In vitro and in vivo evaluation of five low molecular weight proteins of Ehrlichia ruminantium as potential vaccine components

Ultra low molecular weight saccharide fragments (ULMFs) have been obtained from porcine mucosal heparin (PMH) by extraction (e-ULMF) and by bacterial heparinase depolymerization (h-ULMF-8) processes. Both fragments showed a strong anti Xa activity (>2000 u/mg units, Yin and Wessler, J. Lab. Clin. Med. 81, 298, 1973) and possess relatively weak potencies in the US Pharmacoepial (<40 USP u/mg) and other conventional coagulant assays (activated and non activated partial thromboplastin time, thrombin time and whole blood activated recalcification times). Since ULMFs showed a strong anti Xa activity, we evaluated their antithrombotic actions in a modified stasis-thrombosis model (Wessler et. al. J. App. Phys. 14, 943, 1959) challenging the animals with various thrombogenic stimuli; activated and non activated prothrombin complex concentrates, factor Xa concentrates and human serum. h-ULMF-8 at dosage <0.125 mg/kg (<250 Anti Xa u/kg) IV and <1.0 mg/kg (>2000 anti Xa u/kg) SC completely protected the thrombogenic effects of various thrombogenic agents, whereas PMH at these dosages failed to produce any protection in pre and post treatment regiments. Similar studies with e-ULMF showed protection, however, the antithrombotic responses varied among animals. In vitro supplementation of heparin fragments at 5 times the concentration which protected animals against the thrombogenic effects of activated prothrombin complex concentrates failed to produce any elevation of prothrombin time, partial thromboplastin times, thrombin time and other coagulant assays. Our studies suggest that ULMFs are potent antithrombotic agents and may exert their effects involving multiple sites and primarily inhibiting the Xa and the non-thrombin serine proteases formed during activated states.

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Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648988 ◽

1994 ◽

Vol 72 (06) ◽

pp. 942-946 ◽

Cited By ~ 20

Author(s):

Raffaele Landolfi ◽

Erica De Candia ◽

Bianca Rocca ◽

Giovanni Ciabattoni ◽

Armando Antinori ◽

...

Keyword(s):

Molecular Weight ◽

Unfractionated Heparin ◽

Low Molecular Weight Heparin ◽

Primary Source ◽

In Vivo Studies ◽

Low Molecular Weight ◽

Heparin Administration ◽

To Receive

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.

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Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661675 ◽

1986 ◽

Vol 56 (03) ◽

pp. 318-322 ◽

Cited By ~ 18

Author(s):

V Diness ◽

P B Østergaard

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Thrombus Formation ◽

Experimental Models ◽

Protamine Sulfate ◽

Low Molecular Weight ◽

Antithrombotic Effect ◽

Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

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In Vitro and In Vivo Measurement of Percutaneous Penetration of Low Molecular Weight Toxins of Military Interest

10.21236/ada206485 ◽

1989 ◽

Author(s):

B. W. Kemppainen ◽

M. Mehta ◽

R. G. Stafford ◽

R. T. Riley ◽

C. R. Clark

Keyword(s):

Molecular Weight ◽

Percutaneous Penetration ◽

Low Molecular Weight ◽

In Vivo Measurement

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Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽

10.1182/blood-2003-07-2334 ◽

2004 ◽

Vol 103 (4) ◽

pp. 1356-1363 ◽

Cited By ~ 30

Author(s):

Barbara P. Schick ◽

David Maslow ◽

Adrianna Moshinski ◽

James D. San Antonio

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Tandem Repeats ◽

Factor Xa ◽

Low Molecular Weight ◽

Heparin Binding ◽

High Affinity ◽

Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

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Novel Role for Albumin in Innate Immunity: Serum Albumin Inhibits the Growth of Blastomyces dermatitidis Yeast Form In Vitro

Infection and Immunity ◽

10.1128/iai.71.11.6648-6652.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6648-6652 ◽

Cited By ~ 12

Author(s):

Steven Giles ◽

Charles Czuprynski

Keyword(s):

Molecular Weight ◽

Inhibitory Activity ◽

Mammalian Species ◽

Blastomyces Dermatitidis ◽

Low Molecular Weight ◽

Rat Serum ◽

Yeast Form ◽

Domain Iii

ABSTRACT In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

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