Inactivation and binding of human plasma kallikrein by antithrombin III and heparin.

Purified human antithrombin III was found to inactivate the esterase, the kininogenase, and the plasminogen activator activity of purified plasma kallikrein. The reaction rate was increased by heparin. Kinetic studies indicated second-order kinetics for the reaction between kallikrein and antithrombin III, and a catalytic effect of heparin. SDS polyacrylamide gel electrophoresis revealed that three new bands were generated during the inactivation of kallikrein by antithrombin III. Heparin did not influence the factor XII-dependent activation of prekallikrein in human plasma, whereas the inactivation of kallikrein elapsed slightly more rapidly in plasma samples where heparin was added. It is concluded that antithrombin III probably is of minor importance for the inactivation of kallikrein in plasma under physiological circumstances.     

Comparison between the effect of pentosan polysulphate heparin and antithrombin III injections in antithrombin III deficient patients

Pentosan polysulphate is an heparin analogue which acts via an antithrombin III (AT III) independent pathway. We compared the effect of this drug to that of heparin and AT III infusions in AT III deficient patients. Four patients with AT III congenital deficiency received on three different occasions: (i) an infusion of human AT III concentrate (20 U/kg or 40 U/kg), (ii) an intramuscular injection of pentosan polysulphate (2 mg/kg), (iii) a subcutaneous calcium heparin injection (100 U/kg). AT III infusion inhibits the excessive thrombin generation (46% of inhibition) observed in the plasma of AT III deficient patients during at least 12 hours, but does not modify the factor Xa formation. On the contrary, pentosan polysulphate has a marked effect on both thrombin (62% of inhibition) and factor Xa generation (57% of inhibition) still present 8 hours after injection. Heparin injection has the same effect, more prolonged, as pentosan polysulphate on thrombin generation but is not so effective on impairing factor Xa generation (27% of inhibition). The marked effect of pentosan polysulphate on thrombin and factor Xa generation in these patients is due to its AT III independent mechanism of action.     

Decreased binding of heparin to antithrombin following the interaction between antithrombin and thrombin

A complex formed between bovine antithrombin and bovine thrombin was shown to elute from a heparin-agarose column at a lower ionic strength than free antithrombin. Moreover, it was demonstrated that less heparin remained attached to the antithrombin-thrombin complex than to free antithrombin, when mixtures of high-affinity heparin and either complex or antithrombin were separated by gel electrophoresis or gel chromatography under identical conditions. These results suggest that the interaction between antithrombin and thrombin leads to a decreased affinity of heparin for the antithrombin moiety of the complex. This decrease may be an essential feature of the proposed function of heparin as a catalyst in the antithrombin-thrombin reaction.     

Simultaneous determination of plasma prothrombin and antithrombin III (heparin cofactor).

A method for the simultaneous determination of plasma prothrombin and antithrombin III has been developed. It involves activation of a dilute plasma solution with Taipan snake venom and measurement of the heparin-mediated loss of thrombin activity after short incubation. The antithrombin III content is quantitated on the basis of results with known mixtures of normal and antithrombin III-deficient plasmas.     

Further characterization of a pathological isoantithrombin with no affinity for heparin (antithrombin III Roma)

A molecular antithrombin III variant (Antithrombin III Roma) with an abnormal pattern of crossed immunoelectrofocusing was further investigated in order to identify the pathological isoforms. AT III crossed immunoelectrofocusing of the whole plasma from the affected patients showed a loop overlapping the peak normally present at pH 4.8-4.6. Affinity chromatography demonstrated the presence of an AT III fraction totally lacking in heparin affinity. Crossed immunoelectrophoresis on heparin-agarose (H-CIE) and crossed immunoelectrofocusing (CIEF) runs performed on the fractions obtained by heparin-agarose affinity chromatography confirmed that the functional defect was exclusively related to the pathological isoantithrombin (pH 4.8-4.6), which was also devoid of any progressive activity. The AT III fraction with normal affinity to heparin displayed H-CIE and CIEF patterns identical to the control AT III.     

Interaction of heparin with histidine-rich glycoprotein and with antithrombin III

The interaction between heparin, histidine-rich glycoprotein and antithrombin III was studied in purified systems. Histidine-rich glycoprotein binds heparin and thereby interferes with its interaction with antithrombin III, resulting in neutralization of the anticoagulant activity. This interaction occurs with clinical grade heparin as well as with high affinity (for antithrombin III) heparin and with a high affinity heparin fragment with Mr 4,300. Low affinity heparin competes with high affinity heparin for the binding to histidine-rich glycoprotein which results in an apparent increase of the anticoagulant activity of high affinity heparin. The interaction between heparin and histidine-rich glycoprotein is counteracted by Ca2+-binding anticoagulants, indicating that it is dependent on the presence of divalent metal ions. Ethylenediaminetetraacetate is a much more potent inhibitor of the interaction between heparin and histidine-rich glycoprotein than citrate.     

Elimination of intravenously administered radiolabelled antithrombin III and heparin in humans

Antithrombin III was purified from normal plasma by DEAE-Sephadex chromatography and heparin affinity chromatography; the protein was subsequently radiolabelled with 125I. 125I-antithrombin III alone and 125I-antithrombin III in the presence of high affinity 35S-heparin fractions were injected into normal humans. 125I-radiolabel and protein bound 35S-radioactivity were followed separately. In semilogarithmic plots 125I-antithrombin III disappeared according to a double exponential curve with a half-life in the second phase of 56.8 hr in the absence of heparin and of 33.7 hr in the presence of heparin. Protein bound 35S-radioactivity disappeared much faster than the 125I-radiolabel. These data support the concept that heparin disappears as free heparin from the equilibrium heparin - antithrombin III in equilibrium heparin + antithrombin III. Immuno-reactive antithrombin III decreased from 100% to 85-90% immediately after injection of 125I-antithrombin III in the presence of heparin and returned to normal values within 30 min. This suggests that antithrombin III is transiently sequestered, possibly in trimolecular complexes consisting of antithrombin III, heparin and either lipases or other vascular bound proteins.     

Activity of antithrombin III and effect of heparin on coagulation in shock

In 16 patients admitted for DIC and shock,series of coagulation tests were carried out prior to and during heparin therapy. Plasma heparin concentrations measured were corresponding to expected levels. In some cases however,considerably higher or lower levels were found. This could be partly explained by release of PF 4 from platelets and by a decreased turnover of heparin because of a malfunction of the kidneys or of the liver. A relation between the effect of heparin on coagulation and the activity of antithrombin III could be established. A diminution of this activity resulted in a diminished or even missing effect of heparin. In some instances,the PTTand the thrombin clotting time were considerably prolonged when FDP were present or when procoagulant factors were diminished. These tests therefore,did not reflect true heparin concentrations in shock. For this reason,regular assays of antithrombin III and of heparin are proposed.     

Antithrombin III microheterogeneity in antithrombin III deficiency and in the antithrombin III abnormality, "antithrombin III Toyama"

Antithrombin III (AT III) microheterogeneity was investigated in 12 cases of congenital AT III deficiency and 2 cases of congenital AT III abnormality by isoelectric focusing (IEF) and immunofixation. In congenital AT III deficiency, IEF and immunofixation revealed AT III as 8 bands which was indistinguishable from normal control in terms of the number of bands and the isoelectric point (pI) of each band. In the proband of the congenital AT III abnormality, however, IEF and immunofixation showed AT III as 8 bands which shifted slightly but definitely to the acidic side compared to those of normal subjects. This change in pI of the abnormal AT III was considered to reflect the amino acid replacement in the polypeptide chain of the abnormal AT III molecule.     

The relative antithrombotic effectiveness of heparin, a low molecular weight heparin, and a pentasaccharide fragment in an animal model

The antithrombotic efficacy of unfractionated heparin (UFH), a low molecular weight heparin (LMWH) and a synthetic pentasaccharide (PENTA) has been compared in an animal model for stasis thrombosis. We have also compared the relative ability of these three agents to impair thrombin generation in vitro and in vivo, and measured their effects on anti-Xa activity and thrombin clotting times. UFH, LMWH and PENTA all had the capacity to impair thrombogenesis, although there were marked differences in their relative effectiveness. Reduction of thrombin generation to 20% of control values was closely correlated with the prevention of thrombosis after 20 minutes' stasis, but this was only achieved with UFH. The same dry weight dose of LMWH or PENTA reduced thrombin generation to about half control values, and neither significantly impaired thrombus formation after 20 minutes' stasis. Impaired thrombin generation correlated better than anti-Xa activity with prevention of stasis thrombosis. In this model, UFH was clearly superior to LMWH and PENTA as an antithrombotic agent.     

Homozygous variant of antithrombin III that lacks affinity for heparin, AT III Kumamoto

Abnormal antithrombin III (AT III) was found in the plasma of a 31-year-old female who suffered from recurrent thrombotic episodes. Heparin cofactor activity was 28% of normal and undetectable when measured by inhibition of thrombin and factor Xa (F.Xa), while both progressive antithrombin and antifactor Xa activities were normal. The concentration of plasma AT III antigen was 37 mg/dl. Analysis by crossed-immunoelectrophoresis (CIE) in the presence of heparin and affinity chromatography on heparin-Sepharose revealed that the propositus' AT III did not bind to heparin. When heparin cofactor II (HC II) was removed from propositus' plasma, heparin cofactor activity of AT III was not detected. Thus, HC II seemed to account for the plasma heparin cofactor activity found in the presence of thrombin. The patient's parents and three of her brothers demonstrated qualitative abnormality of AT III; heparin cofactor activity was 30-50% of normal levels in the presence of both thrombin and F.Xa. These findings indicate that the propositus' AT III lacks affinity for heparin and the mode of its inheritance seems to be autosomal dominant and, hence, the propositus would be a homozygote. For this variant, the name of AT III Kumamoto is proposed.     

Identification and characterisation of an antithrombin III mutant (AT Dublin 2) with marginally decreased heparin reactivity

The antithrombin III (ATIII) isoform patterns of plasma and serum samples from cancer patients and controls were analysed by isoelectric focusing and immunoblotting. A novel ATIII banding pattern was identified in two individuals: a patient with breast carcinoma who developed deep venous thrombosis and a blood donor. Family studies in the patient showed the abnormal pattern to be due to a mutant form of ATIII (AT Dublin 2). The coagulation properties of AT Dublin 2 heterozygotes were normal. Immunologic and activity levels of ATIII, measured by standard techniques, were normal. Mutant plasma ATIII showed reduced thrombin reactivity at low concentrations of thrombin and demonstrated decreased reactivity with heparin over a range of heparin concentrations. This was confirmed using a modified ATIII heparin cofactor activity assay with varying heparin concentrations. The abnormal ATIII was also found to elute from heparin agarose at a lower ionic strength than normal ATIII. Two dimensional gel electrophoresis showed the abnormal ATIII to have similar molecular size distribution to normal ATIII. Neuraminidase treatment of normal and mutant plasma reduced the ATIII isoforms to one in both samples. The possible role of AT Dublin 2 in predisposing to hypercoagulation is discussed.     

Safety of virus inactivated antithrombin III concentrate ANTITHROMBIN III IMMUNO (AT III)

In a prospective clinical trial the risk of infection after application of virus inactivated antithrombin III concentrate ANTITHROMBIN III IMMUNO (AT III) was investigated in patients undergoing cardiovascular surgery. The study was conducted according to the recommendations of the International Committee on Thrombosis and Hemostasis (ICTH), with the exception that most patients required additional blood products as well as AT III.

Twenty-seven patients were eligible to test for the risk of acquiring hepatitis B. Twenty-six patients could be evaluated in terms of hepatitis NANB transmission considering ALT-levels whereas 20 patients could be tested for anti-HCV one year after surgery. Samples from 78 patients could be monitored for anti-HIV-1. None of these patients showed any signs of infection. AT III IMMUNO seems to be an antithrombin III concentrate with low or absent infectivity.     

A functional abnormal antithrombin III (AT III) deficiency : AT III Charleville

An antithrombin III (AT III) functional defect (AT III Charleville) was discovered in a patient presenting with recurrent venous thrombosis. Both anti-activated factor X (anti Xa) and antithrombin activity were decreased, in the absence and in the presence of heparin, while protein concentration was normal in an immunological assay. The abnormal AT III copurified with functional AT III using insolubilized heparin affinity chromatography. Polyacrylamide gel electrophoresis (PAGE) and high pressure liquid chromatography (HPLC) on a TSK column suggest that AT III Charleville forms unstable complexes with thrombin from which a modified protein is rapidly released.     

Anticoagulant activities and effects on platelets of a heparin fragment with high affinity for antithrombin

A fragment of heparin containing 10–16 sugar units and retaining ability to bind to antithrombin III has been prepared by degrading standard heparin with nitrous acid. This fragment greatly potentiated the inhibition of factor X_a by antithrombin III but had virtually no effect on the inhibition of thrombin. Studies on heparin neutralization showed that the fragment was affected to a much lesser extent than standard heparin by heparin neutralizing components in plasma. The heparin-potentiated aggregation of platelets by low concentrations of ADP was measured for a number of heparin fractions and the fragment. The molecular weight of the heparin was found to be the most important factor determining the platelet aggregation activity, low molecular weight fractions including the fragment being much less active than high molecular weight ones.