Heparin-mimetic sulfated peptides with modulated affinities for heparin-binding peptides and growth factors

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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Growth factors in the regulation of reparative response in the presence of peritoneal damage

Pleura and Peritoneum ◽

10.1515/pp-2020-0114 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Irina A. Shurygina ◽

Мichael G. Shurygin ◽

Lubov V. Rodionova ◽

Nataliya I. Ayushinova

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Extended Period ◽

Tissue Growth ◽

Tissue Response ◽

Heparin Binding ◽

Lower Quadrant ◽

Male Wistar Rats ◽

The Right ◽

Endothelial Growth Factor

AbstractObjectivesTo study the expression of growth factors in the regulation of tissue repair after peritoneal damage tissue response to peritoneal damage.MethodsExperimental study in 35 male Wistar rats determining the evolution over time of the tissue response to aseptic peritoneal damage. A standardized bowel and peritoneal lesions were created in the right lower quadrant by laparotomy. Then, tissular expression of growth factors was evaluated by multiplex polymerase chain reaction at seven timepoints between 6 h and 30 days, postoperatively.ResultsTissular responses of granulocyte-stimulating factors (Csf2, Csf3), connective tissue growth factor (Ctgf), epidermal growth factors and receptor (Egf, Egfr), fibroblast growth factors (Fgf2, 7 and 10), heparin binding EGF-like growth factor (Hbegf), hepatocyte growth factor (Hgf), insulin-like growth factor-1 (Igf1), mitogenic transforming growth factors (Tgfa, Tgfb1, Tgfbr3), and vascular endothelial growth factor A (Vegfa) were biphasic with a first expression peak at day 3, followed by a more pronounced peak at day 14.ConclusionsWe observed a long-lasting, widespread response of tissular growth factors for at least two weeks after peritoneal damage. To be clinically effective, the prophylaxis of postoperative adhesions might be needed for an extended period of time.

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CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor.

The Journal of Cell Biology ◽

10.1083/jcb.128.4.687 ◽

1995 ◽

Vol 128 (4) ◽

pp. 687-698 ◽

Cited By ~ 269

Author(s):

K L Bennett ◽

D G Jackson ◽

J C Simon ◽

E Tanczos ◽

R Peach ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factors ◽

Growth Factor ◽

Binding Proteins ◽

Vascular Endothelial Cells ◽

Heparin Binding ◽

Binding Studies ◽

Cd44 Isoforms ◽

Cultured Endothelial Cells ◽

Alternatively Spliced

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall.

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Heparin stimulates the proliferation of intestinal epithelial cells in primary culture

Journal of Cell Science ◽

10.1242/jcs.107.2.401 ◽

1994 ◽

Vol 107 (2) ◽

pp. 401-411

Author(s):

N. Flint ◽

F.L. Cove ◽

G.S. Evans

Keyword(s):

Growth Factors ◽

Heparan Sulphate ◽

Primary Cultures ◽

Cell Types ◽

Heparin Binding ◽

Epithelial Proliferation ◽

Inhibition Of Proliferation ◽

Trophic Effect ◽

Heparin Binding Growth Factors

Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminoglycans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysaccharides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.

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