Heparin binding peptides co‐purify with glycosaminoglycans from human plasma

Chevanne, Marta; Caldini, Riccardo; Manao, Giampaolo; Ruggiero, Marco; Vannucchi, Simonetta

doi:10.1016/s0014-5793(99)01620-8

Cited by 7 publications

(8 citation statements)

References 18 publications

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“…Although the number of tested participants was limited (n ¼ 3), the results concerning the amount of HSGAGs recovered in plasma are consistent with those reported in the literature [4][5][6][7]. The authors also suggest that their binding to specific plasma proteins (and/or peptides resulting from proteolysis) could play a role in the hemostatic balance.…”

Section: Discussionsupporting

confidence: 88%

“…All plasmatic glycosaminoglycans circulate in association with a number of proteins. The association between HSGAGs and plasma proteins was the object of several studies, but this notwithstanding, little is known about the physiological role of this strong association [3,6]. Indeed, plasmatic glycosaminoglycans, in particular HSGAGs, are so strongly associated with proteins that complete purification of HSGAGs from healthy individuals is quite a long and difficult process, unsuitable for routine analysis of plasmatic HSGAGs [7].…”

Section: Discussionmentioning

confidence: 99%

“…In human plasma all glycosaminoglycans can be found [3], and CSGAGs and HSGAGs are the most abundant [4,5]. Both classes of glycosaminoglycans interact with plasma proteins; HSGAGs, however, show the tightest association with plasma proteins, and this characteristic renders their extraction and purification from plasma quite difficult [6,7].…”

Section: Introductionmentioning

confidence: 99%

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Heparin/heparan sulfate anticoagulant glycosaminoglycans in human plasma of healthy donors: preliminary study on a small group of recruits

Cecchi

Pacini

Gulisano

et al. 2008

Blood Coagulation &Amp; Fibrinolysis

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a Glycosaminoglycans in normal human plasma, mainly represented by chondroitin sulfates and heparan sulfates/ heparin (HSGAGs), show a specific distribution in the Cohn-Oncley fractions of human plasma. In the present study we investigated their effects on coagulation. Plasma was fractionated following the procedure of Cohn-Oncley, and each fraction was treated for extraction of glycosaminoglycans after extensive proteolysis; the anticoagulant activity in the extracted samples was measured by activated partial thromboplastin time (APTT). The effects of the samples containing HSGAGs on factor II and factor X activities, before and after treatment with heparinase I, were also measured. The molecular weight of HSGAGs was determined by polyacrylamide gelelectrophoresis. Cryoprecipitate and fraction I, fraction IIRIII, and fraction IV-1 (the fractions containing HSGAGs) prolonged the APTT, whereas fractions IV-4 and V had no effect on the APTT. Fractions containing HSGAGs showed effects on factor II and factor X activities that were sensitive to heparinase I treatment. The molecular weight of HSGAGs recovered in cryoprecipitate and fraction I was 15-18 kDa; that of HSGAGs recovered in fraction IV-1 was 12.0 kDa. In conclusion, these results demonstrate that HSGAGs of different molecular weight, endowed with anticoagulant activity, circulate in normal human plasma in association with specific proteins involved in the regulation of hemostasis; and that endogenous HSGAGs play a role in maintaining the antithrombotic/hemostatic balance in normal human plasma.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Heparin/heparan sulfate anticoagulant glycosaminoglycans in human plasma of healthy donors: preliminary study on a small group of recruits

Cecchi

Pacini

Gulisano

et al. 2008

Blood Coagulation &Amp; Fibrinolysis

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“…Anticoagulant activity of the fraction eluted with 1.0 M GdnHCl was as follows: anti-Xa 256.2 B 34.8 units/ml; anti-IIa 262.8 B 29.1 units/ml; APTT 265.5 B 18.1 units/ml. The ratio of anti-Xa to anti-IIa was quite similar to that of standard heparin of high molecular mass [12,23]. The fraction eluted at 1.0 M GdnHCl, when submitted to gradient PAGE, evidenced a metachromatic spot of approximate mean molecular mass of 12 kD on the basis of comparison with standard heparin of known molecular mass ( fig.…”

Section: Resultsmentioning

confidence: 57%

“…Among the recovered endogenous GAGs, an N-sulfated GAG species comigrating with standard heparin, endowed with significant anticoagulant activity and sensitive to nitrous acid treatment was recognizable. The pattern of anticoagulant activity of this N-sulfated GAG species was indistinguishable from that of standard heparins [12,23].…”

Section: Discussionmentioning

confidence: 72%

Isolation of Endogenous Anticoagulant N-Sulfated Glycosaminoglycans in Human Plasma from Healthy Subjects

Ruggiero

Melli²,

Parma³

et al. 2002

Pathophysiol Haemos Thromb

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Endogenous N-sulfated glycosaminoglycans (GAGs) comigrating with standard heparin and sensitive to nitrous acid treatment were isolated from plasma of healthy donors. The amount of these compounds was 7–10 µg/ml, and activated partial thromboplastin time, anti-Xa and anti-IIa activities were similar to those of standard heparin of high molecular mass. Analysis with gradient PAGE of the putative endogenous heparin showed a mean molecular mass of 12 kD. These N-sulfated GAGs could be isolated only after removal of binding peptides that impaired purification by ion-exchange chromatography. We used SDS-PAGE as a tool to separate peptides from endogenous GAGs. N-sulfated GAGs exited the gel before peptides when the electrophoresis was overrun. Endogenous GAGs could be recovered by ion-exchange chromatography of the SDS-PAGE buffer, ‘free’ from associating peptides. These results strongly support the hypothesis that endogenous heparin is associated in vitro with a variety of proteins and that this association could be responsible for modification of both heparin and protein activities.

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Heparin inhibits osteoclastic differentiation and function

Ariyoshi

Takahashi

Kanno

et al. 2008

J of Cellular Biochemistry

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We investigated the effects of Glycosaminoglycans (GAGs) on mouse monocytic cell line in regard to their differentiation, proliferation, and function in vitro. RAW 264.7 cells were cultured with receptor activator of NF-kappaB ligand (RANKL) and various GAGs. Osteoclastic cells were visualized by staining for tartrate-resistant acid phosphatase (TRAP) and detected using a phenyl-phosphate substrate method. RAW 264.7 cells were also cultured with stimulants contained in BD BioCoat OSTEOLOGIC(TM) kit, and bone resorption activity was assessed by counting the numbers of resorption pits. We also examined the effect of heparin on cell growth using MTT assay, while the expression level of c-Src protein was determined by immunoblot analysis. Heparin suppressed TRAP-positive multinucleated cell formation and TRAP activity induced by RANKL, whereas the other GAGs showed no effects on osteoclast differentiation. Heparin also inhibited the formation of resorption pits, while the others did not. In the MTT assay, none of the tested GAGs had an influence on RAW 264.7 cell proliferation. However, heparin reduced the level of c-Src protein in RAW 264.7 cells stimulated with RANKL. To determine the affinity of heparin and RANKL, they were subjected by HiTrap heparin column chromatography and each fraction was collected. Western blotting analysis revealed the expression of RANKL in the fraction bound to heparin. The binding of RANKL and heparin was confirmed by quartz-crystal microbalance. These results indicate that the inhibitory effect of heparin toward osteoclastogenesis induced by RANKL is due to the binding of heparin to RANKL.

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Heparin binding peptides co‐purify with glycosaminoglycans from human plasma

Cited by 7 publications

References 18 publications

Heparin/heparan sulfate anticoagulant glycosaminoglycans in human plasma of healthy donors: preliminary study on a small group of recruits

Heparin/heparan sulfate anticoagulant glycosaminoglycans in human plasma of healthy donors: preliminary study on a small group of recruits

Isolation of Endogenous Anticoagulant N-Sulfated Glycosaminoglycans in Human Plasma from Healthy Subjects

Heparin inhibits osteoclastic differentiation and function

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