TISSUE THROMBOPLASTIN-INDUCED REVERSIBLE DIC AS AN IN VIVO MODEL OF THROMBIN GENERATION AND INHIBITION

1987 ◽  
Author(s):  
G A Marbet ◽  
B Zbinden ◽  
P Satiropas ◽  
F Duckert
Keyword(s):  
Thrombin Generation ◽  
Dermatan Sulfate ◽  
Order Rate Constant ◽  
Thrombin Inhibitors ◽  
In Vivo Model ◽  
Heparin Cofactor Ii ◽  
First Order ◽  
Thrombin Inhibition ◽  
Tissue Thromboplastin

We have tried a kinetical approach to characterize the dynamics of thrombin generation, thrombin action and heparin-enhanced inhibitors in vivo. Integrals (φ) of free thrombin concentration over time were calculated from fibrinogen decrease to characterize tissue thromboplastin-induced DIC in the dog. DIC with φ < 8nMmin was reversible. Dynamic thrombin inhibition (DTI), measured as pseudo-first order rate constant of thrombin inactivation by plasma (baseline DTI = 6.31± 0.79/min, n=30) increased to 68.69±59.98/min (n=7) with heparin (H) and to 22.48±14.91/min (n=5 with pentosan polysulfate (PPS). DTI correlated significantly with heparin doses (p< 0.002), and with the prolongation of APTT (p<0.0 2) and of prothrombin time (p < 0.05). The efficiency β of tissue thromboplastin (Tp) to trigger DIC ( φ per ml TP) was reduced from β =3.81±3.16nMmin/ml to 0.74±0.52nMmin/ml by H (p < 0.01) and to 1.16±1.10nMmin/ml (n.s.) by PPS. As expected from the product DTI.φ = 23.4 ± 13.8nM there was no detectable decrease of prothrombin, of the combined activity of antithrombin III + heparin cofactor II (ATHC) or of heparin cofactor II (HC, specific assay by dermatan sulfate activation) in reversible DIC without glycosaminoglycan protection. However, increasing doses of TP at constant PPS protection induced a statistically significant and persistent decrease of prothrombin by 17.6±9.91 and of HC by 20.4±8.81 indicating HC enhancement by PPS in vivo. The model is suitable for the study of glycosa-minoglycans and thrombin inhibitors.

Tissue Thromboplastin Induced Reversible DIC and Heparin-Enhanced Inhibitors in Dogs

1986 ◽  
Vol 55 (01) ◽  
pp. 078-085 ◽  
Author(s):  
German A Marbet ◽  
Michael J Griffith
Keyword(s):  
Antithrombin Iii ◽  
Ex Vivo ◽  
Order Rate Constant ◽  
Thrombin Inhibitors ◽  
Heparin Cofactor Ii ◽  
First Order ◽  
Tissue Thromboplastin ◽  
Pseudo First Order ◽  
Homologous Tissue

SummaryReversible acute disseminated intravascular coagulation (DIC) has been induced in dogs by intravenous injection of homologous tissue thromboplastin. There was no measurable consumption of antithrombin III and heparin cofactor II even if fibrinogen was reduced during DIC by more than 80% of its baseline. The prothrombin level remained practically constant. These data correspond to the generation of a few nanomoles of thrombin in vivo with subsequent pseudo-first order inactivation by the major thrombin inhibitors. An ex vivo measure of the pseudo-first order rate constant (dynamic thrombin inhibitory capacity, DTIC) was a sensitive probe of circulating heparin. There was no change of DTIC during DIC in the absence of exogenous heparin suggesting that heparin-like endogenous glycosaminoglycans were not released in substantial amounts. Pretreatment with heparin efficiently inhibited the development of tissue thromboplastin induced DIC. This animal model may serve as a tool for the study of glycosaminoglycan anticoagulants in vivo.

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.

Role of Thrombin Exosites in Protection from Inhibition by Antithrombin in the Presence of Heparin and Fibrin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1721-1721
Author(s):  
James C. Fredenburgh ◽  
Alan R. Stafford ◽  
Jeffrey I. Weitz
Keyword(s):  
Ternary Complex ◽  
Dermatan Sulfate ◽  
Order Rate Constant ◽  
Thrombin Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Heparin Cofactor Ii ◽  
Fold Reduction ◽  
The Individual ◽  
Two Populations

Abstract The γ-chain of fibrinogen exists in two forms, γA and γ′, such that circulating fibrinogen consists of two populations, γA/γA (90%) and γA/γ′ (10%). The 16 amino acid extension at the COOH-terminus of the γ′ chain contains numerous negatively-charged residues. This alteration endows γA/γ′-fibrin (Fn) with a greater capacity to bind thrombin (IIa), a feature that may render thrombi prothrombotic. The purpose of this study was to explore how the various mechanisms by which IIa binds to Fn impact on IIa protection from inactivation by antithrombin/heparin. IIa binds weakly to γA/γA-Fn utilizing exosite 1. IIa binds with higher affinity to γA/γ′-Fn due to the additional exosite 2/γ′ interaction. In the presence of heparin, IIa can bind to γA/γA-Fn with high affinity by forming a ternary complex wherein heparin bridges IIa to Fn via exosite 2, thereby heightening the interaction of IIa with Fn via exosite 1. Consequently, the amount of IIa bound to γA/γA-Fn clots increases in the presence of heparin. Formation of the γA/γA-Fn/heparin/IIa ternary complex reduces the second order rate constant of IIa inhibition by antithrombin (AT) 11-fold compared with the heparin-catalyzed rate of inhibition of free IIa (1.1x108 M−1 min−1). This reduction reflects the inability of AT-bound heparin to access exosite 2. When γA/γ′-Fn is used in place of γA/γA-Fn, the heparin-catalyzed rate is reduced 55-fold. The enhanced protection with γA/γ′-Fn is due to the exosite 2-mediated interaction of IIa with the γ′-chain because addition of an antibody against the γ′ sequence that blocks this interaction reduces the protection from AT to the level seen with γA/γA-Fn. Thus, both γA/γ′-Fn/IIa and γA/γA-Fn/heparin/IIa complexes restrict the access of heparin to exosite 2, thereby impairing inhibition by heparin/AT. Heparin cofactor II (HCII) also utilizes heparin to bridge to IIa, but, unlike AT, HCII must also directly engage exosite 1 to effect IIa inhibition. Consequently, the heparin-catalyzed rate of IIa inhibition by HCII is reduced 27-fold in the presence of γA/γA-Fn compared with the 11-fold reduction with AT. To examine the contribution of the heparin-Fn interaction to this phenomenon, dermatan sulfate (DS) was used to catalyze HCII because, unlike heparin, DS does not bind Fn. γA/γA-Fn produced only a 5-fold reduction in the DS-catalyzed rate of IIa inhibition by HCII. This suggests that in the absence of heparin, occupation of exosite 1 by γA/γA-Fn only modestly impairs inhibition by HCII. In contrast, γA/γ′-Fn produced a 28-fold reduction in the DS-catalyzed rate of IIa inhibition by HCII. This protection is abolished by addition of the antibody that blocks binding of IIa to the γ′ chain. Therefore, although exosite 1 mediates IIa binding to both γA/γA- and γA/γ′-Fn, interaction of exosite 2 with the γ′-chain in γA/γ′-Fn heightens exosite 1-mediated binding. These findings provide independent confirmation that ligation of both exosites on IIa accentuates the affinity of the individual exosite 1 and 2 interactions. Thus, reactants that require access to either exosite will be restricted when IIa binding is mediated by both exosites as occurs with γA/γA-Fn/heparin or with γA/γ′-Fn. These results confirm that Fn can serve as a reservoir of active IIa and that bound IIa is protected from inhibition by circulating inhibitors. Our data also highlight the limitations of physiological inhibitors of IIa and validate the need for development of direct thrombin inhibitors.

scholarly journals Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 742-747 ◽  
Author(s):  
FA Ofosu ◽  
GJ Modi ◽  
LM Smith ◽  
AL Cerskus ◽  
J Hirsh ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Intrinsic Pathway ◽  
Heparin Cofactor Ii ◽  
Normal Plasma ◽  
Normal Vessel

Abstract Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4- sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.

scholarly journals Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 742-747
Author(s):  
FA Ofosu ◽  
GJ Modi ◽  
LM Smith ◽  
AL Cerskus ◽  
J Hirsh ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Thrombin Generation ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Intrinsic Pathway ◽  
Heparin Cofactor Ii ◽  
Normal Plasma ◽  
Normal Vessel

Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4- sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.

Molecular and functional characterization of a natural homozygous Arg67His mutation in the prothrombin gene of a patient with a severe procoagulant defect contrasting with a mild hemorrhagic phenotype

Blood ◽  
2002 ◽  
Vol 100 (4) ◽  
pp. 1347-1353 ◽  
Author(s):  
Sepideh Akhavan ◽  
Raimondo De Cristofaro ◽  
Flora Peyvandi ◽  
Silvia Lavoretano ◽  
Raffaele Landolfi ◽  
...  
Keyword(s):  
Dermatan Sulfate ◽  
Functional Characterization ◽  
Factor Xa ◽  
Order Rate Constant ◽  
Hemorrhagic Diathesis ◽  
Thrombin Inhibition ◽  
Factor Va ◽  
Km Value ◽  
Prothrombin Gene

In a patient who presented with a severe coagulation deficiency in plasma contrasting with a very mild hemorrhagic diathesis a homozygous Arg67His mutation was identified in the prothrombin gene. Wild-type (factor IIa [FIIa]-WT) and mutant Arg67His thrombin (FIIa-MT67) had similar amidolytic activity. By contrast, the kcat/Km value of fibrinopeptide A hydrolysis by FIIa-WT and FIIa-MT67 was equal to 2.1 × 107M−1s−1 and 9 × 105M−1s−1. Decreased activation of protein C (PC) correlated with the 33-fold decreased binding affinity for thrombomodulin (TM; Kd = 65.3 nM vs 2.1 nM, in FIIa-MT67 and in FIIa-WT, respectively). In contrast, hydrolysis of PC in the absence of TM was normal. The Arg67His mutation had a dramatic effect on the cleavage of protease-activated G protein–coupled receptor 1 (PAR-1) 38-60 peptide (kcat/Km = 4 × 107M−1s−1 to 1.2 × 106M−1s−1). FIIa-MT67 showed a weaker platelet activating capacity, attributed to a defective PAR-1 interaction, whereas the interaction with glycoprotein Ib was normal. A drastic decrease (up to 500-fold) of the second-order rate constant pertaining to heparin cofactor II (HCII) interaction, especially in the presence of dermatan sulfate, was found for the FIIa-MT67 compared with FIIa-WT, suggesting a severe impairment of thrombin inhibition by HCII in vivo. Finally, the Arg67His mutation was associated with a 5-fold decrease of prothrombin activation by the factor Xa-factor Va complex, perhaps through impairment of the prothrombin-factor Va interaction. These experiments show that the Arg67His substitution affects drastically both the procoagulant and the anticoagulant functions of thrombin as well as its inhibition by HCII. The mild hemorrhagic phenotype might be explained by abnormalities that ultimately counterbalance each other.

The Venous Antithrombotic Profile of Naroparcil in the Rabbit

1994 ◽  
Vol 72 (06) ◽  
pp. 874-879 ◽  
Author(s):  
Jean Millet ◽  
Jocelyne Theveniaux ◽  
Neil L Brown
Keyword(s):  
Oral Administration ◽  
Bleeding Time ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Bleeding Risk ◽  
Thrombin Time ◽  
Heparin Cofactor Ii ◽  
Antithrombotic Activity ◽  
Thrombin Inhibition ◽  
Maximal Reduction

SummaryThe venous antithrombotic profile of naroparcil or (4-[4-cyanoben-zoyl]-phenyl)-1.5-dithio-β-D-xylopyranoside was investigated in the rabbit following single i. v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i. v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i. v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same doses of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human a-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay. The results suggest that naroparcil could have a safe venous antithrombotic profile following oral administration (antithrombotic effect compared to bleeding risk). It is probable that part of the mechanism of action of the β-D-xyloside, naroparcil, is due to the induction of chondroitin sulfate-like glycosaminoglycan biosynthesis, this material being detectable in the plasma.

HUMAN MEIZOTHROMBIN 1, ITS ISOLATION AND PROPERTIES

1987 ◽  
Author(s):  
Zbigniew S Latallo ◽  
Craig M Jackson
Keyword(s):  
Rate Constants ◽  
Antithrombin Iii ◽  
Order Rate Constant ◽  
Red Cross ◽  
Apparent Lack ◽  
First Order ◽  
Order Rate ◽  
Matching Grant ◽  
Echis Carinatus

Meizothrombin (MT) and meizothrombin des Fragment 1 (MT1) are intermediates in the conversion of prothrombin to α-thrombin (αTH). Due to their transient character, properties of these enzymes are difficult to establish. Isolation of MT1 was achieved by affinity chromatography on D-Phe-Pro-Arginal (FPRal)immobilized on Affi-Gel 10 as originally employed for thrombin purification (Patel et al. Biochim.Biophys. Acta 748,321 (1983)). Human prethrombin 1 was activated with the purified activator from Echis carinatus venom in the presence of Ca++;, benzamidine and FPRal gel at pH 7.8. After exhaustive washing the MT1 was eluted with 0.1 M hydroxylamine in 0.15 M Na acetate buffer, pH 5.5. Under these conditions the MT1 is stable and can bestored at -70°C. Upon changing the pH of the preparation to 8.0, complete conversion into aTH occurred atroom temperature within 48 hours. Homogeneity of both preparations wasdemonstrated by PAGE. The Km and ke, values for MT1 measured on Tos-Gly-Pro-Arg pNA(0.1 M NaCl, 0.01 M TRIS, 0.01 M HEPES, 0.1% PEG, pH 7.8, 25°C) were 15.7 /iM and 126 s-1. The kinetic con stants for the aTH resulting from autocatalytic degradation of MT1 were indistinguishable from those previously established forαTH obtained by Xa activation i.e. 4.77 /μM and 126 s-1. Clotting activity of MT1 was found to be only one fifth as high as that of the resulting μTH(746 u/mg vs. 3900 u/mg as tested using the NIH standard) .Inhibitionof MTl by antithrombin III was alsomuch less rapid than αTH andmost importantly, it was not affected by high affinity heparin( Mr20,300). Under conditions of the experiment (0.3 M NaCl, 0.0rl M TRIS, 0.01 M HEPES, 2.5 mM EDTA, 0.1% PEG, pH 7.8, 25°C; [ATIII] 100 nM, [E] 10 nM), the pseudo first order rate constants in the absence of heparin were 4.04 × 10-3V1 (MTl) and 1.13 × 10-3V1 (αTH), giving apparent second order rate constants of 4.04 × 103 and 1.13 × 10-4M-1s-1. In the presence of 4.5 nM of heparin the observed first order rate constant for MTl remained unchanged whereas it increased to 6.241 × 10-3s-1 (5.5 fold) for αTH. This apparent lack of an effect of heparin may be of significance in vivo.Supported by a Matching Grant from the American National Red Cross and by the Southeastern Michigan Blood Service.

SECONDARY FIBRINOLYSIS, PROTEIN C ACTIVATION, PLATELET DECREASE, BUT NOT CONTACT ACTIVATION CAN BE CORRELATED TO FREE THROMBIN ACTION IN EXPERIMENTAL DIC

1987 ◽  
Author(s):  
G A Marbet ◽  
P Satiropas ◽  
C Pantaleoni ◽  
F Duckert
Keyword(s):  
Protein C ◽  
Defense Mechanisms ◽  
Dermatan Sulfate ◽  
Correlation Coefficients ◽  
Factor Xii ◽  
Spearman Correlation ◽  
Contact Activation ◽  
Defence Mechanisms ◽  
Tissue Thromboplastin

We have studied the influence of activated coagulation on antithrombotic defence mechanisms in vivo. Conventional coagulation variables, platelets (Tcy), plasminogen (Pig), α2-antiplasmin (AP) , protein C (PC), factor VIII C, factor XII and Cl-inhibitor have been measured before and during reversible tissue thromboplastin-induced DIC in the dog. Free thrombin action as derived from fibrinogen (Fbg) decrease has been expressed as integral of active thrombin concentration over time (φ). Protection by heparin H, pentosan polysulfate PPS or dermatan sulfate DS was studied. DIC had no consistent effect on the behaviour of factor XII and Cl-inhibitor, but led to the consumption (Δ) of the following variables:The Spearman correlation coefficients between and φ in the Δ whole group were all statistically significant and ranged from rs=0.51 (ΔPlg) to rs =0.94 (ΔFbg). The response of major defense mechanisms in vivo quantitatively depends on active thrombin.

An automated approach to characterize, in simple kinetic terms, the generation and inactivation of thrombin and the interaction of fibrinogen with thrombin.

1988 ◽  
Vol 34 (10) ◽  
pp. 1971-1975 ◽  
Author(s):  
D R Hoak ◽  
S K Banerjee ◽  
G Kaldor
Keyword(s):  
Prothrombin Time ◽  
Rate Constant ◽  
Thrombin Generation ◽  
Order Rate Constant ◽  
Clinical Use ◽  
First Order ◽  
Pathological Conditions ◽  
Pseudo First Order ◽  
Instantaneous Concentration

Abstract Here, we used a fully automated, computer-directed centrifugal analyzer (which permitted simultaneous turbidimetry and calculation of results) and purified thrombin, fibrinogen, and various inhibitors to study clot formation. The Km and Vm for these reactions were useful in detecting and partly characterizing anticoagulants. We also explored the generation and inactivation of thrombin, using the two-stage prothrombin time and antithrombin activity tests. The amount of thrombin instantaneously generated and inactivated was monitored under artificially created pathological conditions. The pseudo-first-order rate constant for thrombin generation and inactivation and the instantaneous concentration of enzymatically active and inactive thrombin were used in the characterization of these conditions. We believe this approach is suitable for routine clinical use.

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