Thrombogenicity of the Injured Vessel Wall – Role of Antithrombin and Heparin

1994 ◽  
Vol 71 (01) ◽  
pp. 147-153 ◽  
Author(s):  
Siw Frebelius ◽  
Ulf Hedin ◽  
Jesper Swedenborg
Keyword(s):  
Vessel Wall ◽  
Antithrombin Iii ◽  
Rabbit Aorta ◽  
Continuous Treatment ◽  
Balloon Injury ◽  
Coagulant Activity ◽  
Risk For Thrombosis ◽  
Inhibition Of Thrombin

SummaryThe thrombogenicity of the vessel wall after endothelial denudation is partly explained by an impaired inhibition of thrombin on the subendothelium. We have previously reported that thrombin coagulant activity can be detected on the vessel wall after balloon injury in vivo. The glycosaminoglycans of the subendothelium differ from those of the endothelium and have a lower catalyzing effect on antithrombin III, but inhibition of thrombin can still be augmented by addition of antithrombin III to the injured vessel surface.In this study the effect of antithrombin III and heparin on thrombin coagulant activity on the vessel wall was studied after in vivo balloon injury of the rabbit aorta using biochemical and immunohistochemical methods and thrombin was analysed after excision of the vessel. Continuous treatment with heparin, lasting until sacrifice of the animal, or treatment with antithrombin III resulted in significant reduction of thrombin coagulant activity on the injured aorta. Heparin given only in conjunction with the injury did not prevent thrombin coagulant activity or deposition of fibrin on the surface.The capacity of the injured vessel wall to inhibit thrombin in vitro was improved on aortic segments obtained from animals receiving antithrombin III but not from those given heparin. It is concluded that treatment with antithrombin III interferes with thrombin appearance on the vessel wall after injury and thereby reduces the risk for thrombosis.

Radiolabeled r-Hirudin as a Measure of Thrombin Activity at, or within, the Rabbit Aorta Wall In Vitro and In Vivo

1994 ◽  
Vol 71 (04) ◽  
pp. 499-506 ◽  
Author(s):  
Mark W C Hatton ◽  
Bonnie Ross-Ouellet
Keyword(s):  
Rabbit Aorta ◽  
Balloon Injury ◽  
Recombinant Hirudin ◽  
Aorta Wall ◽  
Thrombin Activity ◽  
Before And After ◽  
The Matrix

SummaryThe behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit aorta wall has been studied in vitro and in vivo to determine its usefulness as an indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to either sulfopropyl-Sephadex or heparin-Sepharose bound >95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased substantially if the tissue was pre-treated with thrombin; the quantity of l25I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quantity of bound 131I-thrombin (p <0.01). Aortas balloon-injured in vivo were measured for thrombin release from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p <0.001). Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury. After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endo- thelialized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (± 2.5) and 25.6 (±18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thrombin is measured in this way.

scholarly journals On the interaction of rabbit antithrombin III with the luminal surface of the normal and deendothelialized rabbit thoracic aorta in vitro

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 878-886
Author(s):  
MW Hatton ◽  
SL Moar ◽  
M Richardson
Keyword(s):  
Thoracic Aorta ◽  
Antithrombin Iii ◽  
Balloon Catheter ◽  
Rabbit Aorta ◽  
Luminal Surface ◽  
High Affinity ◽  
Antithrombin Activity ◽  
Thrombin Antithrombin Iii Complex

Pure rabbit antithrombin III was isotope labeled (with 125I or 3H) by two different methods; neither procedure caused a loss of antithrombin activity although both methods affected the affinity of the protein for Sepharose-heparin. From segments from freshly excised rabbit aorta, the uptake of isotope-labeled antithrombin III by the endothelium was rapid and saturable, although relatively small compared to the uptake of thrombin; binding of 3H-antithrombin III to the endothelium resembled that of 125I-antithrombin III. Transendothelial passage of antithrombin III into the subendothelial layers (intima-media) was slow and progressive. Endothelium binding was not affected by pretreating the vessel with either heparin, thrombin, or glycosaminoglycan-specific enzymes. Endothelium-bound antithrombin III was not selectively displaced by either heparin or thrombin. In contrast, endothelium-bound thrombin was rapidly dislodged by antithrombin III as a thrombin- antithrombin III complex. The surface of the deendothelialized aorta (ie, subjected to a balloon catheter) bound antithrombin III avidly. Pretreatment of the deendothelialized vessel with glycosaminoglycan- specific enzymes, particularly heparitinase, decreased intima-media binding by up to 80%. 125I-antithrombin III, when bound to the deendothelialized vessel surface, was actively displaced by either heparin, thrombin, or by unlabeled antithrombin III. The relatively poor binding of antithrombin III compared with that of thrombin by the endothelium in vitro supports an earlier proposal (Lollar P, Owen WG: J Clin Invest 66:1222–1230, 1980) that thrombin bound to high-affinity sites, possibly pericellular proteoglycan, of the endothelium is inactivated by plasma antithrombin III in vivo. Such a situation probably holds for large arteries at least.

scholarly journals On the interaction of rabbit antithrombin III with the luminal surface of the normal and deendothelialized rabbit thoracic aorta in vitro

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 878-886 ◽  
Author(s):  
MW Hatton ◽  
SL Moar ◽  
M Richardson
Keyword(s):  
Thoracic Aorta ◽  
Antithrombin Iii ◽  
Balloon Catheter ◽  
Rabbit Aorta ◽  
Luminal Surface ◽  
High Affinity ◽  
Antithrombin Activity ◽  
Thrombin Antithrombin Iii Complex

Abstract Pure rabbit antithrombin III was isotope labeled (with 125I or 3H) by two different methods; neither procedure caused a loss of antithrombin activity although both methods affected the affinity of the protein for Sepharose-heparin. From segments from freshly excised rabbit aorta, the uptake of isotope-labeled antithrombin III by the endothelium was rapid and saturable, although relatively small compared to the uptake of thrombin; binding of 3H-antithrombin III to the endothelium resembled that of 125I-antithrombin III. Transendothelial passage of antithrombin III into the subendothelial layers (intima-media) was slow and progressive. Endothelium binding was not affected by pretreating the vessel with either heparin, thrombin, or glycosaminoglycan-specific enzymes. Endothelium-bound antithrombin III was not selectively displaced by either heparin or thrombin. In contrast, endothelium-bound thrombin was rapidly dislodged by antithrombin III as a thrombin- antithrombin III complex. The surface of the deendothelialized aorta (ie, subjected to a balloon catheter) bound antithrombin III avidly. Pretreatment of the deendothelialized vessel with glycosaminoglycan- specific enzymes, particularly heparitinase, decreased intima-media binding by up to 80%. 125I-antithrombin III, when bound to the deendothelialized vessel surface, was actively displaced by either heparin, thrombin, or by unlabeled antithrombin III. The relatively poor binding of antithrombin III compared with that of thrombin by the endothelium in vitro supports an earlier proposal (Lollar P, Owen WG: J Clin Invest 66:1222–1230, 1980) that thrombin bound to high-affinity sites, possibly pericellular proteoglycan, of the endothelium is inactivated by plasma antithrombin III in vivo. Such a situation probably holds for large arteries at least.

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.

The Effects of Heparin and Annexin V on Fibrin Accretion after Injury in the Jugular Veins of Rabbits

1993 ◽  
Vol 69 (03) ◽  
pp. 227-230 ◽  
Author(s):  
J Van Ryn-McKenna ◽  
H Merk ◽  
T H Müller ◽  
M R Buchanan ◽  
W G Eisert
Keyword(s):  
Vessel Wall ◽  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Specific Effect ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Jugular Veins ◽  
Prothrombinase Complex ◽  
Wall Injury

SummaryWe compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely deendothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p <0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins. We conclude that, i) mild vessel wall injury (selective de-endothelialization) in veins results in a thrombogenic vessel wall; ii) the thrombogenecity of which is not inhibited by prophylactic doses of heparin; but iii) is inhibited by annexin V, which binds to injured vessel wall surface, and inhibits thrombin generation independently of antithrombin III.

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.

Heparin

1985 ◽  
Vol 05 (03) ◽  
pp. 121-126
Author(s):  
L. B. Jaques
Keyword(s):  
Lipoprotein Lipase ◽  
Antithrombin Iii

ZusammenfassungIn vivo bewirkt Heparin das Auftreten einer Lipoprotein-Lipase, einer Diaminoxydase (Histaminase) und anderer Enzyme. In Tierversuchen konnten viele günstige Wirkungen von Heparin und Heparinoiden aufgezeigt werden, wie z.B. Schutzeffekte gegen toxische Medikamente und Prozeduren, gegen Überempfindlichkeitsreaktionen, Änderungen von Hormoneffekten und die Erhöhung der negativen elektrischen Ladung von Körperzellen. Die Einzelwirkungen sind für bestimmte Kettenstrukturen spezifisch. Während Heparin in vitro gerinnungshemmend wirksam ist, zeigt der Vergleich der gerinnungshemmenden Wirkung in der Blutzirkulation mit der chemischen Konzentration im Blut, daß in vivo eine Aktivierung von nicht gerinnungshemmend aktiven Fraktionen bzw. Heparinketten erfolgt. Heparin wird rasch von den Zellen des RES-Systems gegen einen Konzentrationsgradienten aufgenommen, so daß in vivo die Heparinkonzentration im Gefäßendothel lOOOfach höher ist als im Blut.Die Fixierung des Heparins im Endothel vermehrt das elektronegative Potential des Endothels. Diese Wirkung und andere Wirkungen (die Aktivierung von Antithrombin III etc.) sind lokal die Basis der thromboseverhütenden Heparinwirkung. Demnach ist das Endothel das Zielorgan für Heparin.

INCREASED SULFATION IMPROVES THE ABILITY OF VESSEL WALL GLYCOSA-MINOGLYCANS TO REGULATE THROMBIN ACTIVITY AND PROTHROMBIN ACTIVATION IN PLASMA

1987 ◽  
Author(s):  
F A Ofosu ◽  
G J Modi ◽  
M A Blajchman ◽  
M R Buchanan ◽  
E A Johnson
Keyword(s):  
Vessel Wall ◽  
Dermatan Sulfate ◽  
Heparin Cofactor Ii ◽  
Thrombin Inhibition ◽  
Antithrombotic Effects ◽  
The Relationship ◽  
Functional Attribute ◽  
Prothrombin Activation

Studies have shown that dermatan sulfate (DS), heparan sulfate (HS) and chondroitin-4-sulfate (C4S), have antithrombotic properties. The sulfate to carboxylate ratios of these three glycosaminoglycans (GAGs) are approximately half that of heparin (HEP) and the gravimetric dose of each of the three GAGs required to achieve antithrombotic effects in vivo comparable to HEP can be 10 times or more than that of HEPT Since antithrombotic effects depend on the ability of a GAG to catalyse thrombin inhibition and/or to inhibit prothrombin activation, we determined the relationship between the extent of sulfation of various GAGs and their effects on these two reactions in normal plasma. In addition to the three GAGs, DS, HS and C4S were resulfated in vitro to yield DS-S, HS-S and C4S-S, each with a sulfate to carboxylate ratio comparable to that of heparin. As summarized below, increased sulfation improved the ability of a GAG to catalyse thrombin inhibition and to inhibit prothrombin activation. Increasing the degree of sulfation primarily improved the ability of a GAG to accelerate the inhibition of thrombin by heparin cofactor II. The degree of sulfation, therefore, appears to be an important functional attribute of the ability of vessel wall GAGs to regulate the formation and activity of thrombin in plasma.

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.

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.

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