their heparin requirements varied widely. In 77 normal blood donors, normal ranges (mean +/- 2 SD) were 82-122% for AT III and 65-145% for HC II. When compared with these controls 82 dialyzed CRF patients had a subnormal AT III activity and a significantly (p less than 0.001) lower HC II activity. To evaluate the effect of hemodialysis we compared AT III, HC II and total proteins in plasma before and after dialysis in 24 patients (12 with normal and 12 with low basal HC II activity). AT III and HC II activities significantly (p less than 0.001) increased in absolute value. When related to total plasma proteins, in order to suppress the influence of hemoconcentration induced by dialysis, AT III decreased significantly (p less than 0.01) whereas HC II increased slightly but significantly (p less than 0.01) in the 12 patients with low initial HC II activity. The decrease of AT III induced by heparin administrated during dialysis is likely to account for this relative decrease of AT III activity. A modification of the distribution of both HC II and heparin between the vascular wall and the circulating blood is evoked to explain the relative increase in HC II activity and the need for higher heparin dosage in patients with low HC II levels.

Antithrombin III Alger: a new homozygous AT III variant.

A qualitative abnormality of antithrombin III (AT III) was found in the plasma of a 41-year old patient. The plasmatic AT III antigen concentration was 130% and the progressive anti-F IIa and anti-F Xa activities were normal (105% and 137%). The plasma heparin cofactor activity was less than 10%, when measured by F IIa or F Xa inhibition. Crossed immunoelectrophoresis of AT III in the presence of heparin revealed in the plasma an abnormal slow-moving peak. When tested by affinity chromatography on heparin Sepharose, this abnormal AT III did not bind to heparin. Among the investigated relatives, 5 subjects had normal AT III levels, whatever the test used, the nine others having reduced levels of antithrombin heparin cofactor activity (45-61%) but normal levels of immunoreactive AT III (97-122%). Consanguinity was found in the family history. We therefore considered our patient as homozygous for an AT III molecular abnormality affecting the binding site for heparin.

Antithrombin III antigen in human platelets.

Immunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 10(9) platelets with a mean value of 70.3 +/- 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.

In vivo release of a heparin-like factor in dogs during profound hypothermia.

Experimentally induced hypothermia (20 degrees C) for 60 minutes in dogs provokes a significant decrease in the platelet count, which reverses during subsequent rewarming, and the constant release of a heparin-like factor, which reacts as a specific inhibitor of factor Xa. This phenomenon is also rapidly reversible, and heparin values are not significantly different from control levels after 90 minutes of rewarming. The mean maximal concentration of heparin-like material is 0.54 U/ml, or about double control levels. Its half-life is approximately 90 minutes. The level of circulating antithrombin III was not modified during hypothermia and rewarming.

Antithrombin III patterns in disseminated intravascular coagulation.

Antithrombin III-heparin cofactor has now been recognized as a major inhibitor of thrombin and other serine proteases in the blood coagulation system. Since the reaction between antithrombin III and serine proteases is irreversible, one would expect antithrombin III consumption in the face of pathologic intravascular coagulation and attendant generation of thrombin, IXa, Xa, XIa, XIIa, and plasmin. Using a new assay system for antithrombin III that is unaffected by heparin or fibrino (geno) lytic degradation products, antithrombin III was monitored before and during therapy in 38 patients who had acute or chronic disseminated intravascular coagulation. It was found that early and significant decreases in anththrombin III occur in disseminated intravascular coagulation and thus may serve as a useful diagnostic tool. It was further found that monitoring antithrombin III during therapy reflected a cessation of antithrombin III consumption and, thus, served as an indicator of the efficacy of therapy in stopping the clotting process. Since the assay system is unaffected by fibrino(geno)lytic degradation products and heparin, it proved useful in monitoring the efficacy of heparin therapy for disseminated intravascular coagulation. In addition for this group of patients, it appeared that mini-heparin therapy and large doses of heparin were equally efficacious in correcting other laboratory abnormalities of disseminated intravascular coagulation, and in controlling clinical hemorrhage in disseminated intravascular coagulation.

Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum.

1. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions, amino-terminal sequences, immunological cross-reactivities, and inhibitions of human thrombin were studied. 2. Human, porcine, rabbit, and rat antithrombin III are single-chain glycoproteins containing hexose, glucosamine, and neuraminic acid. 3. The total carbohydrate contents were 17, 16, 14, and 15% for human, porcine, rabbit, and rat antithrombin III, respectively. 4. Molecular weights estimated from the migration in sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis were 59,000, 58,000, 63,000, and 63,000 for human, porcine rabbit, and rat antithrombin III, respectively. 5. These four proteins have similar amino acid compositions, although some minor differences were noted. 6. Human, porcine, and rabbit antithrombin III have a histidine residue at the amino-terminus, while rat antithrombin III contains an amino-terminal asparagine residue. 7. The amino-terminal sequences up to the first 17 residues showed high homology among the four proteins. 8. Some immunological cross-reactivity was observed only between human and porcine antithrombin III. 9. The apparent dissociation constants (KI) for the complexes between human thrombin and human, porcine, rabbit, and rat antithrombin III were about 1.2 x 10(-10) M, 9.5 X 10 (-9) M, 1.4 X 10(-7) M, and 2.8 X 10(-9) M, respectively.

Studies of protease inhibitors in the sera of patients with amyotrophic lateral sclerosis.

Serum levels of 4 protease inhibitors, alpha-1-antitrypsin, C1-inactivator, alpha-2-macroglobulin and antithrombin-III were measured in 11 patients with amyotrophic lateral sclerosis (6 males and 5 females) and a control group without neurologic disease. Our results indicated no significant differences in the level of serum alpha-2-macroglobulin between the 2 groups. We found slight but significantly lower levels of serum antithrombin-III in ALS. The possibility of the participation of proteases or protease inhibitors in the pathogenesis of ALS is discussed.

Coagulation and fibrinolysis in chronic subdural hematoma.

In 19 patients with chronic subdural hematoma, coagulation and fibrinolysis in venous blood taken at the time of surgery and in the hematoma contents aspirated from chronic subdural hematoma were studied. Compared with coagulation results for venous blood, the hematoma contents demonstrated marked prolongation of the recalcification time, prothrombin time, and activated partial thromboplastin time, and marked reduction of clotting factor V, the hepaplastin test, prothrombin, and fibrinogen. Antithrombin III was also decreased, and fibrinopeptide A was increased in the hematomas. Fibrinolytic results demonstrated that both plasminogen and alpha 2-plasmin inhibitor were decreased, and both fibrinopeptide B beta 15-42 and fibrin and fibrinogen degradation products were increased in the hematomas. These findings indicate excessive activation of the clotting system, thrombin generation, and increased fibrinolytic activity occurring in the hematomas. From these results, excessive activation of both the clotting and fibrinolytic systems is emphasized to be the possible etiological factor for the origin and development of chronic subdural hematoma.

Platelet and coagulation functions during triphasic oestrogen-progestogen treatment.

The purpose of this investigation was the longitudinal evaluation of the hemostatic system before and after 1, 3, and 6 months of treatment with a triphasic oestrogen-progestogen combination. No changes of circulating platelet aggregates, as an index of in vivo platelet aggregability, and of megathrombocytes, an indirect evaluation of accelerated thrombocytopoiesis, were observed. A very slight, but significant, increase of Fibrinopeptide A (FPA), a reliable index of thrombin formation, was found only after 1 month of treatment; after 3 and 6 months, the increase of FPA was not homogeneous and not significant. Antithrombin III activity (AT III) showed no modifications after the first month; after 3 months AT III increased to a small extent, and after 6 months it was similar to basal values. Our findings indicate that the triphasic combination does not modify platelet functions and induces a low-degree activation of coagulation counteracted by an increased activity of the physiological inhibitors of blood clotting.

Laboratory determination of heparin cofactor II.

Heparin cofactor II (HC II) is a recently characterized protein that is capable of neutralizing thrombin but not activated factor X. Recent evidence suggests that it may be a physiologically important regulator of thrombin activity. We evaluated and modified a method for clinical laboratory determination of this protein and then utilized the method to analyze HC II activity in various clinical samples. Low levels were associated with liver disease, consumptive coagulopathy, and preeclampsia; normal levels were seen with uncomplicated pregnancy, oral anticoagulant therapy, hereditary antithrombin III (AT III) deficiency, and in 31 patients evaluated for a thrombotic tendency. Except in hereditary AT III deficiency, decreased HC II activity was associated with decreased AT III activity. The potential clinical role of this assay is discussed.

Plasma prekallikrein, functional kallikrein inhibition, antithrombin III, plasminogen, kallikrein activity and proenzyme functional inhibition index in 172 acute ill surgical patients on admission.

The present study was performed in order to study disturbances in plasma proteolysis in acute ill surgical patients on admission. The paper describes the distribution of values for plasma PKK, KKI, AT III, Plg, KK and PFI-index. In early stages of the disease process rather minimal information can be obtained by these data in addition to clinical examination. The study indicates that chromogenic peptide substrate assays should be reserved for surgical patients treated in the intensive care unit.

Significance of the whole blood activated clotting time in cardiopulmonary bypass.

Recent publications have described a poor correlation between whole blood activated clotting time (WBACT) values and plasma heparin levels during cardiopulmonary bypass (CPB). A prospective, controlled study was undertaken to investigate the variables which may influence the WBACT in this situation. Antithrombin III levels over a range of 35-93 u/dl did not influence either the WBACT value or plasma heparin level. However, reduced platelet function following infusion of prostacyclin (10 ng/kg/min prior to CPB and 20 ng/kg/min thereafter); platelet number (range 63-287 X 10(9)/l) and packed cell volume (range 16-30%) were found to correlate with the WBACT. It is concluded that in addition to the circulating plasma heparin level, the wide variations in platelet number, platelet function and packed cell volume which are frequently observed during cardiopulmonary bypass may also influence the WBACT value.