NEUTRALIZATION OF HEPARIN-LIKE SACCHARIDES BY COMPLEMENT S PROTEIN/VITRONECTIN

1987 ◽  
Author(s):  
D A Lane ◽  
A M Flynn ◽  
G Pejler ◽  
U Lindahl ◽  
K Preissner
Keyword(s):  
Heparan Sulfate ◽  
Anticoagulant Activity ◽  
Protein A ◽  
Membrane Attack Complex ◽  
Factor Xa ◽  
Molar Ratio ◽  
Platelet Factor ◽  
S Protein ◽  
Heparin Activity ◽  
Heparin Fractions

S protein, a major inhibitor of the assembly of the membrane attack complex of complement, has recently been shown to be identical to the serum spreading factor vitronectin. It has also been demonstrated to have anti-heparin properties. We have studied the heparin neutralizing properties of S protein/vitronectin using heparin, heparan sulfate and heparin oligosaccharides with high affinity for antithrombin. The ability of heparin fractions, Mr 7300-18800, and of the International Heparin Standard, to accelerate the inactivation of thrombin and Factor Xa by anti thrombin was readily neutralized by S protein/vitronectin. Addition of the protein to the various saccharide fractions at a molar ratio 1-3/1 produced 50/ neutralization, while complete neutralization was achieved at a molar ratio of <10/1. Moreover, S protein/vitronectin efficiently neutralized oligosaccharides of Mr 2400-7200, unlike the two other physiologically occur ing heparin neutralizing proteins histidine-rich glycoprotein <HRG) and platelet factor 4 < PF4) <Lane et al (1986) J. Biol. Chem.261, 3980; Lane et al (1984) Biochem. J. 218, 725). Like PF4, but unlike HRG, S protein/vitronectin readily neutralized the anticoagulant activities of heparan sulfate of Mr ˜20000. These findings indicate that S protein/vitronectin requires little more than the antithrombin-binding pentasaccharide with which to interact in order to express its anti-heparin activity. Furthermore, the results suggest that S protein/vitronectin may be a physiological1y important modulator of the anticoagulant activity of heparin-like material on or near the vascular endothelium.

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.

scholarly journals Platelet Factor 4 (PF4) and Protamine Neutralisation of Heparin in Plasma

1977 ◽  
Author(s):  
R. Michalski ◽  
D.A. Lane ◽  
D. Pepper ◽  
V.V. Kakkar
Keyword(s):  
Intravenous Injection ◽  
Factor Xa ◽  
Weight Basis ◽  
Platelet Factor 4 ◽  
Assay System ◽  
Platelet Factor ◽  
Protamine Sulphate ◽  
Heparin Activity ◽  
Plasma Heparin

The ability of PF4 and protamine sulphate to neutralise heparin in plasma has been studied using a specific anti-Factor Xa assay and a KCCT assay to measure residual heparin. When heparin is added to plasma in vitro PF4 and protamine neutralise almost equivalent amounts of heparin on a weight basis, 1.0 unit of heparin being neutralised by approximately 20 μg of PF4 and 15 μg of protamine. Similar results are obtained using either of the heparin assays. However, following intravenous injection of heparin only about one half of the circulating heparin could be neutralised in vitro by PF4 or protamine when it was measured by anti-Factor Xa assay. Total neutralisation was obtained with both neutralising agents in the KCCT assay system. These results demonstrate that the choice of assay is important when a protamine titration is used to measure plasma heparin levels, and that PF4 and protamine are unable to totally neutralise circulating antithrombotic heparin activity.

Metabolism of Sodium Pentosan Polysulphate in Man Measured by a New Competitive Binding Assay for Sulphated Polysaccharides – Comparison with Effects Upon Anticoagulant Activity, Lipolysis and Platelet α-Granule Proteins

1985 ◽  
Vol 53 (03) ◽  
pp. 411-414 ◽  
Author(s):  
I R MacGregor ◽  
J Dawes ◽  
D S Pepper ◽  
C V Prowse ◽  
J Stocks
Keyword(s):  
Binding Assay ◽  
Peak Plasma ◽  
Anticoagulant Activity ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Competitive Binding ◽  
Platelet Factor ◽  
Competitive Binding Assay ◽  
Sulphated Polysaccharides ◽  
Pentosan Polysulphate

SummaryThree human volunteers were injected with a range of doses of pentosan polysulphate, SP54, i.v. or s.c. A competitive binding assay (CBA) for sulphated polysaccharides was used to detect circulating SP54 after doses as low as 1 mg i.v. and a linear relationship was observed between the peak plasma concentration of SP54 measured by CBA and the administered dose. A comparison was made between the clearance of SP54 measured by CBA and its anticoagulant and lipolytic activities. SP54 was detectable by CBA after doses which caused no alteration in activated partial thromboplastin time (APTT) or anti-factor Xa activity but after which a small increase of lipase activity was measurable. After SP54 at 10 mg i.v. or 100 mg s.c. anti-factor Xa activity was 4-6 times greater than would be expected from the in vitro activity of the concentrations of SP54 measured by CBA. Like heparin and other heparin analogues, SP54 caused an increase in plasma concentrations of platelet factor 4 (PF4) without a concomitant rise in p-thromboglobulin (β-TG).It is concluded that the newly developed CBA will provide a more sensitive means than conventional bioassays for the determination of plasma concentrations of SP54.

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.

Dual Effect of Platelet Factor Four on the Activities of Factor Xa

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1034-1034
Author(s):  
Martine Marie Fiore ◽  
Ian J Mackie
Keyword(s):  
Factor Xa ◽  
Fold Increase ◽  
Peptide Hydrolysis ◽  
Dual Effect ◽  
Platelet Factor ◽  
Paradoxical Effects ◽  
Heparin Activity ◽  
High Concentrations ◽  
Prothrombin Activation

Abstract Platelet Factor 4 (PF4) is a cationic molecule that binds to heparin with high affinity and neutralises the activity of the latter. Our recent studies indicate that heparin can promote an interaction of fXa with PF4 since neutralization of heparin activity by PF4 was dependent on the concentration of protease. To examine the contribution of PF4 in protease function, fXa activity was determined in chromogenic assays. Upon preincubation with fXa and heparin, PF4 (at a concentration of 100 nM) decreased the kcat of S2765 peptide hydrolysis 4-fold and that of prothrombin activation about 2-fold. These results suggested an effect of PF4 on the primary specificity of the protease. In fact, PF4 exerted a mild effect (30 % decrease) on the Na+ dependence of fXa, consistent with linkage between Na+ and S1. PF4 preincubation with fXa also prevented the binding of the S1 probe p-aminobenzamidine (pAB) while simultaneous addition of PF4 and pAB diminished the contribution of PF4. In the presence of excess fVa (relative to fXa), kinetic parameters measuring fXa amidolytic activity in the presence of PF4 were restored to control values in the absence of PF4. Interestingly, high concentrations of PF4 (&gt; 1 μM) totally restored fXa activity toward peptidyl substrate and strongly enhanced prothrombin activation, indicating a dual effect of PF4 on fXa activities. The inhibitory contribution of PF4 during prothrombin activation was due to a three-fold decreased affinity of fXa for fVa while enhancement of prothrombin activation was accompanied by a three-fold increase in fVa-dependent cofactor activity. Thus, the effects of PF4 possibly involved a region of the heparin/fVabinding exosite that is linked to the S1 and Na+ sites. These findings suggest that PF4 is a probe of fVa-dependent changes occurring in the active site of fXa and provide an explanation for the in vivo paradoxical effects of PF4 reported in the literature.

scholarly journals On the molecular-weight-dependence of the anticoagulant activity of heparin

1979 ◽  
Vol 181 (1) ◽  
pp. 241-243 ◽  
Author(s):  
L Thunberg ◽  
U Lindahl ◽  
A Tengblad ◽  
T C Laurent ◽  
C M Jackson
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Molecular Weights ◽  
High Affinity ◽  
Polysaccharide Chain ◽  
Molecular Weight Dependence ◽  
Heparin Fractions

The inactivation of thrombin and factor Xa by antithrombin was determined in the presence of heparin fractions of different molecular weights and with high affinity for antithrombin. The ability to potentiate the inactivation of either coagulation factor increased with increasing length of the polysaccharide chain.

scholarly journals Binding of platelet factor 4 to heparin oligosaccharides

1983 ◽  
Vol 209 (2) ◽  
pp. 455-460 ◽  
Author(s):  
J Denton ◽  
D A Lane ◽  
L Thunberg ◽  
A M Slater ◽  
U Lindahl
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Factor Xa ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Physiological Conditions ◽  
Heparin Fractions ◽  
Specific Activities ◽  
Binding Sequence ◽  
Heparin Oligosaccharides

Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.

Neutralization of the Antiheparin Activity of Platelet Factor 4 by a Monoclonal Antibody

1992 ◽  
Vol 67 (01) ◽  
pp. 137-143 ◽  
Author(s):  
Leopoldo Saggin ◽  
Flavia Cazzola ◽  
Giuseppe Corona ◽  
Emanuela Salvatico ◽  
Giuseppe Cella ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Factor Xa ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Molar Ratio ◽  
Rabbit Platelet ◽  
Platelet Factor ◽  
Chromogenic Assay ◽  
Antiheparin Activity ◽  
Human Molecule

SummaryWe have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4’s ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/ PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

scholarly journals ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis

2020 ◽  
Vol 117 (17) ◽  
pp. 9311-9317 ◽  
Author(s):  
Ryan J. Weiss ◽  
Philipp N. Spahn ◽  
Alejandro Gómez Toledo ◽  
Austin W. T. Chiang ◽  
Benjamin P. Kellman ◽  
...  
Keyword(s):  
Mast Cells ◽  
Heparan Sulfate ◽  
Zinc Finger Protein ◽  
Cultured Cells ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Binding Motif ◽  
Promoter Regions ◽  
Transcription Factor Binding Motif ◽  
Mammalian Cell Lines

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non–heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.

scholarly journals The limited importance of factor Xa inhibition to the anticoagulant property of heparin in thromboplastin-activated plasma

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 2048-2052 ◽  
Author(s):  
J Pieters ◽  
T Lindhout
Keyword(s):  
Thrombin Generation ◽  
Ex Vivo ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Factor Va ◽  
Factor Xa Inhibition ◽  
Heparin Fractions ◽  
Antifactor Xa ◽  
Antithrombotic Efficacy ◽  
Inhibition Of Thrombin

Abstract The antifactor Xa activities of heparin fractions are widely used as an ex vivo index of their antithrombotic efficacy. Its clinical meaning, however, remains speculative. In the study reported, we measured the effects of standard heparin, a synthetic pentasaccharide heparin (antifactor Xa activity only), and a low molecular weight heparin (LMWH) on factor Xa, factor Va, and thrombin generation in thromboplastin-activated plasma. We clearly demonstrated that the antifactor Xa activity of heparin contributed little in its anticoagulant activity. The inhibition of factor Va generation, dependent on the heparin antithrombin activity only, is of prime importance to the inhibition of thrombin generation in plasma. The inhibition of thrombin generation by the LMWH was comparable with that of standard heparin on the basis of their respective antithrombin specific activities, but not on the basis of their antifactor Xa activities.

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