Localization of the Inhibitory Site(s) of Pentosan Polysulphate in Blood Coagulation

SummaryWe studied the inhibitory effect of pentosan polysulphate (PPS, Hémoclar®) on thrombin formation in blood coagulation. In contrast to a current hypothesis (1) the anti thrombin III independent effect of PPS on blood coagulation is not caused by preventing the binding of the factors IX, IXa, X, Xa, VIII, V, Va and II onto procoagulant phospholipids.We investigated the activation by thrombin of factors I, V and VIII. A strong inhibitory effect of PPS on factor VIII activation could be observed. Inhibition of the activation of factor V to the same extent requires about 30-fold higher concentrations of PPS, whereas the activation (clotting) of fibrinogen is not inhibited. The effect of PPS on factor VIIIa is two-fold: A) it inhibits its formation and B) it inhibits its function probably by the formation of a factor VIIIa-PPS complex.Prothrombinase, constituted of purified factors Xa, Va and phospholipids was not inhibited by PPS, neither were incomplete forms of this enzyme, lacking phospholipids or factor Va. The complete factor X activating enzyme (factors IXa, VIIIa and phospholipids), however, was strongly inhibited, but incomplete forms, lacking factor VIII, were not. The inhibition of the complete enzyme can be explained by reversible binding of PPS to factor VIIIa (causing an inhibition of its function) and it is not an effect on the enzymatic function of the complete enzyme. On saturation of the enzyme with an excess of factor VIIIa no inhibition by PPS is noticed.We postulate therefore that the antithrombin III independent inhibitory effect of PPS on thrombin generation on blood coagulation is by interaction with factor VIIIa. This effect is additional to the heparin-like action of PPS, i.e. potentiation of the activity of antithrombin III and/or heparin cofactor II. At concentrations attained during therapeutic use the action of pentosan polysulphate on factor VIIIa is the only one that will significantly inhibit the coagulation mechanism.

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HEPARIN IS NOT AN EFFICIENT INHIBITOR OF THE FACTOR Xa-DEPENDENT ACTIVATION OF FACTOR V AND FACTOR VIII

10.1055/s-0038-1642931 ◽

1987 ◽

Author(s):

F A Ofosu ◽

G J Modi ◽

M R Buchanan ◽

J Hirsh ◽

M A Blajchman

Keyword(s):

Factor Viii ◽

Antithrombin Iii ◽

Specific Inhibitor ◽

Factor Xa ◽

Factor V ◽

Factor X ◽

Inhibitory Effects ◽

Efficient Inhibitor ◽

System 1 ◽

Prothrombin Activation

We have previously proposed that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent activation of factor V and factor VIII. This observation was based on the demonstration that therapeutic concentrations of heparin or 1μM of the thrombin specific inhibitor, phe-pro-arg CH2Cl (PPACK) completely inhibited the activation of prothrombin when contact-activated plasma (CAP) was recalcified for up to 1 min. Under similar conditions, heparin and PPACK only partially inhibited the activation of factor X. Moreover, the addition of thrombin (lOnM) to CAP 1 min before that of heparin or PPACK reversed their inhibitory effects. We now provide further support for our hypothesis by showing that when the activity of thrombin is suppressed by heparin or PPACK, efficient activation of radiolabelled prothrombin occurs only when the factor Xa then present activates factor V and factor VIII. We compared the effects of HEP of PPACK on the following four systems for initiating the activation of prothrombin: (1) CAP; (2) CAP + lOnM thrombin; (3) CAP + InM Xa and (4) unactivated plasma + InM Xa + InM Va + coagulant phospholipids. In each system, the enzymes were added 1 min before the heparin or PPACK. In the absence of heparin or PPACK, all four systems generated the same amount of thrombin activity in 45s. Complete inhibition of prothrombin activation by heparin and PPACK was observed only in system 1 which did not contain exogenous thrombin or factor Xa. No inhibition by heparin or PPACK was observed when thrombin or factor Xa was added to CAP in systems (2) and (3). Only partial inhibition was observed in system (4) which contained exogenous prothrombi-nase complex. Factor Xa thus provides an effective by-pass mechanism for the activation of factor VIII and factor V in plasma containing therapeutic concentrations of heparin. Our data provide further evidence that the heparin-antithrombin III system is not effective in inactivating factor Xa. These results support the hypothesis that in unactivated normal plasma, the primary anticoagulant effect of heparin is the inhibition of the thrombin-dependent activation of factor V and factor VIII.

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The inhibition of thrombin-dependent positive-feedback reactions is critical to the expression of the anticoagulant effect of heparin

Biochemical Journal ◽

10.1042/bj2430579 ◽

1987 ◽

Vol 243 (2) ◽

pp. 579-588 ◽

Cited By ~ 75

Author(s):

F A Ofosu ◽

P Sie ◽

G J Modi ◽

F Fernandez ◽

M R Buchanan ◽

...

Keyword(s):

Antithrombin Iii ◽

Anticoagulant Activity ◽

Factor Xa ◽

Factor V ◽

Factor X ◽

Intrinsic Pathway ◽

Sulphated Polysaccharide ◽

Pathway Activation ◽

Pentosan Polysulphate ◽

Inhibition Of Thrombin

Heparin catalyses the inhibition of two key enzymes of blood coagulation, namely Factor Xa and thrombin, by enhancing the antiproteinase activities of plasma antithrombin III and heparin cofactor II. In addition, heparin can directly inhibit the activation of Factor X and prothrombin. The contributions of each of these effects to the anticoagulant activity of heparin have not been delineated. We therefore performed experiments to assess how each of these effects of heparin contributes to its anticoagulant activity by comparing the effects of heparin, pentosan polysulphate and D-Phe-Pro-Arg-CH2Cl on the intrinsic pathway of coagulation. Unlike heparin, pentosan polysulphate catalyses only the inhibition of thrombin by plasma. D-Phe-Pro-Arg-CH2Cl is rapid enough an inhibitor of thrombin so that when added to plasma no complexes of thrombin with its inhibitors are formed, whether or not the plasma also contains heparin. Heparin (0.66 microgram/ml) and pentosan polysulphate (6.6 micrograms/ml) completely inhibited the intrinsic-pathway activation of 125I-prothrombin to 125I-prothrombin fragment 1 + 2 and 125I-thrombin. On the addition of thrombin, a good Factor V activator, to the plasma before each sulphated polysaccharide, the inhibition of prothrombin activation was demonstrable only in the presence of higher concentrations of the sulphated polysaccharide. D-Phe-Pro-Arg-CH2Cl also completely inhibited the intrinsic-pathway activation of prothrombin in normal plasma. The inhibitory effect of D-Phe-Pro-Arg-CH2Cl was reversed if thrombin was added to the plasma before D-Phe-Pro-Arg-CH2Cl. The inhibition of the activation of prothrombin by the three agents was also abolished with longer times with re-added Ca2+. Reversal of the inhibitory effects of heparin and pentosan polysulphate was associated with the accelerated formation of 125I-thrombin-antithrombin III and 125I-thrombin-heparin cofactor complexes respectively. These results suggest that the anticoagulant effects of heparin and pentosan polysulphate are mediated primarily by their ability to inhibit the thrombin-dependent activation of Factor V, thereby inhibiting the formation of prothrombinase complex, the physiological activator of prothrombin.

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Coagulability of Blood and Fibrinolysis Related to Age and Lipids

10.1055/s-0039-1689669 ◽

1975 ◽

Author(s):

T. Matsuda

Keyword(s):

Factor Viii ◽

Antithrombin Iii ◽

Normal Subjects ◽

Factor Vii ◽

Factor V ◽

Factor X ◽

Progressive Decline ◽

Cholesterol Levels ◽

Α2 Macroglobulin ◽

Older Subjects

Levels of factor VIII, factor VII-X, factor X, factor V, fibrinogen, antithrombin III, euglobulin lysis times, plasminogen, α2-macroglobulin, α1-antitrypsin and FDP were determined in 487 healthy normal subjects from age 20 to 94. In subjects over age 60, serum lipids were determined simultaneously.In the subjects over age 60, levels of factor VIII, fibrinogen, antithrombin III, α2-maeroglobulin, α1-antitrypsin and FDP were higher, factor V activity was lower, and euglobulin lysis times were shorter than in subjects under age 50. In the older subjects, levels of factor VII-X, factor X fibrinogen, antithrombin III, plasminogen and FDP showed slight but progressive decline with age. In these subjects over age 60, serum cholesterol levels were significantly correlated with levels of factor VII-X, factor X, antithrombin III, plasminogen and FDP; antithrombin III concentration was significantly correlated with factor X activity but not with factor VII-X activity; euglobulin lysis times were correlated with obesity.

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Self-Damping Mechanism in Blood Coagulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647672 ◽

1974 ◽

Vol 32 (01) ◽

pp. 057-064 ◽

Cited By ~ 3

Author(s):

Y Nemerson ◽

S.A Silverberg ◽

J Jesty

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Calcium Ions ◽

Control Mechanism ◽

Factor Vii ◽

Damping Factor ◽

Factor V ◽

Factor X ◽

Extrinsic Pathway ◽

Two Systems

SummaryTwo reactions of the extrinsic pathway of coagulation, the activations of Factor X and prothrombin, have been studied in purified systems and shown to be self-damping. Factor X was activated by the tissue factor - Factor VII complex, and prothrombin by two systems: the coagulant protein of Taipan venom, and the physiological complex of activated Factor X, Factor V, lipid, and calcium ions. In each case the yield of enzyme, activated Factor X or thrombin, is a function of the concentration of activator. These and other observations are considered as a basis for a control mechanism in coagulation.

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The Natural Course of Defibrination Syndrome Caused by Echis Colorata Venom in Man

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649181 ◽

1974 ◽

Vol 31 (03) ◽

pp. 420-428 ◽

Cited By ~ 3

Author(s):

M Fainaru ◽

S Eisenberg ◽

N Manny ◽

C Hershko

Keyword(s):

Factor Viii ◽

Blood Coagulation ◽

Major Bleeding ◽

Renal Damage ◽

Specific Treatment ◽

Natural Course ◽

Factor V ◽

Thrombocyte Count

SummaryThe natural course of defibrination syndrome caused by Echis colorata venom (ECV) in five patients is reported. All patients developed afibrinogenemia within six hours after the bite. Concomitantly a depression in factor V was recorded. Factor VIII and thrombocyte count in blood were normal in most patients. In the light of the known effects of ECV on blood coagulation in vivo and in vitro it is concluded that the afibrinogenemia is due to intravascular clotting.Four patients had transient renal damage, manifested by oliguria, azotemia, albuminuria and cylindruria, ascribed to microthrombi in the renal glomeruli.After the bite, the natural course was benign, no major bleeding was observed, and all signs of coagulopathy reverted to normal within 7 days. Therefore we recommend no specific treatment for this condition. In the case of heavily bleeding patients, administration of antiserum against ECV and/or heparin should be considered.

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Unfractionated heparin inhibits thrombin-catalysed amplification reactions of coagulation more efficiently than those catalysed by factor Xa

Biochemical Journal ◽

10.1042/bj2570143 ◽

1989 ◽

Vol 257 (1) ◽

pp. 143-150 ◽

Cited By ~ 69

Author(s):

F A Ofosu ◽

J Hirsh ◽

C T Esmon ◽

G J Modi ◽

L M Smith ◽

...

Keyword(s):

Factor Viii ◽

Antithrombin Iii ◽

Factor Xa ◽

Activate Factor ◽

Factor V ◽

Inhibitory Effects ◽

Exogenous Factor ◽

Additional Support ◽

Prothrombin Activation ◽

Inhibit Factor

We have proposed previously that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent amplification reactions, and that prothrombinase is formed in heparinized plasma only after Factor Xa activates Factor VIII and Factor V. These propositions were based on the demonstration that both heparin and Phe-Pro-Arg-CH2Cl completely inhibited 125I-prothrombin activation for up to 60 s when contact-activated plasma (CAP) was replenished with Ca2+. Furthermore, the addition of thrombin to CAP before heparin or Phe-Pro-Arg-CH2Cl completely reversed their inhibitory effects. Additional support for the above hypotheses is provided in this study by demonstrating that, when the activity of thrombin is suppressed by heparin (indirectly) or by Phe-Pro-Arg-CH2Cl (directly), exogenous Factor Xa reverses the ability of these two agents to inhibit prothrombin activation. Prothrombin activation was initiated by adding Factor Xa (1 nM) or thrombin (1 or 10 nM) simultaneously with CaCl2 to CAP. In the absence of heparin or Phe-Pro-Arg-CH2Cl, prothrombin activation was seen 15 s later in either case. Heparin failed to delay, and Phe-Pro-Arg-CH2Cl delayed for 15 s, prothrombin activation in CAP supplemented with Factor Xa. In contrast, heparin and Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation for at least 45 s in CAP supplemented with 1 nM-thrombin. Heparin failed to delay prothrombin activation in CAP supplemented with 10 nM-thrombin, whereas Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation in this plasma for 45 s. These results suggest that in CAP: (1) Factor Xa can effectively activate Factor VIII and Factor V when the proteolytic activity of thrombin is suppressed; (2) heparin-antithrombin III is less able to inhibit Factor Xa than thrombin; (3) suppression of the thrombin-dependent amplification reactions is the primary anticoagulant effect of heparin.

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Membrane-binding properties of the Factor VIII C2 domain

Biochemical Journal ◽

10.1042/bj20101797 ◽

2011 ◽

Vol 435 (1) ◽

pp. 187-196 ◽

Cited By ~ 20

Author(s):

Valerie A. Novakovic ◽

David B. Cullinan ◽

Hironao Wakabayashi ◽

Philip J. Fay ◽

James D. Baleja ◽

...

Keyword(s):

Factor Viii ◽

Structural Integrity ◽

Membrane Binding ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Binding Motif ◽

C2 Domain ◽

Factor X ◽

Factor Ixa ◽

Factor Viiia

Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa–Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.

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THE MODE OF ACTION OF PENTOSAN POLYSULPHATE IN PLASMA

10.1055/s-0038-1643249 ◽

1987 ◽

Author(s):

S Béguin ◽

H C Hemker

Keyword(s):

Factor Viii ◽

Linear Increase ◽

Factor Ix ◽

Factor X ◽

Heparin Cofactor Ii ◽

Normal Plasma ◽

Viii And Ix ◽

Pentosan Polysulphate ◽

Lag Times ◽

Prothrombinase Activity

We developed a method which enables as to compute the course of prothrombinase activity in clotting plasma (H.C. Hemker, G.M. Willems, S. Béguin: Thromb. Haemostas. 56, 9-17, 1986) and used this for a study of the effect of pentosan polysulphate (PPS) on thrombin generation.When added to normal plasma in the concentration range of 0-8 μg/ml PPS induces a linear increase of the pseudo first order decay constant of endogenous thrombin like heparin does, 1 ug of PPS being equivalent to 0.045 Aig of heparin. Contrary to heparin this action is partly (∼ 65%) dependent upon AT III and partly (∼ 35%) upon heparin cofactor II.In normal plasma PPS causes an inhibition of both extrinsic and intrinsic prothrombinase formation. Only in the intrinsic system an increase of the lag time of prothrombinase appearance is observed. Unlike heparin, PPS does not inhibit factor IXa induced thrombin formation neither does it inhibit prothrombinase formation in the presence of preactivated factor VIII. The prolongation of the lag times must therefore be ascribed to inhibition by PPS of the activation of factor VIII.The inhibition of extrinsic prothrombinase formation by PPS increases with progressive dilution of thromboplastin and is not seen in haemophilia A or B plasma. This demonstrates the existance of a factor VIII and IX dependent process in extrinsic coagulation that gains in importance when the potency of factgr VII-tissue factor complex decreases, i.e. the Josso pathway.PPS, but also heparin causes an unexplained increase of prothrombinase action in haemophIIic plasma. The same phenomenon may be expected to exist in normal plasma, be it obscured by a concomitant inhibition. This, together with the incomplete inhibition of factor VIII activation by PPS makes that we cannot use this inhibitor as a means to quantitate the Josso pathway. The best estimate that we can obtain is that, in the presence of 2% thromboplastin, the factor IX dependent activation of factor X contributes more then 20% to prothrombinase generation.

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Evolutionary Adaptations in Pseudonaja Textilis Venom Factor X Induce Zymogen Activity and Resistance to the Intrinsic Tenase Complex

Thrombosis and Haemostasis ◽

10.1055/s-0040-1715441 ◽

2020 ◽

Vol 120 (11) ◽

pp. 1512-1523

Author(s):

Mark Schreuder ◽

Geraldine Poenou ◽

Viola J. F. Strijbis ◽

Ka Lei Cheung ◽

Pieter H. Reitsma ◽

...

Keyword(s):

Factor Viia ◽

Intrinsic Factor ◽

Salt Bridge ◽

Original Form ◽

Factor V ◽

Factor X ◽

Prothrombinase Complex ◽

Factor Viiia ◽

Protease Activation ◽

Activation Peptide

AbstractThe venom of the Australian snake Pseudonaja textilis comprises powerful prothrombin activators consisting of factor X (v-ptFX)- and factor V-like proteins. While all vertebrate liver-expressed factor X (FX) homologs, including that of P. textilis, comprise an activation peptide of approximately 45 to 65 residues, the activation peptide of v-ptFX is significantly shortened to 27 residues. In this study, we demonstrate that exchanging the human FX activation peptide for the snake venom ortholog impedes proteolytic cleavage by the intrinsic factor VIIIa–factor IXa tenase complex. Furthermore, our findings indicate that the human FX activation peptide comprises an essential binding site for the intrinsic tenase complex. Conversely, incorporation of FX into the extrinsic tissue factor–factor VIIa tenase complex is completely dependent on exosite-mediated interactions. Remarkably, the shortened activation peptide allows for factor V-dependent prothrombin conversion while in the zymogen state. This indicates that the active site of FX molecules comprising the v-ptFX activation peptide partially matures upon assembly into a premature prothrombinase complex. Taken together, the shortened activation peptide is one of the remarkable characteristics of v-ptFX that has been modified from its original form, thereby transforming FX into a powerful procoagulant protein. Moreover, these results shed new light on the structural requirements for serine protease activation and indicate that catalytic activity can be obtained without formation of the characteristic Ile16–Asp194 salt bridge via modification of the activation peptide.

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Feiba Immuno: A Preparation with Factor Eight Inhibitor Bypassing Activity

10.1055/s-0039-1682501 ◽

1977 ◽

Author(s):

F. Elsinger

Keyword(s):

Factor Viii ◽

Factor Xa ◽

Coagulation Factors ◽

Active Principle ◽

Factor Viii Inhibitor ◽

Factor V ◽

In Vitro Experiments ◽

Factor X ◽

Viii Inhibitor

FEIBA IMMUNO is a preparation in which a new activity is generated capable of bypassing factor VIII. The preparation which is used to treat patients with inhibitors (especially inhibitors to factor VIII) is standardized in FEIBA units, i.e. in terms of its in vitro capacity to shorten the activated PTT of a factor VIII inhibitor plasma.It could be concluded from different in vitro experiments that none of the classic’ activated coagulation factors is responsible for the factor VIII bypassing reaction; FEIB-activity seems to be correlated to a new complex of coagulation factors.To get an answer to the question which coagulation factors are essential for FEIB-activity, we tried to generate this activity from different deficient plasmas; from these experiments the following conclusions could be drawn:, the presence of at least factors VII, IX, and X is essential for the generation of the molecular species responsible for factor VIII as well as factor X bypassing activity, but factor V is not bypassed. This activity is not factor Xa itself. Factors VIII and V are not necessary for the generation of this active principle, but factor V is finally needed for its bypassing action.

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