scholarly journals An Antithrombin-Heparin Complex Increases the Anticoagulant Activity of Fibrin Clots

2008 ◽  
Vol 2008 ◽  
pp. 1-4 ◽  
Author(s):  
Lesley J. Smith ◽  
Tracy Anne Mewhort-Buist ◽  
Leslie R. Berry ◽  
Anthony K. C. Chan
Keyword(s):  
Anticoagulant Activity ◽  
Procoagulant Activity ◽  
Surrounding Environment ◽  
Covalently Linked ◽  
Inhibition Of Thrombin ◽  
Surprising Observation ◽  
Fibrin Clots

Clotting blood contains fibrin-bound thrombin, which is a major source of procoagulant activity leading to clot extension and further activation of coagulation. When bound to fibrin, thrombin is protected from inhibition by antithrombin (AT) + heparin but is neutralized when AT and heparin are covalently linked (ATH). Here, we report the surprising observation that, rather than yielding an inert complex, thrombin-ATH formation converts clots into anticoagulant surfaces that effectively catalyze inhibition of thrombin in the surrounding environment.

S PROTEIN/VITRONECTIN NEUTRALIZES THE ANTICOAGULANT ACTIVITY OF GLYCOSAMINOGLYCANS IN THE INHIBITION OF THROMBIN BY HEPARIN COFACTOR II

1987 ◽  
Author(s):  
K T Preissner ◽  
P Sie
Keyword(s):  
Dermatan Sulfate ◽  
Enzyme Inhibitor ◽  
Anticoagulant Activity ◽  
Coagulation System ◽  
Binary Complex ◽  
Heparin Cofactor Ii ◽  
S Protein ◽  
Adhesive Protein ◽  
Inhibition Of Thrombin

The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin against fast inactivation by antithrombin III. The interference of S protein with glycos-aminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in a purified system. In the presence of 0.3 μg/ml heparin, or 0.5 μg/ml pentosan polyphosphate (SP 54), or 2 μg/ml dermatan sulfate, S protein induced a concentration-dependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo-first order rate constants by about 17-fold (heparin), or about 7-fold (SP 54), but only by about 2-fold for dermatan sulfate at a physiological ratio of S protein to heparin cofactor II. Likewise, S protein significantly counteracted the anticoagulant activity of heparin and SP 54 bot not of dermatan sulfate when tested in an inhibition assay using various concentrations of glycosaminoglycans. For heparin, the activity of S protein at the point of 50% inhibition of thrombin was expressed in the range 0.06-0.6 μg/ml (0.01-0.1 U/ml) and for SP 54 in the range 0.3-2 pg/ml. Exposure of the glycos-aminoglycan-binding region of S protein by reduction and carb-oxymethylation of the protein even increased the neutralizing activity of S protein towards heparin and SP 54. S protein not only was found together with thrombin in a binary complex. S protein also became incorporated into a ternary complex with thrombin and heparin cofactor II as judged by crossed immunoelectrophoresis, regardless whether complex formation was initiated by heparin or dermatan sulfate. These findings underline the role of S protein as potent glycosaminoglycan-neutral-izing protein in plasma and as scavenger protein which may bind to enzyme-inhibitor complexes of the coagulation system.

Procoagulant activity of erythrocytes and platelets through phosphatidylserine exposure and microparticles release in patients with nephrotic syndrome

2012 ◽  
Vol 107 (04) ◽  
pp. 681-689 ◽  
Author(s):  
Chunyan Gao ◽  
Rui Xie ◽  
Chengyuan Yu ◽  
Qin Wang ◽  
Fangfang Shi ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Cell Injury ◽  
Anticoagulant Activity ◽  
Minimal Change Nephrotic Syndrome ◽  
Significant Inhibition ◽  
Minimal Change ◽  
Procoagulant Activity ◽  
Clotting Time ◽  
Thrombolytic Activity ◽  
Inhibition Effect

SummaryRecent studies showed that an imbalance of prothrombotic and antithrombotic factors and impaired thrombolytic activity contribute to the thrombophilia of the nephrotic syndrome (NS). However, it is not clear whether blood cell injury and/or activation is involved in hypercoagulability in NS patients. Our objectives were to study the increase in microparticle (MP) release and phosphatidylserine (PS) exposure on the outer membrane of MP-origin cells in NS patients, and to evaluate their procoagulant activity (PCA). The subjects were patients with membranous nephropathy (MN), minimal change nephrotic syndrome (MCNS) and healthy controls. Analyses of MPs and PS exposure were performed using a flow cytometer. PCA was determined by clotting time and purified coagulation complex assays. We found that lactadherin+ MPs, which derived from red blood cells (RBC), platelet and endothelial cell, increased in NS patients. Moreover, PS exposure on RBCs and platelets in each NS group, especially in MN, are higher than that in controls. MP shedding and PS exposure of RBCs/platelets were highly procoagulant in NS patients. However, blockade of PS with lactadherin inhibited over 90% of PCA while an anti-tissue factor antibody had no significant inhibition effect. Our results demonstrate that the thrombophilic susceptibility of NS may be partly ascribed to MP release and PS exposure of RBCs, platelets and endothelial cells. Lactadherin is a sensitive probe for PS that has high anticoagulant activity.

Relieving Inhibition of Prothrombinase By TFPIα: a Procoagulant Activity of Unfractionated, Low Molecular Weight, and Nonanticoagulant Heparins

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1478-1478
Author(s):  
Jeremy P Wood ◽  
Lisa M Baumann Kreuziger ◽  
Rodney M. Camire ◽  
Umesh R Desai ◽  
Alan E. Mast
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Research Funding ◽  
Thrombin Generation ◽  
Size Dependence ◽  
Procoagulant Activity ◽  
Low Molecular Weight ◽  
Direct Inhibition ◽  
Acidic Region ◽  
Inhibition Of Thrombin

Abstract Introduction: Prothrombinase, the complex of factor Xa (FXa) and factor Va (FVa), is inhibited by tissue factor pathway inhibitor (TFPI)α during the initiation of coagulation (Wood JP et al, PNAS 2013). Efficient inhibition of thrombin generation by prothrombinase requires an interaction between the TFPIα basic C-terminus and an acidic region of the FVa B-domain. This acidic region is present in FXa-activated FVa and FVa released from activated platelets, but is rapidly removed by thrombin. Thus, prothrombinase inhibition only occurs during the initiation phase of thrombin generation. As the exosite interaction is charge-dependent, large negatively charged molecules, including unfractionated heparin (UFH), block it, prevent prothrombinase inhibition, and promote thrombin generation. Studies using the negatively charged molecule polyphosphate have suggested a size requirement for blocking this TFPIα activity (Smith SA et al, Blood2010). A similar size-dependence may exist with heparins and could have clinical implications, as currently-used heparins range from long (unfractionated heparin; UFH) to medium (low molecular weight heparins; LMWHs) to short (the antithrombin-binding pentasaccharide fondaparinux). Studies were performed to assess the ability of the LMWHs enoxaparin and dalteparin, fondaparinux, and the nonanticoagulant heparin 2-O, 3-O desulfated heparin (ODSH) to block TFPIα and promote thrombin generation through this mechanism. Methods: TFPIα inhibition of thrombin generation by prothrombinase, assembled with a form of FVa containing the acidic region of the B domain, was measured in the absence or presence of UFH, enoxaparin, dalteparin, fondaparinux, and ODSH. The effect of these compounds on the direct inhibition of FXa by TFPIα was measured using a FXa chromogenic substrate. The effect of these compounds on thrombin generation in plasma was measured by calibrated automated thrombography using human plasma immunodepleted of antithrombin III and heparin cofactor II (AT3/HCII-depleted plasma). Results: TFPIα inhibited prothrombinase activity (IC50 = 6.8 nM), and UFH blocked this inhibition (IC50 = 12.5 nM or 14.9 nM at 0.5 or 1 U/mL, respectively). Enoxaparin (0.8 U/mL; IC50 = 30.3 nM) and dalteparin (1 U/mL; IC50 = 29.7 nM) appeared to be more effective at reversing TFPIα inhibition. The reason for this apparent enhanced effect of LMWHs compared to UFH is not clear, as UFH and the LMWHs similarly enhanced the direct inhibition of FXa by TFPIα, and the differential activity was also observed when heparins were normalized to saccharide concentration. The same pattern was observed when measuring thrombin generation in AT3/HCII-depleted plasma, with LMWHs being more procoagulant than UFH. Consistent with TFPIα inhibition being charge-dependent, ODSH promoted thrombin generation similarly to LMWHs in both purified systems and AT3/HCII-depleted plasma. In contrast, clinical doses of fondaparinux had no effect in any assay. In a purified system, ~1000 times the clinical dose of fondaparinux was required to promote thrombin generation. Conclusion: There is a size-dependence for blocking TFPIα inhibition of prothrombinase using heparins, as the pentasaccharide has no effect. However, both LMWHs and UFH are sufficiently long to express this procoagulant activity at therapeutic doses. In addition, the nonanticoagulant heparin ODSH blocks prothrombinase inhibition by TFPIα. This procoagulant activity is likely most clinically relevant under conditions of antithrombin deficiency, which may result from sepsis, liver failure, or administration of L-asparaginase. Under any of these conditions, UFH, LMWHs, and ODSH may have unanticipated procoagulant activity mediated by blocking TFPIα. Disclosures Camire: Pfizer: Consultancy, Patents & Royalties, Research Funding. Mast:Novo Nordisk: Research Funding.

A Low Molecular Weight Heparin Alters the Fetal Coagulation System in the Pregnant Sheep

1986 ◽  
Vol 55 (03) ◽  
pp. 342-346 ◽  
Author(s):  
M Andrew ◽  
F Ofosu ◽  
F Fernandez ◽  
A Jefferies ◽  
J Hirsh ◽  
...  
Keyword(s):  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation System ◽  
Dermatan Sulphate ◽  
Protamine Sulphate ◽  
Pregnant Sheep ◽  
Pregnant Ewe ◽  
Inhibition Of Thrombin

SummaryStandard heparin and a LMWH, CY222 do not cross the placenta nor alter fetal coagulation when injected into the pregnant ewe. We found that another LMWH, Pharmuka-10169 (PK-10169) alters fetal coagulation without crossing the placenta in the pregnant sheep. To characterize this anticoagulant we measured the in vitro and in vivo effects of 125I-PK-10169 in maternal and fetal plasmas following administration of PK-10169 to the mother or fetus. The fetal anticoagulant activity was not neutralizable by protamine sulphate and was attributable to the inhibition of thrombin but not factor Xa. In vitro, the fetal anticoagulant activity had properties similar to dermatan sulphate : both catalyzed the inhibition of thrombin but not factor Xa by sheep plasma; and neither was neutralizable by protamine sulphate. These effects were due to the enhanced neutralization of thrombin by heparin cofactor II. We conclude that PK-10169 does not cross the placenta, but does induce the release of an endogenous dermatan sulphate-like substance which alters fetal coagulation.

Modulation of Heparin Cofactor II Function by S Protein (Vitronectin) and Formation of a Ternary S Protein-Thrombin-Heparin Cofactor II Complex

1988 ◽  
Vol 60 (03) ◽  
pp. 399-406 ◽  
Author(s):  
Klaus T Preissner ◽  
Pierre Sié
Keyword(s):  
Dermatan Sulfate ◽  
Enzyme Inhibitor ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Binding Domain ◽  
Coagulation System ◽  
Pentosan Polysulfate ◽  
Heparin Cofactor Ii ◽  
S Protein ◽  
Inhibition Of Thrombin

SummaryThe complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin as well as factor Xa against fast inactivation by antithrombin III. The interference of S protein with glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in these studies. S protein significantly counteracted the anticoagulant activity of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of 0.3 μg/ml heparin, 0.5 μg/ml pentosan polysulfate, or 2 μg/ml dermatan sulfate, S protein induced a concentrationdependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin), or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and carboxymethylation of the protein increased the neutralizing activity of S protein towards heparin and pentosan polysulfate. The results of these functional experiments correlated well with the demonstration of direct binding of S protein to both polysaccharides but not to dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective in neutralizing other dermatan sulfate compounds with varying degree of sulfation, a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding domain of S protein, counteracted their anticoagulant activity. Independent on the polysaccharide used, S protein was found incorporated within a ternary complex with thrombin and heparin cofactor II during the inhibition reaction as judged by crossed immunoelectrophoresis, ultracentrifugation as well as ELISA analysis, emphazising the function of S protein as scavenger protein for enzyme-inhibitor complexes of the coagulation system. These findings demonstrate the role of S protein as effective neutralising plasma protein of the anticoagulant activity of various glycosaminoglycans also with respect to heparin cofactor II. Although the glycosaminoglycan-binding domain of S protein readily neutralized different dermatan sulfate compounds, physiological modulation of heparin cofactor-II-dependent inhibition of thrombin by native S protein appears to be restricted to the vascular compartments, where other glycosaminoglycans than dermatan sulfate appear to be operative.

Design and characterization of dipetacompinR10H, a dipetalogastin II-derived, classical competitive thrombin inhibitor

2007 ◽  
Vol 97 (01) ◽  
pp. 139-145
Author(s):  
Mercedes López ◽  
Goetz Nowak ◽  
Thomas Bitter
Keyword(s):  
Serine Proteases ◽  
Anticoagulant Activity ◽  
Tight Binding ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Coagulation Assays ◽  
Competitive Kinetics ◽  
Inhibition Of Thrombin

SummaryThe design of small chimeric thrombin inhibitors based on the structure of dipetalogastin II has been previously described. These proteins are effective inhibitors of thrombin showing slow binding or slow, tight-binding kinetics. We report here about dipetacompinR10H, a new dipetalogastin II-derived chimeric thrombin inhibitor, which exhibits classical competitive kinetics. The dissociation constant Ki of dipetacompinR10H was determined to be 17.1 ± 0.8 pM. In various coagulation assays it showed a comparable anticoagulant activity like r-hirudin and r-dipetalogastin II. DipetacompinR10H’s inhibition of thrombin was specific, since no inhibition of other serine proteases like factor Xa, plasmin, trypsin or chymotrypsin has been observed.

Identification of the Multimerin Binding Region within the C2 Domain of Human Factor V Using Constructs Generated by Alanine-Scanning and Site-Directed Mutagenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 124-124
Author(s):  
Samira B. Jeimy ◽  
Rachael A. Woram ◽  
Nola Fuller ◽  
Mary Anne Quinn-Allen ◽  
Gerard Nicolaes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Light Chain ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Factor Va ◽  
Covalently Linked

Abstract Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p< 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p<0.01). In addition, thrombin cleaved factor Va had significantly reduced multimerin-binding in assays using antibodies against the factor Va heavy chain and light chain (p<0.01). Recently, our lab identified that platelets contain forms of factor V covalently linked to multimerin via cysteine 1085 in the factor V B-domain. After recombinant factor V was activated by thrombin, there was no detectable binding of the liberated B-domain to multimerin (p<0.001). Nonetheless, the B domain of factor V appeared to enhance factor V binding to multimerin, as factor V constructs synthesized without the B-domain had reduced multimerin binding even after conversion to factor Va, compared to wild-type factor V. Based on the overlap between multimerin-binding and procoagulant, PS binding regions in the C2 domain of factor V, we assessed the effect of multimerin on factor V procoagulant activity in one stage and two stage prothrombinase assays. However, multimerin did not neutralize factor V procoagulant activity when tested in molar excess. Our study indicates that multimerin binding of factor V is modulated by conformational changes in factor V upon activation, and that the factor V B-domain may function to enhance binding to multimerin. The dissociation of multimerin-factor V complexes by thrombin suggests multimerin might be important for delivering and localizing factor V onto platelets, prior to prothrombinase assembly.

Anticoagulant Activity and Mechanism of Non-Anticoagulant Sulfated Polysaccharides

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1208-1208 ◽  
Author(s):  
Michael Dockal ◽  
Susanne Till ◽  
Sabine Knappe ◽  
Hartmut J. Ehrlich ◽  
Friedrich Scheiflinger
Keyword(s):  
Structural Properties ◽  
Brown Algae ◽  
Biological Activities ◽  
Anticoagulant Activity ◽  
Procoagulant Activity ◽  
The Other ◽  
Sulfated Polysaccharides ◽  
Heparin Cofactor Ii ◽  
Thrombin Inhibition ◽  
Thrombin Generation Assay

Abstract Abstract 1208 Fucoidans are sulfated polysaccharides which are extracted from brown seaweeds and echinoderms and have a wide variety of biological activities. Described as non-anticoagulant polysaccharides (NASPs), they have been demonstrated to improve clotting in FVIII- and FIX-deficient plasma (Liu et al., 2006), making them good candidates for hemophilia treatment. However, fucoidans have also been extensively studied for their anticoagulant effects (Pereira et al., 1999), which usually occur at much higher concentrations than the procoagulant activity. This opens a large procoagulant window where procoagulant activities exceed anticoagulant effects. When analyzed by a global hemostatic thrombin generation assay in hemophilia plasma the onset of procoagulant activity is observed at concentrations as low as 0.01 μg/mL with optimal activity at about 1 μg/mL. Reversal of procoagulant activity is seen at concentrations higher than 10 μg/mL. Fucoidans and other sulfated polysaccharides activate different anticoagulant mechanisms depending on their structural properties. Branched fucoidans extracted from brown algae have been shown to directly inhibit thrombin, while linear fucoidan from echinoderms activates antithrombin III (ATIII) or heparin cofactor II (HCII)-mediated thrombin inhibition (Pereira et al., 1999). Sulfated galactans also have serpin-dependent and -independent anticoagulant activities (Glauser et al., 2009). In this study we analyzed fucoidans from several brown algae species for their anticoagulant properties and mode of action to identify the candidate with the best procoagulant and lowest anticoagulant activity. NASPs from several brown algae species including L. japonica (L.j.), F. vesiculosus (F.v.), and U. pinnatifida (U.p.) showed different anticoagulant activities in an activated partial thromboplastin time (aPTT) assay. U.p. fucoidan was about twice as anticoagulant as the other fucoidan preparations, increasing clotting time by 50% at a concentration of 4 μg/mL. In addition, NASPs were analyzed in ATIII- and HCII-thrombin model assays. L.j. fucoidan activated ATIII-mediated thrombin inhibition, whereas the other fucoidans showed no effect on ATIII. Fucoidans from L.j. and F.v. had a direct effect on thrombin, starting at about 10 μg/mL. By contrast, U.p. fucoidan did not directly affect thrombin. However, all preparations increased HCII-mediated thrombin inhibition at concentrations below 1 μg/mL. This suggests that HCII is the main target for the anticoagulant activity of fucoidans. Nevertheless, we observed substantial differences between the fucoidan candidates which will be correlated to structural properties. Our work describes the assessment of anticoagulant activities of a variety of fucoidan species to better understand their intertwined pro- and anticoagulant effects. This provides important mechanistic insights for the development of hemophilia therapies. Disclosures: Dockal: Baxter Innovations GmbH: Employment. Till:Baxter Innovations GmbH: Employment. Knappe:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter BioScience: Employment.

scholarly journals Characterization of L-amino Acid Oxidase Derived from Crotalus adamanteus Venom: Procoagulant and Anticoagulant Activities

2019 ◽  
Vol 20 (19) ◽  
pp. 4853
Author(s):  
Vance G. Nielsen
Keyword(s):  
Amino Acid ◽  
Snake Venom ◽  
Serine Proteases ◽  
Anticoagulant Activity ◽  
Procoagulant Activity ◽  
Acid Oxidase ◽  
Contact Activation ◽  
Amino Acid Oxidase ◽  
Plasmatic Coagulation ◽  
Hydrogen Peroxide Generation

Snake venom enzymes of the L-amino acid oxidase (LAAO) class are responsible for tissue hemorrhage, edema, and derangement of platelet function. However, what role, if any, these flavoenzymes play in altering plasmatic coagulation have not been well defined. Using coagulation kinetomic analyses (thrombelastograph-based), it was determined that the LAAO derived from Crotalus adamanteus venom displayed a procoagulant activity associated with weak clot strength (no factor XIII activation) similar to thrombin-like enzymes. The procoagulant activity was not modified in the presence of reduced glutathione, demonstrating that the procoagulant activity was likely due to deamination, and not hydrogen peroxide generation by the LAAO. Further, unlike the raw venom of the same species, the purified LAAO was not inhibited by carbon monoxide releasing molecule-2 (CORM-2). Lastly, exposure of the enzyme to phenylmethylsulfonyl fluoride (PMSF) resulted in the LAAO expressing anticoagulant activity, preventing contact activation generated thrombin from forming a clot. In sum, this investigation for the first time characterized the LAAO of a snake venom as both a fibrinogen polymerizing and an anticoagulant enzyme acting via oxidative deamination and not proteolysis as is the case with thrombin-like enzymes (e.g., serine proteases). Using this thrombelastographic approach, future investigation of purified enzymes can define their biochemical nature.

EFFECT OF RABBIT THROMBOMODULIN ON THE INHIBITION OF THROMBIN BY ANTITHROMBIN IN PRESENCE OF EXOGENOUS HEPARIN: ROLE OF THE ACIDIC DOMAIN OF THROMBOMODULIN

1987 ◽  
Author(s):  
M C Bourin ◽  
I Bjôrk ◽  
M C Boffa ◽  
U Lindahl
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Proteolytic Degradation ◽  
High Affinity ◽  
Acidic Domain ◽  
Acidic Form ◽  
Cell Membrane Protein ◽  
A Site ◽  
Inhibition Of Thrombin

Thrombomodulin (TM) is an endothelial cell membrane protein chat acts as a cofactor for Protein C (PC) activation by thrombin (T). Rabbit TM also prevents fibrinogen clotting by T (direct anticoagulant activity) and accelerates T inhibition by antithrombin (AT-dependent anticoagulant activity). Rabbit TM was previously found to contain an acidic domain (presumably a heparin-like polysaccharide), separated from the PC cofactor site but required for the direct and AT-dependent anticoagulant activities. Although binding of TM to T modifies the specificity of T, it does not involve a modification of the catalytic site or a global conformational change of the enzyme.A non-acidic form of rabbit TM was obtained by limited proteolytic degradation of the acidic form. The non-acidic form retained only the PC cofactor activity. The acidic and non-acidic forms of rabbit TM were compared with regard to their effects on the inhibition of T by AT in presence of exogenous heparin (hep). The AT-heparin complex was preformed by incubation for 5 min at 37°C of AT (0.1 μM) with high-affinity heparin (HA-hep, 250 ng/ml) and then added to a solution of T (90 nM) or T preincubated with either the non-acidic form (250 nM) or the acidic form (160 nM) of TM. Residual T activity was determined after various times for up to 5 min. Controls were performed by substituting buffer for TM. It was found that T bound to the non-acidic form of TM was inhibited at the same rate as free T (90% T inhibited within 30 sec), whereas T bound to the acidic form of TM was inhibited at a much slower rate (40 to 50% T inhibited after 5 min). Similar results were obtained even when HA-hep was used in excess (up to 750 ng/ml). The data suggest that the acidic domain of rabbit TM is primarily responsible for the retardation of T-AT complex formation in presence of exogenous hep. It is proposed that the polyanionic (endogenous heparin-like) component of rabbit TM blocks a site on T required for binding of T to hep in the ternary T-Hep-AT complex.

Sign in / Sign up

Export Citation Format

Share Document