Biochemical and Pharmacologic Studies on the Protamine Interactions with Heparin, Its Fractions and Fragments

Racanelli, A.; Fareed, Jawed; Walenga, Jeanine M.; Coyne, Erwin

doi:10.1055/s-2007-1004373

Cited by 48 publications

(17 citation statements)

References 27 publications

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“…Peptides containing single consensus sequences (AKKARA (q) or ARKKAAKA (E)) do not bind heparin with a measurable affinity; in contrast, significant heparin binding was seen with peptides containing multiple heparinbinding consensus sequences and increased as a function of peptide M r . ϫ, (AKKARA) 2 tween adjacent consensus sequences were altered. Both increasing(ARKKAAKA-AAAA-ARKKAAKA-AAAA-ARKKAA-KA) and decreasing (ARKKAAKA-RKKAAKA-RKKAAKA) the distance between consensus sequences resulted in decreased heparin binding affinity (K d Х 250 and 450 nM, respectively).…”

Section: Resultsmentioning

confidence: 99%

“…The CD spectra measurements of (AKKARA) 2 in the absence of heparin (1:0 (q)) indicate a charged coil conformation (peaks at 195 and 216 nm, crossover at 210 nm). However, in contrast to the heparin-induced conformational change seen for the high affinity heparin-binding peptide (AKKARA) 6 , (AKKARA) 2 , which binds heparin weakly, remains a charged coil in the presence of heparin can sometimes be achieved by constraining or manipulating peptide conformation (30). In the case of apolioprotein E and apolipoprotein B-100, heparin-binding sites are believed to form ␣-helices upon heparin binding, and molecular modeling illustrates that basic amino acids in the binding sites align to one side of the helix to form a region of high positive charge through which heparin binding occurs (6).…”

Section: Figmentioning

confidence: 94%

“…PGs are thus potential targets for therapeutic intervention. For example, heparin antagonists are needed to take the place of protamine, a heterogeneous, sometimes toxic protein mixture commonly used to neutralize the anticoagulant activity of heparin in humans (2,3); in the design of drugs to be targeted to PG-rich tissues, such as cartilage (4); and to be used to promote cell adhesion in a variety of situations, e.g. by promoting binding of cells that express abundant amounts of PGs, such as endothelial cells (5), to synthetic vascular graft surfaces.…”

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confidence: 99%

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Design of Peptides with High Affinities for Heparin and Endothelial Cell Proteoglycans

Verrecchio¹,

Germann²,

Schick³

et al. 2000

Journal of Biological Chemistry

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Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA) n and (ARKKAAKA) n (n ‫؍‬ 1-6). Affinity coelectrophoresis revealed that low M r peptides (600 -1300) had no affinities for low M r heparin, but higher M r peptides (2000 -3500) exhibited significant affinities (K d Х 50 -150 nM), which increased with peptide M r . Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K d Х 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an ␣-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M r peptides exhibited high affinities for total endothelial cell proteoglycans (K d Х 300 nM), and ϳ4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices. Proteoglycans (PGs)1 are composed of a core protein to which are covalently attached one or more sulfated glycosaminoglycans (GAGs). PGs are ubiquitous components of cell surfaces and the extracellular matrix, and their GAG chains contribute to myriad biological functions, such as modulation of enzyme activities, regulation of cell growth, and control of assembly of the extracellular matrix (1). PGs are thus potential targets for therapeutic intervention. For example, heparin antagonists are needed to take the place of protamine, a heterogeneous, sometimes toxic protein mixture commonly used to neutralize the anticoagulant activity of heparin in humans (2, 3); in the design of drugs to be targeted to PG-rich tissues, such as cartilage (4); and to be used to promote cell adhesion in a variety of situations, e.g. by promoting binding of cells that express abundant amounts of PGs, such as endothelial cells (5), to synthetic vascular graft surfaces. To develop a rationale for the design of such reagents, it is useful to examine known features of protein structure required for high affinity interactions with GAGs. Thus, analysis of the structural features of many known heparin-and heparan sulfate (HS)-binding proteins has revealed the presence of conserved motifs, through which GAG binding has been postulated to occur. Cardin and Weintraub (6) identified two clusters of basic charge in known heparin-binding proteins in which amino acids tend to be arranged in the patterns XBBXBX or XBBBXXBX, where B represents an ...

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Section: Resultsmentioning

confidence: 99%

Section: Figmentioning

confidence: 94%

mentioning

confidence: 99%

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Design of Peptides with High Affinities for Heparin and Endothelial Cell Proteoglycans

Verrecchio¹,

Germann²,

Schick³

et al. 2000

Journal of Biological Chemistry

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“…The antagonization of LMW heparins was studied by several groups using protamine sulfate 74,[76][77][78][79][80] or protamine chloride 36,[63][64][65] in vitro (all) and in vivo. 36 ' 63 " 65 ' 78 ' 80 The results are all rather similar in several respects:…”

Section: Fig 2 the Correlation Of The Molecular Weight Mw Of The Lmmentioning

confidence: 99%

Comparative Human Pharmacology of Low Molecular Weight Heparins

Harenberg¹,

Stehle²,

Augustin³

et al. 1989

Semin Thromb Hemost

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The pharmacodynamics of LMW heparins differ consistently from those of normal heparin. The relative bioavailability is greater than 90% and for some LMW heparins more than 100% after subcutaneous administration, as measured by the anti-Factor Xa activity. This indicates release of endothelial bound glycosaminoglycans with anti-Factor Xa-like activities. Direct determination of the LPL and HTGL activities demonstrate higher capacities of two and a lower capacity of three LMW heparins to release these enzymes from their endothelial glycoprotein receptors into the bloodstream. Thus, all LMW heparins are characterized by improved pharmacodynamics compared with normal heparin, but their anti-Xa to APTT and anti-Xa to anti-IIa ratios and half-lives differ among each other. The neutralization of the anticoagulant effects of LMW heparins by protamine are similar in vitro and in vivo at equigravimetric doses. However, unfractionated heparin and LMW heparins are not completely antagonized by protamine on a whole blood clotting test (thrombelastography). This inhibition of Factor Xa of LMW heparins is counteracted not completely by protamine. This may be due to a release of other glucosaminoglycans by LMW heparins. The present overview demonstrates that a comparative human pharmacology gives useful devices for prophylactic and therapeutic regimen for LMW heparins in patients in addition to the data obtained from animal models.

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“…The protamines are a family of basic proteins rich in arginine (7), which ionically bind heparin and inactivate heparin's anticoagulant activity (9,(28)(29)(30)(31). We now demonstrate that protamine can negate the vascular antiproliferative effects of heparin as well as the antithrombotic effects.…”

Section: Methodsmentioning

confidence: 99%

Protamine and protamine-insulins exacerbate the vascular response to injury.

Edelman¹,

Pukac²,

Karnovsky³

1993

J. Clin. Invest.

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Endothelial cells and smooth muscle cells produce heparinlike compounds that are growth inhibitory for vascular smooth muscle cells, and it has been suggested that these compounds play a regulatory role that is perturbed with vascular injury. Indeed, exogenous heparin preparations effectively suppress smooth muscle cell proliferation following injury imposed on vascular endothelium. We now report that protamine, an agent that binds heparin and negates its anticoagulant properties, has potent stimulatory effects on vascular smooth muscle cell proliferation. The administration of protamine, alone or as part of commonly used insulin preparations, stimulated the proliferation of cultured smooth muscle cells, exacerbated vascular smooth muscle cell proliferative lesions in laboratory rats, and interfered with the growth-inhibitory effects of heparin in culture and in vivo. These results confirm the importance of endogenous heparinlike compounds in arterial homeostasis and may require reconsideration of protamine use following' vascular reparative procedures and in diabetics. (J. Clin. Invest. 1993.

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Biochemical and Pharmacologic Studies on the Protamine Interactions with Heparin, Its Fractions and Fragments

Cited by 48 publications

References 27 publications

Design of Peptides with High Affinities for Heparin and Endothelial Cell Proteoglycans

Design of Peptides with High Affinities for Heparin and Endothelial Cell Proteoglycans

Comparative Human Pharmacology of Low Molecular Weight Heparins

Protamine and protamine-insulins exacerbate the vascular response to injury.

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