Adverse Effect of Heparin in Thrombin-Antithrombin III Interaction

1975 ◽  
Vol 34 (03) ◽  
pp. 748-762 ◽  
Author(s):  
Ewa Marciniak
Keyword(s):  
Adverse Effect ◽  
Antithrombin Iii ◽  
Enzyme Inhibitor ◽  
Active Form ◽  
Factor Xa ◽  
Heparin Therapy ◽  
Inhibitory Capacity ◽  
Antithrombin Iii Activity

SummaryThrombin, while reacting in the presence of heparin, impairs the inhibitory capacity of antithrombin III so that subsequent inhibition of thrombin or factor Xa is decreased or abolished. This adverse effect of heparin has been observed directly with at least 1.5 Iowa units of thrombin per each unit of purified human antithrombin III participating in the reaction. The inhibitory capacity was then totally destroyed and some residual thrombin remained in the active form. With a lower enzyme/inhibitor ratio inactivation of thrombin in the presence of heparin was fast and complete, however, a significant decrease of inhibitory capacity below that found in reaction without heparin, has been established by measuring the residual antithrombin III activity. In defibrinated human plasma at least 2 units of thrombin per each antithrombin III unit were required to demonstrate directly the adverse effect of heparin, but a fast depletion of inhibitory capacity has been also observed after repeated additions of small thrombin portions into plasma heparinized in vitro or in vivo. Portions of enzyme initially added disappeared with great velocity; subsequent portions, however, accumulated building up a high thrombin level not seen in the absence of heparin. The accumulation of residual enzyme was more extensive in plasma containing about 1 heparin unit per ml than anticoagulant at lower concentrations and was particularly noticeable in antithrombin III deficient plasma. These results may have some bearings on the approach to heparin therapy in the event when thrombin continuously generates or when a marked deficiency of antithrombin III exists.

Adverse Effect of Heparin on Thrombin Inactivation

1975 ◽  
Author(s):  
E. Marciniak
Keyword(s):  
Adverse Effect ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Binding Properties ◽  
Inhibitory Capacity ◽  
Antithrombin Iii Deficiency ◽  
Final Amount ◽  
Native Plasma

In the presence of heparin thrombin, although fast inactivated, impairs the inhibitory capacity of antithrombin III, in result of which the final amount of neutralized enzyme markedly decreases. This adverse effect of heparin was found during the reaction of purified thrombin with both purified human antithrombin III and native plasma hepariniz purified thrombin with both purified human antithrombin III and native plasma heparinized in vitro or in vivo. In the absence of heparin, at concentration equal to that in normal plasma antithrombin III binds 450 Iowa units of thrombin; in the presence of heparin (at 1 unit concentration) this binding is reduced to 145 thrombin units. A fast depletion of inhibitory capacity is also evident after a stepwise addition of thrombin in small installments into a medium containing antithrombin III and heparin. Portions of enzyme initially added disappear with great velocity; subsequent additions, however, accumulate building up a high thrombin level not seen in the absence of heparin. The escalation of thrombin is reversely proportional to the reacting antithrombin III level, thus especially noticeable in antithrombin III deficient plasma. Residual thrombin left in the presence of heparin disappears at a fast rate upon a new addition of antithrombin III. No decrease in anticoagulant properties of heparin is observed during these interactions. Binding of factor Xa to antithrombin III which reacted with thrombin and heparin is also decreased or abolished.These results indicate that in the presence of heparin thrombin not only exposes rapidly a binding site on the inhibitor, but also causes a further change leading to the deletion of antithrombin III binding properties. This may explain adverse, thrombotic effect of heparin sporadically seen in vivo, and suggests that heparin should be applied with caution in patients with antithrombin III deficiency.

Heparin Therapy In Dispholidus Typus Envenomation: An Experimental Study

1981 ◽  
Author(s):  
B A Bradlow ◽  
P M Atkinson ◽  
M Rebello ◽  
M C Gaillard
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Therapy ◽  
In Vivo Studies ◽  
Intrinsic Pathway ◽  
Heparin Resistance ◽  
Very High ◽  
Direct Activator

The coagulant action of Dispholidus typus venom was relatively resistant to inhibition by heparin in vitro. Heparin concentrations that inhibited coagulation due to either intrinsic pathway or Russell’s viper venom activation had little effect on coagulation due to D. Typus venom. At very high heparin to venom ratios, similar to ratios attainable in vivo this resistance could be overcome. The resistance could not be attributed to an abnormal thrombin produced by the venom since the thrombin produced from purified prothrombin by venom action reacted similarly to the thrombin produced by Factor Xa activation with purified antithrombin III. Thrombin produced from whole plasma by venom action also reacted similarly to physiological thrombin with antithrombin III in a crossed immunoelectrophoresis system. Incubation of venom with heparin and with antithrombin III did not alter the activities of these inhibitors. The heparin resistance may therefore be due to the fact that the venom is a direct activator of prothrombin. In vivo studies in rabbits indicated that heparin administered simultaneously with venom delayed the onset and reduced the severity of disseminated intravascular coagulation. Heparin administered later was much less effective. Early heparin therapy may be of value in human victims when specific antivenom is not available.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Thrombin-Based Antithrombin Assays Show Overestimation of Antithrombin III Activity in Patients on Heparin Therapy due to Heparin Cofactor II Influence

1994 ◽  
Vol 71 (03) ◽  
pp. 280-283 ◽  
Author(s):  
Jürgen Bohner ◽  
Klaus-Werner von Pape ◽  
Mathias Blaurock
Keyword(s):  
Plasma Levels ◽  
Antithrombin Iii ◽  
Clinical Chemistry ◽  
Factor Xa ◽  
Heparin Therapy ◽  
Heparin Cofactor Ii ◽  
Activity Method ◽  
Antithrombin Iii Activity

SummaryAn extensive comparison has been performed on the clinical chemistry automate Hitachi 717 between thrombin-and Factor Xa-based methods for determination of antithrombin III activity. In 460 patients who did not receive any heparin therapy the agreement between assays was in general close although the thrombin-based methods resulted in slightly higher assignments of 0.3—2.6% antithrombin III activity. The discrepancy was, however, substantial in plasmas from patients receiving heparin of >20000 IU/day, resulting in plasma levels of heparin of 0.8-1.2 IU/ml. Thus, analysis of 102 patients showed that the thrombin-based methods resulted in, on average, 7-16% higher assignment of antithrombin III activity as compared to the Factor Xa-based method used.Addition of antibodies to antithrombin III and heparin cofactor II revealed that the discrepancy was primarily due to contribution of heparin cofactor II activity in the thrombin-based methods. The results thus suggest that the Factor Xa-based antithrombin III activity method provides more valid results in patients on heparin therapy.

Bleeding in Uremic Patients after Carbenicillin

1976 ◽  
Vol 36 (01) ◽  
pp. 115-126 ◽  
Author(s):  
K Andrassy ◽  
E Weischedel ◽  
E Ritz ◽  
T Andrassy
Keyword(s):  
Platelet Function ◽  
Antithrombin Iii ◽  
Serum Levels ◽  
Coagulation Factors ◽  
Coagulation System ◽  
Hemorrhagic Diathesis ◽  
Thrombin Time ◽  
Antithrombin Iii Activity

SummaryHemorrhagic diathesis was observed in patients with renal insufficiency after carbenicillin at serum levels > 300 μg/ml. Normal coagulation factors (F. I, II, V, VII, VIII, X), normal PTT, normal platelet counts, negative ethanol gelation test (fibrin monomers) were found as well as a prolongation of thromboplastin time (Quick), thrombin time, reptilase time and thrombin coagulase time. Platelet function was disturbed. In addition, the plasmatic system was involved: inhibition of fibrinogen-fibrin conversion (Belitser assay) and enhanced antithrombin III activity; in vivo the latter was ascribed to a heparin-like activity. In vitro, abnormal fibrinogen-fibrin conversion and a modified electrophoretic mobility of antithrombin III was seen: however an enhanced antithrombin III activity in vitro was not found with carbenicillin and various penicillin derivatives.This study demonstrates that carbenicillin, in addition to its known effect on platelet function, also disturbs the plasmatic coagulation system. This additional effect of carbenicillin is clinically important since protamin chloride effectively blocks bleeding without interfering with antibacterial activity.Both penicillin and penicillin derivatives have been shown to interfere with hemostasis and to cause clinically manifest hemorrhagic diathesis (Fleming and Fish 1947, Lurie et al. 1970a, b, McClure et al. 1970, Yudis et al. 1972, Demos 1971, Waisbren et al. 1971). Carbenicillin interferes with ADP-, collagen- or thrombin-induced platelet aggregation and with the release reaction both in vivo (McClure et al. 1970, Cazenave et al. 1973) and in vitro (McClure et al. 1970, Cazenave et al. 1973). In addition Lurie and colleagues (1970b) concluded that an inhibition of the conversion of fibrinogen to fibrin is involved although no experimental details were given. Later Brown and colleagues (1974) concluded that carbenicillin at usual dose levels “only affects the platelet component of hemostasis and has little effect on fibrin formation or other phases of coagulation in patients with normal renal function”.

Effect of Heparin on Antithrombin III Activity During Delivery and Early Puerperium

1975 ◽  
Author(s):  
I. Rákóczi ◽  
B. Garadnay ◽  
L. Arnold ◽  
I. Gáti
Keyword(s):  
Pregnant Women ◽  
Blood Coagulation ◽  
Antithrombin Iii ◽  
Modified Method ◽  
Heparin Treatment ◽  
At Iii ◽  
Antithrombin Iii Activity

It is known that antithrombin III (AT III) is the main inhibitor of blood coagulation. It inactivates thrombin and activated × factor. Heparin increases the AT III activity in vitro as well as in vivo. The authors have studied the AT III activity in sera of 30 pregnant women, at term, before labour started (2–24 hrs) and 30 as well as 60 min after the birth of placenta and daily for five days after delivery. The AT III activity was determined using the modified method of Gerendas. Out of the 30 women 15 were given 5000 IU heparin subcutaneously 1½—2 hours before delivery. In the 15 women with no heparin treatment AT III activity of serum significantly decreased after birth of placenta compared with the predelivery value and became normal on the fifth postpartum day. Heparin given subcutaneously prevented development of this decrease.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

1990 ◽  
Vol 63 (02) ◽  
pp. 220-223 ◽  
Author(s):  
J Hauptmann ◽  
B Kaiser ◽  
G Nowak ◽  
J Stürzebecher ◽  
F Markwardt
Keyword(s):  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Factor Xa Inhibitors ◽  
Venous Stasis ◽  
Clotting Time ◽  
Thrombosis Model ◽  
Activity Factor ◽  
Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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