Effect of Calcium and Synthetic Peptides on Fibrin Polymerization

Furlan, Miha; Rupp, C; Beck, Eugene A.; Svendsen, L

doi:10.1055/s-0038-1657143

Cited by 53 publications

(33 citation statements)

References 18 publications

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“…The GHRP peptide mimicking the amino terminus of the fibrin ␤ chain offered no protection at all in the absence of Ca 2ϩ . This is in agreement with results obtained by others with fibrinogen (39,42) or (desAA)fibrin monomers (15). The longer peptides (encoding the true sequence of the ␣ chain, as opposed to the peptide analog GPRP, which binds to fibrinogen more tightly than does the wild-type sequence) were markedly less protective than was GPRP.…”

Section: Discussionsupporting

confidence: 92%

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The Polymerization Pocket “a” within the Carboxyl-terminal Region of the γ Chain of Human Fibrinogen Is Adjacent to but Independent from the Calcium-binding Site

Côté

Pratt

Davie

et al. 1997

Journal of Biological Chemistry

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The carboxyl-terminal region of the ␥ chain of fibrinogen is involved in calcium binding, fibrin polymerization, factor XIIIa-mediated cross-linking, and binding to the platelet fibrin(ogen) receptor. to Leu 427 (␥C30) from the carboxyl end of the ␥ or ␥ chains, respectively, of human fibrinogen were expressed in yeast (Pichia pastoris) and characterized as to their cross-linking by factor XIIIa, polymerization pocket, and calcium-binding site. rFbg␥C30 and ␥C30 were both readily cross-linked by factor XIIIa, but only rFbg␥C30 was capable of inhibiting thrombin-induced platelet aggregation. Two mutants, ␥C30-Q329R and ␥C30-D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbg␥C30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized. rFbg␥C30 inhibited (desAA)fibrin polymerization in a dose-dependent manner, while the two mutant forms did not. Similarly, rFbg␥C30 and ␥C30 were protected from plasmin degradation by the presence of Ca 2؉ or the peptide Gly-Pro-Arg-Pro, indicating that a functional Ca 2؉ -binding site and polymerization pocket are contained within each of these fragments. The mutant fragments, however, were protected from plasmin only by metal ions, while no protective effect was conferred by GPRP or by any other peptide tested. These results indicate that the polymerization pocket "a", which binds the peptide GPRP, functions independently from the nearby calcium-binding site and that amino acids Gln 329 and Asp 364 play a crucial role in fibrin polymerization.Fibrinogen is a large glycoprotein composed of six polypeptide chains (␣, ␤, ␥) 2 held together by disulfide bonds. By electron microscopy, the molecule appears as a trinodular structure (1) in which the central nodule contains the amino termini of all six chains (2). The two distal nodules contain the carboxyl-terminal regions of the ␥ and ␤ chains. Upon cleavage by thrombin, fibrinopeptides A and B are released from the amino termini of the ␣ and ␤ chains, resulting in fibrin monomers that polymerize spontaneously. The newly exposed amino termini constitute polymerization sites A and B that are complementary to polymerization pockets "a" and "b", respectively (3-6). The "a" pocket is located within the carboxyl region of the ␥ chain (7, 8), while the location of the "b" pocket remains controversial. It has been proposed to arise upon the alignment of the D domains of two fibrin molecules (6), to be contained in the carboxyl-terminal region of the ␤ chain (9, 10), and to involve the carboxyl-terminal region of the ␣ chain (11).Calcium promotes the polymerization of fibrin monomers (12-15) and the cross-linking of fibrin by factor XIIIa (16). It also protects fibrinogen fragment D against plasmic degradation (17). Fibrinogen binds three calcium ions per molecule (18,19), including one in the carboxyl-terminal region of each ␥ chain. The third calcium is present in the central nodule, provided that the ␣ chains are intact (20). The precise loc...

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Section: Discussionsupporting

confidence: 92%

“…Calcium promotes the polymerization of fibrin monomers (12)(13)(14)(15) and the cross-linking of fibrin by factor XIIIa (16). It also protects fibrinogen fragment D against plasmic degradation (17).…”

mentioning

confidence: 99%

The Polymerization Pocket “a” within the Carboxyl-terminal Region of the γ Chain of Human Fibrinogen Is Adjacent to but Independent from the Calcium-binding Site

Côté

Pratt

Davie

et al. 1997

Journal of Biological Chemistry

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“…7 Because the propositus is a heterozygous carrier of abnormal fibrinogen, the purified fibrinogen preparation contained normal and abnormal A␣-and ␥-chains. Normal fibrinogen was isolated from a plasma pool of healthy donors.…”

Section: Purification Of Fibrinogenmentioning

confidence: 99%

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

Bolliger-Stucki

Lord

Furlan

2001

Blood

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Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: A␣ R16C and ␥ G165R. As seen previously with other heterozygous A␣ R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to A␣ 16 C. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the ␣-helix content in the variant D3 fragment. It is concluded that the A␣-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the ␥-chain induced a conformational change in the D3 fragment. IntroductionFibrinogen, a soluble plasma glycoprotein of 340 kd, is made up of 2 copies of 3 different polypeptide chains (A␣ 2 , B␤ 2 ,␥ 2 ), linked together with 29 interchain and intrachain disulfide bridges. The molecule is organized in a dimeric fashion consisting of a central E domain containing the amino termini of all 6 polypeptide chains and 2 outer D domains. In the final stage of blood coagulation, thrombin cleaves the fibrinopeptides A and B in a sequential manner from the amino termini of the A␣-and B␤-chains. Cleavage occurs between residues R16 (single-letter amino acid abbreviations) and G17 of the A␣-chain and residues R14 and G15 of the B␤-chain, exposing the A and B polymerization sites. Resultant monomers join together to form 2-stranded, halfstaggered protofibrils. It has been shown that protofibrils result from longitudinal D-D interactions and noncovalent contacts between the A and the a sites. The a site is formed by residues 329, 330, 340, and 364 in the carboxy-terminal part of the ␥-chain. 1,2 Subsequently, the growing fibrils aggregate in a lateral fashion to form fibers that increase progressively in thickness and develop branch points. The evolving network is finally stabilized with covalent bonds by the activated factor XIII, resulting in a clot resistant to mechanical disruption.Dysfibrinogenemia is a heritable disorder characterized by structural mutations in any of the 3 polypeptide cha...

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“…This enhances the rate and extent of lateral association of protofibrils (10). Cleavage of the ␣ chain alone by proteases such as batroxobin, however, also leads to gel formation (11), indicating that the initial association of the complementary ''a'' and A sites is sufficient to promote clot formation.…”

mentioning

confidence: 99%

“…The peptides GPRP and GPRPamide bind even more tightly to the fibrinogen ''a'' site than does a peptide corresponding to the ␣ chain native sequence GPRV (15). The complex between GPRP and fibrinogen has an increased resistance to degradation by plasmin (16), and the polymerization reaction is depressed by the addition of excess peptide (11,12). The binding of calcium also stabilizes fibrinogen against plasmin digestion (17).…”

mentioning

confidence: 99%

The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro

Pratt

Côté

Chung

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

157

133

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After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the ␣ chain of one fibrin molecule and the C-terminal region of a ␥ chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) ␥ chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the ␣ chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

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Effect of Calcium and Synthetic Peptides on Fibrin Polymerization

Cited by 53 publications

References 18 publications

The Polymerization Pocket “a” within the Carboxyl-terminal Region of the γ Chain of Human Fibrinogen Is Adjacent to but Independent from the Calcium-binding Site

The Polymerization Pocket “a” within the Carboxyl-terminal Region of the γ Chain of Human Fibrinogen Is Adjacent to but Independent from the Calcium-binding Site

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

The primary fibrin polymerization pocket: Three-dimensional structure of a 30-kDa C-terminal γ chain fragment complexed with the peptide Gly-Pro-Arg-Pro

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