scholarly journals Neutralization of heparin activity by neutrophil lactoferrin

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 421-428 ◽  
Author(s):  
HF Wu ◽  
RL Lundblad ◽  
FC Church
Keyword(s):  
Defense Mechanisms ◽  
Biochemical Characterization ◽  
Proteinase Inhibitors ◽  
Inflammatory Diseases ◽  
Anticoagulant Activity ◽  
Serine Proteinase ◽  
Heparin Binding ◽  
Necrosis Factor Alpha ◽  
Platelet Factor ◽  
Thrombin Inhibition

Abstract Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin- induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.

scholarly journals Neutralization of heparin activity by neutrophil lactoferrin

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 421-428
Author(s):  
HF Wu ◽  
RL Lundblad ◽  
FC Church
Keyword(s):  
Defense Mechanisms ◽  
Biochemical Characterization ◽  
Proteinase Inhibitors ◽  
Inflammatory Diseases ◽  
Anticoagulant Activity ◽  
Serine Proteinase ◽  
Heparin Binding ◽  
Necrosis Factor Alpha ◽  
Platelet Factor ◽  
Thrombin Inhibition

Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin- induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.

HEPARIN DIRECTS THE INACTIVATION OF ANTITHROMBIN BY ELASTASE

1987 ◽  
Author(s):  
R E Jordan ◽  
J Kilpatrick ◽  
J Nelson ◽  
J O New gren ◽  
M A Fournel
Keyword(s):  
Alternative Explanation ◽  
Anticoagulant Activity ◽  
Molar Ratio ◽  
Heparin Binding ◽  
Apparent Contradiction ◽  
Platelet Factor ◽  
Complete Inactivation

In apparent contradiction to its anticoagulant activity, we have observed a previously undetected, and potentially opposing function for heparin: a distinct heparin-dependency for the in vitro inactivation of highly-purified human antithrombin by neutrophil elastase. Similar to its ability to accelerate antithrombin-mediated inhibition of coagulation enzymes, anticoagulantly-active heparin was also found to stimulate the rate of inactivation of antithrombin by the neutrophil enzyme.In the absence of heparin, or in the presence of the heparin antagonists platelet factor 4 or polybrene, little or no inactivation of antithrombin occurred. Catalytic amounts of heparin and elastase caused the complete inactivation of antithrombin (approximate molar ratio of 1:1:400 respectively) in 5-10 minutes. The loss of heparin binding affinity by the elastase-cleaved form of antithrombin permitted its separation from active antithrombin by heparin-agarose chromatography.The purified elastase-inactivated antithrombin was injected into rabbits for determination of its comparative clearance behavior. In contrast to intact, functional antithrombin (t 1/2 >30 hours) and the thrombin-antithrombin (T-AT) complex (t 1/2 previously shown to be minutes), elastase-inactivated antithrombin circulated for approximately 13 hours. This prolonged clearance relative to the T-AT complex may suggest an alternative explanation for the circulating, non-functional antithrombin observed in certain coagulopathic states. In summary, these results point to a potential and unexpected role for heparin in directing the inactivation of antithrombin and suggest a possible in vivo mechanism for neutralizing the usually non-thrombogenic nature of the vascular lining.

Trypanosoma cruzi heparin-binding proteins present a flagellar membrane localization and serine proteinase activity

Parasitology ◽  
2012 ◽  
Vol 140 (2) ◽  
pp. 171-180 ◽  
Author(s):  
F. O. R. OLIVEIRA-JR ◽  
C. R. ALVES ◽  
F. S. SILVA ◽  
L. M. C. CÔRTES ◽  
L. TOMA ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Binding Proteins ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Biochemical Properties ◽  
Heparin Binding ◽  
Flagellar Membrane ◽  
The Stability ◽  
Ph Range

SUMMARYHeparin-binding proteins (HBPs) play a key role in Trypanosoma cruzi-host cell interactions. HBPs recognize heparan sulfate (HS) at the host cell surface and are able to induce the cytoadherence and invasion of this parasite. Herein, we analysed the biochemical properties of the HBPs and also evaluated the expression and subcellular localization of HBPs in T. cruzi trypomastigotes. A flow cytometry analysis revealed that HBPs are highly expressed at the surface of trypomastigotes, and their peculiar localization mainly at the flagellar membrane, which is known as an important signalling domain, may enhance their binding to HS and elicit the parasite invasion. The plasmon surface resonance results demonstrated the stability of HBPs and their affinity to HS and heparin. Additionally, gelatinolytic activities of 70 kDa, 65·8 kDa and 59 kDa HBPs over a broad pH range (5·5–8·0) were revealed using a zymography assay. These proteolytic activities were sensitive to serine proteinase inhibitors, such as aprotinin and phenylmethylsulfonyl fluoride, suggesting that HBPs have the properties of trypsin-like proteinases.

scholarly journals Rapid assembly and profiling of an anticoagulant sulfoprotein library

2019 ◽  
Vol 116 (28) ◽  
pp. 13873-13878 ◽  
Author(s):  
Emma E. Watson ◽  
Jorge Ripoll-Rozada ◽  
Ashley C. Lee ◽  
Mike C. L. Wu ◽  
Charlotte Franck ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Anticoagulant Activity ◽  
Functional Characterization ◽  
Serine Proteinase ◽  
One Pot ◽  
Tyrosine Residues ◽  
Thrombin Inhibition ◽  
Protein Library ◽  
Target Molecules

Hematophagous organisms produce a suite of salivary proteins which interact with the host’s coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure–activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

Binding of Platelet Factor 4 to Cultured Human Endothelial Cells

1979 ◽  
Author(s):  
C. Busch ◽  
J. Dawes ◽  
D.S. Pepper ◽  
Å Wasteson
Keyword(s):  
Endothelial Cells ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Human Endothelial Cells ◽  
Platelet Factor ◽  
Platelet Release

Platelet factor 4(PF-4) is a product of the platelet release reaction. A well known property of PF-4 is to interact with sulphated glycosaminoglycans (CAGs), including heparin; binding to heparin leads to neutralization of the anticoagulant activity.This study was undertaken to examine the possible binding of PF-4 to monolayers of cultured human endothelial cells(EC). The EC surface has been shown to expose GAG in particular heparan sulphate. Cultures incubated at 37°C with various amounts of 125I-PF-4 bound 125I-radioactivity in a dose-dependent manner. Maximum binding was about 500 ng per 0-7 cm2 dish (0.4x106 cells). About 50% of this was associated with the cells. In the presence of 200 ug/ml of unlabelled PF-4 or 6 ug/ml of heparin the binding was less than 5%. Chondroitin-4-sulphate (30 ug/ml) had little influence on the binding.The observed EC-PF-4-binding in vitro may reflect a similar interaction in vivo. Such a phenomenon may influence the non-thrombogenic properties of the vascular lining. Furthermore it could affect the reliability of the PF-4 measurements as indicators of intravascular platelet release.

Evaluation of Recombinant Platelet Factor 4 and Protamine Sulfate for Heparin Neutralization: Clotting and Clearance Studies in Rat

1994 ◽  
Vol 71 (05) ◽  
pp. 609-614 ◽  
Author(s):  
Laxminarayana N Korutla ◽  
Gwendolyn J Stewart ◽  
Elizabeth C Lasz ◽  
Theodore E Maione ◽  
Stefan Niewiarowski
Keyword(s):  
Half Life ◽  
Anticoagulant Activity ◽  
Protamine Sulfate ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Liver And Kidney ◽  
Kinetics Of ◽  
Heparin Neutralization

SummaryRecombinant platelet factor 4 (rPF4) efficiently neutralized heparin anticoagulant activity in rats without the adverse effect of protamine sulfate (PS) (Circulation 1992; 85: 1102). This study confirmed that rPF4 and PS neutralized heparin in rats. In vitro addition of excess PS but not rPF4 to plasma prolonged the activated partial thromboplastin time. Injection of rPF4 or PS 2 min following injection of 3H-heparin augmented loss of radioactivity from the circulation over the first 2 min but did not affect the half life of 3H-heparin for the next 58 min. PS was coupled to 4-(p-Azidosalicylamido)butylamine (ASBA), radioiodina- ted and purified by means of heparin-agarose chromatography. Heparin prevented the rapid loss of 125I-rPF4 from the circulation within the first 2 min but modestly increased loss of radioiodinated derivatized PS. Heparin extended the half-life of derivitized radioiodinated PS (measured between 2 and 60 min after injection) while modestly shortening that of 125I-rPF4. Both radioiodinated heparin binding proteins accumulated predominantly in liver and kidney. A greater percentage of radioactivity was found in these organs with rPF4 than with PS but more PS was found in urine. A larger percentage of radioiodinated derivatized PS than 125I-rPF4 was undetected. These results indicate that rPF4 and PS affect the kinetics of heparin clearance similarly but that organ deposition of the two agents may differ and offer an explanation of different physiological effects seen previously.

Pharmacokinetics of Full Length and Two-Domain Tissue Factor Pathway Inhibitor in Combination with Heparin in Rabbits

1993 ◽  
Vol 70 (03) ◽  
pp. 454-457 ◽  
Author(s):  
Claus Bregengaard ◽  
Ole Nordfang ◽  
Per Østergaard ◽  
Jens G L Petersen ◽  
Giorgio Meyn ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Fold Increase ◽  
Volume Of Distribution ◽  
Full Length ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
C Terminus ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus.FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg).Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

Reactivity of a Hereditary Abnormal Antithrombin III Fraction in the Inhibition of Thrombin and Factor Xa

1980 ◽  
Vol 44 (02) ◽  
pp. 092-095 ◽  
Author(s):  
T H Tran ◽  
C Bondeli ◽  
G A Marbet ◽  
F Duckert
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Superficial Thrombophlebitis ◽  
Heparin Concentration ◽  
Heparin Binding Site ◽  
At Iii ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryTwo different AT-III fractions were purified from the plasma of a patient with recurrent superficial thrombophlebitis. The abnormal AT-III fraction (A-AT) was compared to the normal AT-III fraction (N-AT) in the inhibition of thrombin and factor Xa. Without heparin, both inactivate proteases in a similar manner and at the same rate. However, at low heparin concentration the thrombin inhibition proceeds more slowly with A-AT than with N-AT. At high heparin concentration the difference between A-AT and N-AT becomes very small. The inhibition of factor Xa follows a similar pattern. It is suggested that the heparin binding site of A-AT differs from that of N-AT resulting in a decreased heparin cofactor activity.

Comparison of the Non-Specific Binding of Unfractionated Heparin and Low Molecular Weight Heparin (Enoxaparin) to Plasma Proteins

1993 ◽  
Vol 70 (04) ◽  
pp. 625-630 ◽  
Author(s):  
Edward Young ◽  
Benilde Cosmi ◽  
Jeffrey Weitz ◽  
Jack Hirsh
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Plasma Proteins ◽  
Low Molecular Weight Heparin ◽  
Specific Binding ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
Fixed Amount

SummaryThe non-specific binding of anticoagulantly-active heparin to plasma proteins may influence its anticoagulant effect. We used low affinity heparin (LAH) essentially devoid of anti-factor Xa activity to investigate the extent and possible mechanism of this non-specific binding. The addition of excess LAH to platelet-poor plasma containing a fixed amount of unfractionated heparin doubled the anti-factor Xa activity presumably because it displaces anticoagulantly-active heparin from plasma proteins. Although dextran sulfates of varying molecular weights also increased the anti-factor Xa activity, less sulfated heparin-like polysaccharides had no effect. These findings suggest that the ability to displace active heparin from plasma protein binding sites is related to charge and may be independent of molecular size. In contrast to its effect in plasma containing unfractionated heparin, there was little augmentation in anti-factor Xa activity when LAH was added to plasma containing low molecular weight heparin (LMWH), indicating that LMWH binds less to plasma proteins than unfractionated heparin. This concept is supported by studies comparing the anticoagulant activity of unfractionated heparin and LMWH in plasma with that in buffer containing antithrombin III. The anti-factor Xa activity of unfractionated heparin was 2-fold less in plasma than in the purified system. In contrast, LMWH had identical anti-factor Xa activity in both plasma and buffer, respectively. These findings may be clinically relevant because the recovered anti-factor Xa activity of unfractionated heparin was 33% lower in plasma from patients with suspected venous thrombosis than in plasma from healthy volunteers. The reduced heparin recovery in patient plasma reflects increased heparin binding to plasma proteins because the addition of LAH augmented the anti-factor Xa activity. In contrast to unfractionated heparin, there was complete recovery of LMWH added to patient plasma and little increase of anti-factor Xa activity after the addition of LAH. These findings may explain why LMWH gives a more predictable dose response than unfractionated heparin.

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