scholarly journals In Vitro Characterization of Rabbit Thrombin, Antithrombin III, and Thrombin-Antithrombin III Complex, and Determination of Their Survival Times in Vivo

1977 ◽  
Author(s):  
Christine N. Vogel ◽  
Kingdon S. Henry ◽  
Roger L. Lundblad
Keyword(s):  
Gel Electrophoresis ◽  
Half Life ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
In Vivo Studies ◽  
Survival Times ◽  
At Iii

Our intention is to study the interaction of rabbit thrombin with antithrombin III (AT-III) in vitro and in vivo. After activation of crude prothrombin with tissue thromboplastin and CaCl2, thrombin was purified and showed two species of thrombin with molecular weights of 36,000 and 39,000 daltons as determined by sodium dodecyl sulfate discontinuous gel electrophoresis. Rabbit AT-III was purified using a heparin agarose column and had a molecular weight of 55,000 daltons. The inhibition of thrombin by AT-III was followed by fibrinogen clotting assays and an AT-III-thrombin complex was observed on gel electrophoresis. For the in vivo studies both thrombin and AT-III were radiolabelled with Na125i using the solid state lactoperoxidase method and retained 99% of the pre-iodinated specific activity. Radiolabelled thrombin and a radiolabelled AT-III-thrombin complex were injected into different rabbits. The rate of removal of both was very similar with a half-life of approximately 9 hours. When radiolabelled AT-III was injected, the half-life was approximately 60 hours. Since the disappearance rate of thrombin more closely approximates that of the preformed AT-III-thrombin complex and is clearly shorter than the turnover rate of AT-III, the possibility is raised that thrombin combines in vivo with a native inhibitor such as AT-III and may in fact be removed from the circulation as a complex rather than as a native molecule.

Studies of Heparin Affinity to Antithrombin III and Other Proteins in Vitro and in Vivo

1977 ◽  
Vol 38 (03) ◽  
pp. 0685-0695 ◽  
Author(s):  
Y Takeda ◽  
N Kobayashi
Keyword(s):  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Heparin Binding ◽  
Life Values ◽  
Canine Plasma ◽  
Heparin Affinity

SummaryProperties of human, canine, and porcine heparin and S-35-heparin were first studied. Their electrophoretic mobility through 10 g % polyacrylamide gels, specific activity and their filtration patterns through Sephadex G-200 columns were closely similar. Then, S-35-heparin (0.5 mg) and cold heparin (27 mg) were simultaneously injected intravenously into 5 dogs and their plasma behavior was compared. The plasma half-lives of S-35-heparin averaged 1.18 ± 0. 13 (SD) hr and was identical with that of cold heparin, but the half-lives were much shorter and averaged 0.46 ± 0.08 (SD) hr in 5 dogs when S-35-heparin alone was injected. Studies were next made of heparin affinity to proteins including canine antithrombin III (AT) by the use of Sephadex G-200 chromatography, polyacrylamide gel electrophoresis, and S-35-heparin as a tracer. It was found that S-35-heparin-binding to alpha1, beta, and gammaglobulins and fibrinogen was readily separable upon addition of 5 mg cold heparin, but that the binding to AT was inseparable by addition of cold heparin or by electrophoresis. However, in canine plasma, both S-35-heparin and cold heparin were mostly bound to proteins other than AT, suggesting that this might be the case in vivo. To further substantiate this, studies were made of the comparative behavior of I-125-labeled AT (I-125-AT) and S-35-heparin in dogs with the idea that the plasma half-lives of both should be equal if they were irreversibly bound to each other. The plasma half-lives of I-125-AT averaged 2.10 ± 0.05 (SD) days in 5 male dogs and 1.99 ± 0.04 (SD) days in 5 female dogs, and were much different from the half-life values of S-35-heparin as given above. These results indicate that heparin in vitro is more tightly bound to AT than to other proteins, that heparin in vivo is not irreversibly bound to AT and suggest that it is mostly bound to proteins other than AT in vivo.

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.

The Control Of Antithrombotic Prophylaxis And Therapy: A Pro-Coagulant Effect Of Heparin

1981 ◽  
Author(s):  
E Szwarcer ◽  
R Giuliani ◽  
E Martinez Aquino
Keyword(s):  
Blood Coagulation ◽  
Antithrombin Iii ◽  
Fixed Time ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Clotting Factors ◽  
Platelet Poor Plasma ◽  
Normal Platelet

For studying heparin effect on blood coagulation and on inhibitors, the drug was added at increasing amounts to a normal platelet poor plasma (PPP), and to plasmas of patients with variable amounts of clotting factors (cirrhotic, pregnant, etc) -IN VITRO STUDIES-, and infused to the same individuals -IN VIVO STUDIES-. Modifications on two clotting assays (KCCT-TT) were compared to heparin potentiating effect on AntiXa (Denson & Bonnar tech).When studied IN VITRO, the sensibility of KCCT, TT, and AntiXa techniques for heparin measurement was similar. IN VIVO, an apparently greater sensibility using AntiXa technique was observed.For determining if this phenomena was related to a specific enhanced potentiating effect of the inhibitor against Xa, exerted by heparin IN VIVO, experiences were repeated IN VITRO and IN VIVO, measuring heparin effect on KCCT, TT, and on the inhibitor, studied against Xa and thrombin. A personal technique was used for the measurement of Antithrombin III heparin potentiating effect, using diluted platelet poor test plasma, heated (56°C 15’) and incubated with thrombin during a fixed time, and reading residual thrombin on citrated human PPP. IN VITRO, all techniques were similar in their ability to show heparin presence.IN VIVO, the potentiating effect of heparin on the inhibitor, measured against Xa or thrombin, was greater than the changes obtained on KCCT or TT.So, AntiXa-Antithrombin III techniques seem to be more sensitive for heparin measurement IN VIVO.This “dissociation” of results in between the potentiating effect on the inhibitor, that is not simultaneously exerted on global coagulation, is interpreted as a heparin pro-coagulant effect, exerted by the drug IN VIVO.

STUDIES ON THE ANTICOAGULANT AND ANTITHROMBOTIC ACTIONS OF DERMATANS AND THEIR SULFATED DERIVATIVES

1987 ◽  
Author(s):  
D Hoppensteadt ◽  
A Kumar ◽  
J Fareed ◽  
J Mardigian
Keyword(s):  
Antithrombin Iii ◽  
Femoral Vein ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Protease Inhibition ◽  
Thrombosis Model ◽  
Antithrombotic Effects ◽  
Activated Prothrombin Complex Concentrates

Non-antithrombin III mediated effects such as interaction with heparin cofactor II, modulation of endothelium and polymorphonuclear leukocytes contribute to the overall antithrombotic effects of glycosaminoglycans. In order to study the role of these dermatans, we investigated their in vitro anticoagulant effects using the clot based (PT, APTT, TT, and Heptest), antiprotease (anti IIa and anti Xa) and Thromboplastin C activated fibrinopeptide A generation test. The in vivo antithrombotic actions were investigated, against activated and non activated prothrombin complex concentrates, and in combination with Russells viper venom in jugular and femoral vein stasis thrombosis models (rabbit). The dermatans studied consisted of a standard dermatan of porcine intestinal origin and four sulfated dermatans with varying degrees of sulfation. All of the dermatans studied showed weak anticoagulant effects on the routinely performed clot based assays. Marked variability was seen on the protease inhibition (anti Xa and anti IIa) assays. In the in vivo studies all dermatans studied showed varying degrees of antithrombotic actions against various thrombogenic agents in a modified stasis thrombosis model. Sulfation appeared to produce stronger anticoagulant effects as determined by in vitro assays, whereas the intravenous antithrombotic actions of native dermatan were stronger than sulfated derivatives. This data suggests that dermatans produce their antithrombotic actions via non-antithrombin III mediated pathways. Furthermore, in vitro testing methods are of limited value in the evaluation of the biologic actions of dermatans and their derivatives.

In Vitro and In Vivo Characterization of Full-Length rFVIII Modified with PEG Via Coupling to Primary Amino Groups.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3147-3147 ◽  
Author(s):  
Peter L. Turecek ◽  
Jürgen Siekmann ◽  
Herbert Gritsch ◽  
Katalin Váradi ◽  
Rafi-Uddin Ahmad ◽  
...  
Keyword(s):  
Half Life ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Full Length ◽  
Thrombin Inhibitor ◽  
Knock Out ◽  
Knock Out Mice

Abstract Chemical modification of recombinant therapeutic proteins with PEG has been shown to enhance the biological half-life. Here we assess the effect of PEGylation on FVIII. Full-length rFVIII bulk drug substance from protein-free fermentation (Advate process, Baxter) was conditioned into a buffer suitable for coupling to polyethylene glycol succinimidyl succinate (linear PEG, 5 kDa PEG chain length). PEG was covalently bound by amine coupling preferentially to lysine residues of FVIII at neutral pH. PEG was removed by ion-exchange chromatography and the PEG-FVIII derivative was concentrated by ultra-diafiltration. The conjugates thus obtained retained about 30–40% of the activity of non-modified rFVIII. The specific activity decreased with the amount of PEG linked to the FVIII molecule. In SDS-PAGE and immunoblot studies PEGylated rFVIII showed a band pattern similar to unmodified FVIII with full-length, heavy chain fragments of 180 kDa and 120 kDa and the light chain fragment of 80 kDa. PEGylation also occurred to a high extent in the B domain of FVIII. All bands appeared broadened due to the attachment of polymeric PEG. The maintenance of functionality of FVIII was demonstrated by its potential to be activated and inactivated by thrombin. In the assay PEGylated and unmodified FVIII were incubated with 1 nM thrombin. Sub-samples were drawn at intervals up to 40 minutes and added to a mixture of FIXa, FX, phospholipid vesicles and Ca2+ containing a thrombin inhibitor. After 3 minutes incubation at 37°C the amount of activated FX (FXa) was measured using a FXa-specific chromogenic substrate. Unmodified rFVIII showed a typical picture of an immediate increase in FXa activity and a subsequent decline with no further FXa generation after 15 minutes. PEGylated rFVIII was activated to the same extent as unmodified FVIII but the decay in FXa generation was slower and did not reach the zero level, even 40 minutes after incubation. The formation of the typical thrombin cleavage fragments, with unmodified as well as PEGylated rFVIII, was demonstrated in a Western blot analysis. The slower inactivation by thrombin was also seen there. The pharmacokinetic properties of PEGylated rFVIII compared with rFVIII were investigated in hemophilia A knock-out mice. Both preparations were applied at a dose of 200 IU rFVIII/kg and groups of mice (n=5) were exsanguinated at several time points up to 24 hours. Terminal half-life for PEGylated rFVIII was calculated at 4.9 hours compared with 1.9 hours for unmodified rFVIII in hemophilia A knock-out mice. AUC was approximately doubled. These results indicate that rFVIII can be biochemically modified with PEG whilst at least partly retaining its major functions, but at the same time prolonging its survival in the circulation of hemophilic mice.

In Vivo Studies On The Inhibition Of Coagulation By A Heparin Analogue

1981 ◽  
Author(s):  
U Schmitz-Huebner ◽  
F Asbeck ◽  
J van de Loo
Keyword(s):  
Plasma Sample ◽  
Anticoagulant Activity ◽  
Inhibitory Effect ◽  
In Vivo Studies ◽  
At Iii ◽  
In Vivo Effects ◽  
Higher Doses ◽  
Free Plasma

SSHA, a semi-synthetic heparin analogue belonging to the chondroitin family, was reported to induce considerable anti-Xa activity in vivo being practically inactive in vitro. In a study designed to elucidate further the in vivo effects of this drug, SSHA and sodium heparin from porcine intestinal mucosa were injected subcutaneously into six volunteers on separate occasions over a period of three days in a cross-over trial. Before injection and 2,4,6,8 hours afterwards, the heparin-like activity was measured by means of the APTT, the anti -Xa clotting test and two chromogenic substrate assays. The results show that SSHA mediates both anti-Xa and antithrombin activities in vivo. A comparison between the effects of SSHA and heparin is problematical, due to the heterogeneity of different heparin preparations. Low doses of the analogue (45 mg s.c.) induce proportionally higher and longer lasting anti-Xa activities than higher doses (90 mg s.c.). In an attempt to identify the mediator involved in the anticoagulant activity induced by SSHA in vivo, antithrombin III AT III) was removed from a plasma sample of one the subjects obtaining SSHA injections by immunosorption using Sepharose IVb coupled with antibodies against AT III. The AT III free plasma obtained was found to be devoid of heparin-like activity in the anti-Xa clotting test but it maintained its anticoagulant activity in the APTT assay. When purified AT III was added to this plasma its anti-Xa activity was largely restored. In conclusion, the inhibitory effect of SSHA on coagulation seems to involve at least two mechanisms: a direct one which does not depend on AT III and an indirect one, induced in vivo and mediated by AT III.

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.

Evaluation of the safety, recovery, half-life, and clinical efficacy of antithrombin III (human) in patients with hereditary antithrombin III deficiency. Cooperative Study Group [see comments]

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 33-39 ◽  
Author(s):  
D Menache ◽  
JP O'Malley ◽  
JB Schorr ◽  
B Wagner ◽  
C Williams ◽  
...  
Keyword(s):  
Half Life ◽  
Antithrombin Iii ◽  
Clinical Symptoms ◽  
Cooperative Study Group ◽  
Significant Difference ◽  
At Iii ◽  
Thrombotic Complications ◽  
Antithrombin Iii Deficiency

Antithrombin III (Human) (AT III) was administered to 18 patients with documented hereditary AT III deficiency. In eight patients with no ongoing clinical symptoms of thrombosis, the percent increase per unit AT III infused per kilogram of body weight ranged from 1.56% to 2.74%, and the half-life from 43.3 to 77.0 hours. No significant difference was noted between patients receiving and those not receiving coumarin therapy. In clinically ill patients, the in vivo recovery was significantly lower and ranged from 0.64% to 1.90% increase per unit AT III infused/kg. Efficacy of AT III was evaluated in 13 patients for the prevention or treatment of thrombosis. AT III was efficacious as assessed by the absence of thrombotic complications after surgery and/or parturition, and the nonextension and nonrecurrence of thrombosis in patients exhibiting an acute thrombotic episode. No side effects were noted. Follow-up studies indicated no hepatitis B seroconversion and no alanine aminotransferase elevations in patients who were not transfused with other blood products.

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.

Three Types of Hereditary Antiterombin III Deficiency

1979 ◽  
Author(s):  
I. Nagy ◽  
H. Losonczy
Keyword(s):  
Antithrombin Iii ◽  
Clinical Signs ◽  
Type I ◽  
Type Ii ◽  
Type Iii ◽  
Basic Activity ◽  
At Iii ◽  
And Function

The authors detected in the last seven years 15 patients with hereditary antithrombin III/AT III/ abnormality. All of them had typical clinical signs of recurrent arterious and venous thromboembolie. The abnormality inherited as an autosomal trait. Three types of the abnormality could be observed. In Type I both quantity and function of AT III were extremely decreased. In type II AT III is normal in quantity but abnormal in function. In Type III AT III is quantitatively normal and also its function seems normal as far as its basic activity is concerned /activity measured in absence of heparin/, but its abnormality becomes manifest in the presence of heparin in vitro/and also in vivo/. 5 of the patients belonged to Type I, 4 to Type II and 6 to Type III. In 60 examined family members of the 15 patients an abnormal AT III could be observed in 44, clinical signs in 23.The examination of AT III activity in the presence of a given amount of heparin ia of great importance in recognition of the different types of antithrombin III abnormalities.

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