Antithrombotic Effects of Synthetic Pentasaccharide with High Affinity for Plasma Antithrombin III in Non-Human Primates

1993 ◽  
Vol 70 (04) ◽  
pp. 631-635 ◽  
Author(s):  
Yves Cadroy ◽  
Stephen R Hanson ◽  
Laurence A Harker
Keyword(s):  
Antithrombin Iii ◽  
Thrombus Formation ◽  
Factor Xa ◽  
High Dose ◽  
Antithrombin Activity ◽  
Model Combining ◽  
Baboon Model ◽  
Dependent Inactivation ◽  
Antithrombotic Effects

SummaryThe pentasaccharide (PS) comprising the minimal heparin structure capable of binding with antithrombin III (ATIII) and exhibiting anti-factor Xa (anti-fXa) activity in plasma without producing detectable antithrombin activity, has been evaluated for its relative antithrombotic and antihemostatic effects in a baboon model combining both platelet-rich and fibrin-rich thrombosis. Thrombosis was produced in a two-component thrombogenic device incorporated into an exteriorized femoral arteriovenous (AV) shunt in baboons; the proximal component constituted a segment of collagen-coated tubing and induced platelet-rich arterial-type thrombus, while the distal component consisted of an expanded chamber producing static and disturbed flow and initiated fibrin-rich venous-type thrombosis. Thrombus formation was measured as the deposition of 111In-platelets and the accumulation of 125I-fibrin. PS was administered intravenously to maintain plasma anti-fXa activity at three different levels: a) low dose (LD) 0.3 ± 0.1 U/ml; b) intermediate dose (ID) 0.6 ± 0.1 U/ml; and c) high dose (HD) 5.6 ± 0.4 U/ml.In untreated Controls, thrombus formed rapidly, reaching a plateau by 40 min of 2.3 ± 0.2 × 109 platelets and 0.62 ± 0.04 mg fibrin deposited on the collagen segments, and 1.9 ±0.4 × 109 platelets and 3.3 ± 0.4 mg fibrin accumulated in the chambers. PS at HD abolished the formation of fibrin-rich thrombus in the chambers and decreased platelet-rich thrombus on collagen by half (p <0.01), while the ID reduced fibrin-rich thrombus in the chambers by about half (p <0.01) but had no effect on platelet-rich thrombus forming on the segments of collagen-coated tubing (p >0.5). Despite its lack of antithrombin activity, PS also decreased plasma fibrinopeptide A levels in a dose-response manner. However, PS had no effect on platelet hemostatic function in vivo, as measured by template bleeding time (BT) determinations (p >0.5). Despite the ability of PS-ATIII complex to inactivate soluble fXa, the complex lacked significant inhibitory activity for fXa immobilized to thrombus formed in vivo. Thus, PS-dependent inactivation of soluble fXa produces antithrombotic effects, primarily for venous-type thrombosis, that are equipotent to Standard heparin on a gravimetric basis, but more sparing of platelet hemostatic function.

scholarly journals The relative importance of thrombin inhibition and factor Xa inhibition to the antithrombotic effects of heparin

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 198-201 ◽  
Author(s):  
MR Buchanan ◽  
B Boneu ◽  
F Ofosu ◽  
J Hirsh
Keyword(s):  
Dermatan Sulfate ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Sulfated Polysaccharides ◽  
Type Model ◽  
Relative Importance ◽  
Antithrombin Activity ◽  
Antithrombotic Effects

Abstract The relative importance of antithrombin and anti-factor Xa activities of heparin fractions required to achieve optimal antithrombotic effects is unknown. To study this, we measured the effects of standard heparin, an octasaccharide heparin fraction (anti-factor Xa activity only), and dermatan sulfate (antithrombin activity only) on the prevention of thrombosis and related this to their anticoagulant effects in vivo in rabbits. Thrombosis was measured as the incorporation of 125I- fibrinogen into tissue thromboplastin-induced thrombi using a Wessler- type model. Ex vivo changes in thrombin clotting time (TCT) were used as an index of antithrombin activity, and a chromogenic anti-factor Xa assay was used to measure anti-factor Xa activity. In addition, the ability of the three sulfated polysaccharides to simultaneously inhibit the generation of thrombin activity and to enhance the inactivation of the factor Xa added to initiate thrombin generation in plasma was determined. Standard heparin, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 90%, prolonged the TCT by two seconds, and resulted in an anti-factor Xa level of 0.32 U/mL. The octasaccharide heparin fraction, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 41%, had no effect on the TCT, and resulted in an anti-factor Xa level of 0.28 U/mL. Higher doses of the octasaccharide resulted in a further increase in the anti-factor Xa levels but had no further effect on thrombus formation. Dermatan sulfate, in a dose of 500 micrograms/kg, inhibited thrombus formation by 95%, but had no affect on the TCT. These results indicate that the antithrombotic effect achieved by inhibiting factor Xa is limited and that better antithrombotic effects are achieved by heparin or heparin- like substances capable of influencing the inactivation and/or the generation of thrombin.

scholarly journals The relative importance of thrombin inhibition and factor Xa inhibition to the antithrombotic effects of heparin

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 198-201 ◽  
Author(s):  
MR Buchanan ◽  
B Boneu ◽  
F Ofosu ◽  
J Hirsh
Keyword(s):  
Dermatan Sulfate ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Sulfated Polysaccharides ◽  
Type Model ◽  
Relative Importance ◽  
Antithrombin Activity ◽  
Antithrombotic Effects

The relative importance of antithrombin and anti-factor Xa activities of heparin fractions required to achieve optimal antithrombotic effects is unknown. To study this, we measured the effects of standard heparin, an octasaccharide heparin fraction (anti-factor Xa activity only), and dermatan sulfate (antithrombin activity only) on the prevention of thrombosis and related this to their anticoagulant effects in vivo in rabbits. Thrombosis was measured as the incorporation of 125I- fibrinogen into tissue thromboplastin-induced thrombi using a Wessler- type model. Ex vivo changes in thrombin clotting time (TCT) were used as an index of antithrombin activity, and a chromogenic anti-factor Xa assay was used to measure anti-factor Xa activity. In addition, the ability of the three sulfated polysaccharides to simultaneously inhibit the generation of thrombin activity and to enhance the inactivation of the factor Xa added to initiate thrombin generation in plasma was determined. Standard heparin, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 90%, prolonged the TCT by two seconds, and resulted in an anti-factor Xa level of 0.32 U/mL. The octasaccharide heparin fraction, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 41%, had no effect on the TCT, and resulted in an anti-factor Xa level of 0.28 U/mL. Higher doses of the octasaccharide resulted in a further increase in the anti-factor Xa levels but had no further effect on thrombus formation. Dermatan sulfate, in a dose of 500 micrograms/kg, inhibited thrombus formation by 95%, but had no affect on the TCT. These results indicate that the antithrombotic effect achieved by inhibiting factor Xa is limited and that better antithrombotic effects are achieved by heparin or heparin- like substances capable of influencing the inactivation and/or the generation of thrombin.

Impact of Antithrombin Deficiency on Efficacies of DU-176b, a Novel Orally Active Direct Factor Xa Inhibitor, and Antithrombin Dependent Anticoagulants, Fondaparinux and Heparin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1874-1874 ◽  
Author(s):  
Toshio Fukuda ◽  
Yuko Honda ◽  
Chikako Matsumoto ◽  
Nobutoshi Sugiyama ◽  
Tadashi Matsushita ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Factor Xa ◽  
In Vitro Study ◽  
Antithrombotic Effects ◽  
At Deficiency ◽  
Fxa Inhibitor ◽  
Orally Active ◽  
Relative Potencies

Abstract Antithrombin (AT) is a major physiological inhibitor of coagulation factors, primarily inhibiting thrombin and factor Xa (FXa). Binding of heparin and its related pentasaccharides, fondaparinux, to AT dramatically accelerates inhibition of thrombin and FXa. Entire AT-dependency of heparins may result in decreased anticoagulant effects in patients with inherited or acquired AT deficiencies. Objectives: We have developed an orally active direct (i.e. AT-independent) FXa inhibitor, DU-176b. The objectives of this study were to examine the anticoagulant and antithrombotic effects of DU-176b, fondaparinux, and heparin in heterozygous AT deficient (AT+/−) mice (Refs 1, 2), and to determine the impact of AT deficiency on the efficacies of these anticoagulants. Methods: [In vitro study] Plasma obtained from wild type (AT+/+, C57BL/6J) and AT+/− mice were subjected to measurement of levels of AT antigen and activity. The anticoagulant effects on prothrombin time (PT) and activated partial thromboplastin time (APTT) was measured and the drug concentrations were calculated required to double the clotting time (CT2). [In vivo study] Male AT+/+ and AT+/− mice were fasted over night. Thrombosis was induced in the inferior vena cava by applying filter paper (1 x 5 mm) presoaked in 15% FeCl3 for 10 min. Thrombus was removed 60 min after FeCl3 treatment and its protein content was assessed by Bradford method. DU-176b was orally administered 60 min before, fondaparinux was given s.c. 30 min before, and heparin was injected into the jugular vein 3 min before thrombus induction. Relative potencies of antithrombotic effects in AT+/− mice to those in AT+/+ mice were analyzed by parallel line assay. Results: [In vitro study] Plasma levels of AT antigen and activity in AT+/− mice were deceased to 40% compared with AT+/+ plasma. PT-CT2 of DU-176b was 0.72 μM in AT+/+ plasma and 0.74 μM in AT+/− plasma, respectively, indicating that anticoagulant activity of the direct FXa inhibitor was not affected by heterozygous AT deficiency. APTT-CT2 of fondaparinux and heparin in AT+/+ plasma was 3.8 μM and 14 mU/mL, respectively, whereas APTT-CT2 in AT+/− plasma was 9.2 μM and 20 mU/mL, respectively. Therefore, anticoagulant activities of such AT-dependent inhibitors were attenuated in AT+/− plasma. [In vivo study] All three anticoagulants inhibited venous thrombus formation of AT+/+ mice in dose-dependent manners. In AT+/− mice, the antithrombotic effects of fondaparinux and heparin were less potent than those in AT+/+ mice. In contrast, DU-176b prevented thrombus formation equipotently in both mice. Relative potencies of DU-176b, fondaparinux and heparin were 0.84, 0.40, and 0.70, respectively. Conclusion: DU-176b exerts a comparable antithrombotic effect even in individuals with low plasma AT antigens and activities. Thus, DU-176b may be prioritized over AT-dependent agents for use at the fixed dose in patients with lower plasma AT concentrations.

The Relationship between Antithrombin III, Progressive Antithrombin Activity and Anti Xa Activity in Plasma

1975 ◽  
Author(s):  
Katherine Whigham ◽  
P. W. Howie ◽  
C. D. Forbes ◽  
C. R. M. Prentice
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Normal Subjects ◽  
Strong Positive Correlation ◽  
Positive Correlation ◽  
Antithrombin Activity ◽  
Α2 Macroglobulin ◽  
Immunological Assays ◽  
The Relationship

In 30 normal subjects, progressive antithrombin activity, as measured by the rate of thrombin neutralisation in ancrod-defibrinated plasma, was compared with antithrombin III, as measured by radial immunodiffusion. No significant correlation was found between the two methods of antithrombin measurement (r = —0.101). Similarly, no correlation was found between progressive antithrombin activity and immunological measurements of α2 macroglobulin and α1 antitrypsin. These results were not changed by using thrombin purified by Amberlite 1RC50 chromatography in place of commercial thrombin in the clotting test. There was, however, a strong positive correlation between the measurements of progressive antithrombin activity using the commercial and purified forms of thrombin (r = 0.78, p < 0.001). In contrast, there was a positive correlation between antithrombin III and anti-factor Xa activity in plasma (r = 0.48, p < 0.01). There was no correlation between plasma anti Xa activity and α2 macroglobulin or α1 antitrypsin.These results suggest that plasma antithrombin activity is a measure of the activities of several plasma proteins and that antithrombin III may not be the major determinant of antithrombin activity. There is little evidence that immunological assays of antithrombin III reflect total thrombin inhibitory capacity as measured by the biological assay. Caution must be exercised in extrapolating from immunological measurements of antithrombin III to antithrombin activity in-vivo.

scholarly journals Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 639-642 ◽  
Author(s):  
A Gruber ◽  
JH Griffin ◽  
LA Harker ◽  
SR Hanson
Keyword(s):  
Protein C ◽  
Thrombus Formation ◽  
Activated Protein C ◽  
Graft Patency ◽  
Scintillation Camera ◽  
Primate Model ◽  
Baboon Model ◽  
Platelet Deposition ◽  
Antithrombotic Effects

The in vivo antithrombotic properties of human plasma activated protein C (APC), a natural anticoagulant enzyme, were investigated in a baboon model of thrombus formation on prosthetic vascular grafts. Infusion of 0.25 to 1.1 mg/kg/h purified, human, APC inhibited blood clotting, as measured by the activated partial thromboplastin time (APTT), and reduced vascular graft platelet deposition by 40% to 70%, as determined by the real-time scintillation camera imaging of 111In-labeled platelet deposition. APC infusion also preserved graft patency. Hemostatic plug formation remained normal, as measured by the template bleeding times. These results suggest that APC administration may produce immediate antithrombotic effects under arterial flow conditions.

Prolonged Inhibition of Acute Arterial Thrombosis by High Dosing of a Monoclonal Anti-platelet Glycoprotein IIb/IIIa Antibody in a Baboon Model

1995 ◽  
Vol 74 (02) ◽  
pp. 751-757 ◽  
Author(s):  
H F Kotzé ◽  
P N Badenhorst ◽  
S Lamprecht ◽  
M Meiring ◽  
V Van Wyk ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Extended Period ◽  
Platelet Glycoprotein ◽  
High Dose ◽  
Graft Material ◽  
Antithrombotic Effect ◽  
Baboon Model ◽  
Long Acting

SummaryThe in vivo activity of MA-16N7C2, the first monoclonal antibody that contains an echistatin-like RGD-sequence and inhibits platelet glycoprotein (GP)IIb/IIIa function, was determined in baboons. A dosefinding study assessing haemostatic variables such as bleeding time and ex vivo platelet aggregation showed that doses of as low as 0.2-0.3 mg/kg resulted in a pronounced effect. The effects were dose-dependent and lasted for several days, implying that MA-16N7C2 is a potent and long-acting GPIIb/IIIa inhibitor. Following the initial studies, the antithrombotic effect of 0.1 and 0.3 mg/kg of the antibody, given as a bolus, was determined in a baboon model of platelet-dependent, arterial-type thrombus formation. In these studies, a thrombogenic device consisting of Dacron vascular graft material was inserted as extension segments into a permanent arteriovenous shunt. The results confirmed the potent and long-lasting antithrombotic effect of MA-16N7C2. Surprisingly, the antithrombotic effect was stronger 48 h after a dose of 0.3 mg/kg administration than on the day of treatment with 0.1 mg/kg, despite the fact that comparable numbers of GPIIb/IIIa receptors were occupied on resting platelets. We postulate that with the high dose of MA-16N7C2 and after an extended period, occupied GPIIb/IIIa may be internalised by the platelets. Upon platelet activation, these receptors become reexposed but arc unable to participate in thrombus formation. This is in contrast to unoccupied internal GPIIb/IIIa receptors early after a low dose of MA-16N7C2.

scholarly journals Inhibition of platelet-dependent thrombus formation by human activated protein C in a primate model

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 639-642 ◽  
Author(s):  
A Gruber ◽  
JH Griffin ◽  
LA Harker ◽  
SR Hanson
Keyword(s):  
Protein C ◽  
Thrombus Formation ◽  
Activated Protein C ◽  
Graft Patency ◽  
Scintillation Camera ◽  
Primate Model ◽  
Baboon Model ◽  
Platelet Deposition ◽  
Antithrombotic Effects

Abstract The in vivo antithrombotic properties of human plasma activated protein C (APC), a natural anticoagulant enzyme, were investigated in a baboon model of thrombus formation on prosthetic vascular grafts. Infusion of 0.25 to 1.1 mg/kg/h purified, human, APC inhibited blood clotting, as measured by the activated partial thromboplastin time (APTT), and reduced vascular graft platelet deposition by 40% to 70%, as determined by the real-time scintillation camera imaging of 111In-labeled platelet deposition. APC infusion also preserved graft patency. Hemostatic plug formation remained normal, as measured by the template bleeding times. These results suggest that APC administration may produce immediate antithrombotic effects under arterial flow conditions.

Antithrombotic Effects and Bleeding Time Prolongation with Synthetic Platelet GPIIb/llla Inhibitors in Animal Models of Platelet-Mediated Thrombosis

1994 ◽  
Vol 71 (01) ◽  
pp. 095-102 ◽  
Author(s):  
Désiré Collen ◽  
Hua Rong Lu ◽  
Jean-Marie Stassen ◽  
Ingrid Vreys ◽  
Tsunehiro Yasuda ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Bolus Injection ◽  
Cell Injury ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Femoral Vein ◽  
Thrombotic Occlusion ◽  
Antithrombotic Effects

SummaryCyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1→5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-prolyl-L-arginyl-glycyl-L-α-aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1→9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor.The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 μg/kg, ex vivo ADP-induccd platelet aggregation with ID50 of 10 μg/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 ± 9 to 1,100 ± 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 μg/kg, ex vivo platelet aggregation with an ID50 of 50 μg/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 μg/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induccd ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 ± 0.4 to 12 ± 2 min. Infusion of 300 μg/kg of TP9201 over 60 min completely prevented thrombotic occlusion, inhibited ex vivo platelet aggregation, but was not associated with prolongation of the template bleeding time.TP9201, unlike G4120, inhibits in vivo platelet-mediated thrombus formation without associated prolongation of the template bleeding time.

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.

Isolation of Antithrombin III from Normal and α1-Antitrypsin-Deficient Human Plasma

1977 ◽  
Vol 38 (02) ◽  
pp. 0475-0485 ◽  
Author(s):  
Anna D. Borsodi ◽  
Ralph A. Bradshaw
Keyword(s):  
Enzymatic Activity ◽  
Isoelectric Focusing ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Antithrombin Activity ◽  
Bovine Trypsin ◽  
Normal Plasma ◽  
Antitrypsin Deficiency ◽  
Simplified Procedure ◽  
Stimulation Of

SummaryThe plasma of individuals, hetero- or homozygous for α1-antitrypsin deficiency, contains greatly decreased amounts of antithrombin activity as assayed against factor Xa. However, heparin stimulation of the residual antithrombin activity is observed, which is comparable to that of normal plasma. Antithrombins isolated from both normal and α1-antitrypsin deficient plasma by a simplified procedure are indistinguishable in both properties and yields. The microheterogeneity observed on isoelectric focusing of both preparations can be eliminated by treatment with neuraminidase. Neither purified human antithrombin nor α1-antitrypsin, when assayed against bovine trypsin, is stimulated by heparin. These results clearly establish the unique natures of antithrombin and α1-antitrypsin and show that about 75% of the antithrombin activity measured in normal plasma is due to α1-antitrypsin. Estimates of anti thrombin III activity in normal plasma by assays dependent on enzymatic activity can probably be obtained only in the presence of heparin.

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