TWO-SIDE IMMUNOASSAYS OF ENZYME-INHIBITOR COMPLEXES FOR THE DIAGNOSIS OF THROMBOPHILIC STATES

1987 ◽  
Author(s):  
H Bleyl
Keyword(s):  
Heart Surgery ◽  
Enzyme Inhibitor ◽  
Open Heart Surgery ◽  
Coagulation Cascade ◽  
Clotting Factors ◽  
Inhibitor Complex ◽  
At Iii ◽  
Complex Measurement ◽  
Sandwich Assays ◽  
Time Of Admission

The diagnosis of prethrombotic states requires methods which detect products of intravasal activation of the coagulation cascade.Two-side immunoassays for antithrombin complexes with clotting factors were developed (IXi-AT, Xi-AT, IIi- AT). These sandwich assays permit the diagnosis of hypercoagulability in the presence or absence of heparin. The biological half live time of the thrombin-antithrombin-complex was found to be about 15 min. Healthy young men 20-25 years old (n=30) have a thrombin-antithrombin-complex concentration of 0.4 ± 0.2 mU/ml thrombin equivalent (S 2238). Patients with acute myocardial infarction (n=40) showed at the time of admission to the hospital up to 10-fold (n=14), up to 100-fold (n=13) more than 100-fold (n=13) elevated thrombin-antithrombin-complex concentrations. Patients with gastrointestinal cancer showed sometime excessive elevated enzyme-inhibitor complexes.No correlation was found between thromboplastine time (Quick) and complex concentration in patients under anticoagulant therapy with dicumarole. In patients under dialysis as well as in patients under open heart surgery with extracorporal circulation, the biocompatibility can be monitored by inhibitor complex measurement.

Blood Coagulation Studies and Extracorporeal Circulation in Man

1967 ◽  
Vol 18 (03/04) ◽  
pp. 634-646 ◽  
Author(s):  
N Thurnherr
Keyword(s):  
Blood Coagulation ◽  
Extracorporeal Circulation ◽  
Blood Clotting ◽  
Heart Surgery ◽  
Open Heart Surgery ◽  
Fibrinolytic System ◽  
Open Heart ◽  
Clotting Factors ◽  
Blood Clotting Factors

SummaryBlood clotting investigations have been executed in 25 patients who have undergone open heart surgery with extracorporeal circulation. A description of alterations in the activity of blood clotting factors, the fibrinolytic system, prothrombin consumption and platelets during several phases of the operation is given.

Rapid Quantitation of Plasma Heparin and Antithrombin III Levels for Cardiopulmonary Bypass Monitoring, Using Fluorometric Substrate Assays

1983 ◽  
Vol 50 (03) ◽  
pp. 745-748 ◽  
Author(s):  
G F Savidge ◽  
P J Kesteven ◽  
S F Al-Hasani ◽  
P F O’Brien
Keyword(s):  
Cardiopulmonary Bypass ◽  
Heart Surgery ◽  
Open Heart Surgery ◽  
Fluorometric Assay ◽  
Immunological Methods ◽  
Poor Correlation ◽  
At Iii ◽  
Serial Samples ◽  
Plasma Heparin

SummaryPlamsa heparin and AT III levels were determined using recently developed fluorometric substrate techniques on serial samples taken from 9 unselected patients undergoing open-heart surgery on cardiopulmonary bypass. The fluorometric assay for AT III and established clotting and immunological methods showed a highly significant correlation (p <0.001). Heparin recovery was reduced in all cases, and plasma levels (fluorometric assay) demonstrated poor correlation to Whole Blood Activated Clotting Time (Hemochron) estimations. AT III levels were dramatically reduced during bypass, and in 3 cases reached levels below 0.08 iu/ml. Heparin reversal schedules based upon empirical dosage led to excessive protamine administration by a mean factor of 3.3, as assessed by in vitro neutralization of standardized heparin concentrations.

scholarly journals Transferrin plays a central role to maintain coagulation balance by interacting with clotting factors

2019 ◽  
Author(s):  
Xiaopeng Tang ◽  
Zhiye Zhang ◽  
Mingqian Fang ◽  
Yajun Han ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Plasma Concentration ◽  
Mouse Models ◽  
Antithrombin Iii ◽  
Fine Tuning ◽  
Coagulation Cascade ◽  
Molar Ratio ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Normal Plasma ◽  
At Iii

SummaryCoagulation balance is maintained through fine-tuning interactions among clotting factors. Physiological concentrations of clotting factors are huge difference. Especially, coagulation proteases’ concentration (pM to nM) is much lower than their natural inactivator antithrombin III (AT-III, ∼3 μM). Here we show that transferrin (normal plasma concentration ∼40 μM) interacts with fibrinogen, thrombin, FXIIa and AT-III with different affinity to maintain coagulation balance. Normally, transferrin is sequestered by binding with fibrinogen (normal plasma concentration ∼10 μM) with a molar ratio of 4:1. In atherosclerosis, abnormally up-regulated transferrin interacts with and potentiates thrombin/FXIIa and blocks AT-III’s inactivation on coagulation proteases by binding to AT-III, and thus induces hypercoagulability. In mouse models, transferrin-overexpression aggravated atherosclerosis while transferrin-knockdown, anti-transferrin antibody or designed peptides interfering transferrin-thrombin/FXIIa interactions alleviated it. Collectively, these findings identify transferrin as a clotting factor and an adjuster for maintaining coagulation balance and modify the coagulation cascade.

scholarly journals Differential role of fractionated heparin in antithrombin-III proteolysis

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 576-581 ◽  
Author(s):  
E Marciniak
Keyword(s):  
Affinity Chromatography ◽  
Antithrombin Iii ◽  
Enzyme Inhibitor ◽  
Anticoagulant Activity ◽  
Lower Affinity ◽  
Inhibitor Complex ◽  
High Affinity ◽  
Highly Active ◽  
At Iii

Abstract Commercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively, Each fraction, in microgram quantities, was examined in the reaction of alpha-thrombin with a molar excess of 125I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21%, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.

Blood Products in the Treatment of Acute Bleeding Complications Following Open-Heart Surgery

1979 ◽  
Author(s):  
M. Crow ◽  
T.K. Kaul ◽  
S.M. Rajah
Keyword(s):  
Heart Surgery ◽  
Open Heart Surgery ◽  
Bleeding Complications ◽  
Blood Component ◽  
Severe Thrombocytopenia ◽  
Open Heart ◽  
Platelet Concentrates ◽  
Clotting Factors ◽  
Excessive Bleeding ◽  
Number Of Patients

We studied the bleeding patterns in 377 patients undergoing open-heart surgery in the last 18 months. The number of patients with excessive bleeding requiring a coagulation screen was 56 (14.84%), of which 30 (7.95%) had a normal profile, with moderate thrombocytopenia. In 22 of these 30 patients specific bleeding sites were found at re-operation and effective surgical haemostasis was achieved. No specific bleeding sites were found in the remaining 8 (2.12%) and the cause for excessive bleeding was not determined. Treatment with fresh frozen plasma (FFP) gradually controlled the bleeding. Twenty-six (6.89%) had abnormal coagulation findings. Severe thrombocytopenia occurred in 1 patient (0.25%) who was treated with platelet concentrates. Prolonged partial thromboplastin time was recorded in 14 (3.71%) cases and these patients were treated with cryoprecipitate, FFP or fresh blood (depending on the haematocrit and blood volume). The final 11 patients had low levels of all clotting factors, were thrombocytopenic and had significantly raised fibrinogen degradation products, indicating a limited form of intravascular coagulation. These patients were treated with cryoprecipitate, FFP and platelet concentrates. Sixty per cent of the patients with post-operative bleeding required specific blood component therapy besides other medication.

Platelet And Coagulation Behaviour During Open Heart Surgery

1981 ◽  
Author(s):  
A Sturk ◽  
E M G Hoogendijk ◽  
M C L Schaap ◽  
P S Sebel ◽  
J G Bovill ◽  
...  
Keyword(s):  
Platelet Count ◽  
Extracorporeal Circulation ◽  
Heart Surgery ◽  
Antithrombin Iii ◽  
Open Heart Surgery ◽  
Blood Platelet ◽  
Open Heart ◽  
Factor Ii ◽  
At Iii ◽  
Blood Platelet Count

Studies were undertaken in 19 patients (16 m, 3 f) having open heart surgery. Parameters investigated were: hematocrit; whole blood platelet count; β-TG levels in plasma and platelets; aggregation by ADP, adrenalin and ristocetin; glycoprotein analysis of whole platelets; plasma levels factor II, X, plasminogen (plg), antithrombin III (AT III) and α2antiplasmin (ap). Samples were taken before (1) and 5 min after induction (2), five min after opening the sternum (3), at aortic canulation (4), 15 min after start of extracorporeal circulation (5) and at release of aortic crossclamp (6). Measurements in 5 and 6 were corrected for dilution. Platelet count was decreased 20-30% in samples 5 and 6. There was a 350% increase in plasma β-TG levels in sample 6, without a change in the circulating-platelet β-TG content. The increase therefore appears to be due to destruction or release of the non-circulating platelets. ADP-aggregation was normal in all samples, Adrenalin and ristocetin aggregation was absent in samples 5 and 6 due to the pump priming fluid (haemaccel). However, in sample 4 aggregation induced by ristocetin (1 mg/ml) was also absent. This could not be explained by the presence of haemaccel or heparin. Absence of GP IB (the possible ristocetin receptor) also could not be demonstrated. Plasma II, X, pig, AT III and ap did not change in sample 1-4. Factor II and X decreased 8-9% in sample 5 and 6, whereas plg, AT III and ap resp. decreased 12, 14 and 20%. Evidently activation of the coagulation starts with the extracorporal circulation.

Self-Association of Antithrombin III Relates to Multimer Formation of Thrombin-Antithrombin III Complexes

1993 ◽  
Vol 69 (05) ◽  
pp. 422-429 ◽  
Author(s):  
Klaus T Preissner
Keyword(s):  
Conformational Changes ◽  
Antithrombin Iii ◽  
Enzyme Inhibitor ◽  
Binary And Ternary Complexes ◽  
Inhibitor Complex ◽  
Thrombin Inhibition ◽  
At Iii ◽  
Self Association ◽  
Enzyme Complexes ◽  
Concomitant Exposure

SummaryDuring the reaction of antithrombin III (AT III) with target proteases the inhibitor serves as pseudo-substrate and undergoes profound conformational changes, becomes incorporated into a covalent stoichiometric enzyme-inhibitor complex which is, in contrast to native AT III, recognized by monoclonal antibody 4C9. In the absence of the target enzyme thrombin, incubation of AT III with 1–2 M guanidine, 0.6% deoxycholate, heating to 56° C, or buffer at pH 4 resulted in inactivation of the inhibitor with concomitant exposure of the epitope for 4C9 and formation of AT III multimers (from 3.9S to 7.1–7.4 S). Loss of activity, formation of multimers and exposure of neoepitope(s) of AT III occurred in a concerted fashion and followed second order kinetics with an activation energy of E a = 31 kcal/mol. AT III-multimerization induced by treatment with 1 M guanidine (mainly AT III-tetramers with M r of 250,000) and formation of the binary AT III-thrombin complex revealed similar self-association patterns as judged by gel electrophoresis under non-denaturing conditions. In the presence of heparin, even higher multimers of AT III-thrombin complexes were noted. Moreover, self-association products of the ternary vitronectin-thrombin-AT III complex, which is the ultimate reaction product following thrombin inhibition in the circulation, could be recognized and quantitated due to exposure of the 4C9 epitope on AT III, indicating that AT III exists in multimeric forms within binary and ternary complexes. It is proposed that the ability of these complexes to form high M r association products is at least in part mediated by the propensity of AT III to multimerize and that multimeric forms of AT III may be involved during clearance of AT III-enzyme complexes.

scholarly journals Differential role of fractionated heparin in antithrombin-III proteolysis

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 576-581
Author(s):  
E Marciniak
Keyword(s):  
Affinity Chromatography ◽  
Antithrombin Iii ◽  
Enzyme Inhibitor ◽  
Anticoagulant Activity ◽  
Lower Affinity ◽  
Inhibitor Complex ◽  
High Affinity ◽  
Highly Active ◽  
At Iii

Commercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively, Each fraction, in microgram quantities, was examined in the reaction of alpha-thrombin with a molar excess of 125I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21%, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.

Enhanced Effective Fibrinolysis following the Neutralization of Heparin in Open Heart Surgery Increases the Risk of Post-Surgical Bleeding

1990 ◽  
Vol 63 (02) ◽  
pp. 241-245 ◽  
Author(s):  
Jørgen Gram ◽  
Thomas Janetzko ◽  
Jørgen Jespersen ◽  
Hans Dietrich Bruhn
Keyword(s):  
Blood Loss ◽  
Plasminogen Activator ◽  
Heart Surgery ◽  
Degradation Products ◽  
Open Heart Surgery ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Chest Tube Drain ◽  
Median Blood Loss

SummaryThe tissue-type plasminogen activator related fibrinolytic system was studied in 24 patients undergoing cardiopulmonary bypass surgery. The degradation of fibrinogen and fibrin was followed during and after surgery by means of new sensitive and specific assays and the changes were related to the blood loss measured in the chest tube drain during the first 24 postoperative hours. Although tissue-type plasminogen activator was significantly released into the circulation during the period of extracor-poreal circulation (p <0.01), constantly low levels of fibrinogen degradation products indicated that a systemic generation of plasmin could be controlled by the naturally occurring inhibitors. Following extracorporeal circulation heparin was neutralized by protamine chloride, and in relation to the subsequent generation of fibrin, there was a short period with increased concentrations of fibrinogen degradation products (p <0.01) and a prolonged period of degradation of cross-linked fibrin, as detected by increased concentrations of D-Dimer until 24 h after surgery (p <0.01). Patients with a higher than the median blood loss (520 ml) in the chest tube drain had a significantly higher increase of D-Dimer than patients with a lower than the median blood loss (p <0.05).We conclude that the incorporation of tissue-type plasminogen activator into fibrin and the in situ activation of plasminogen enhance local fibrinolysis, thereby increasing the risk of bleeding in patients undergoing open heart surgery

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