The synthetic peptide Gly‐Pro‐Arg‐Pro‐amide limits the plasmic digestion of fibrinogen in the same fashion as calcium ion

Yamazumi, Kensuke; Doolittle, Russell F.

doi:10.1002/pro.5560011220

Cited by 29 publications

(18 citation statements)

References 6 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%

“…Fragment D is protected against plasmin degradation by the presence of calcium ions (17) or a peptide resembling the amino terminus of the fibrin ␣ chain (GPRP) (39). Several metal ions and synthetic peptides were tested for their ability to protect rFbg␥C30, ␥ЈC30, and the mutants against plasmin digestion.…”

Section: Resultsmentioning

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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“…The experiment was repeated in the presence of the peptide GPRP (0, 2, and 5 mM) with 1 mM EDTA added to all reactions. 12 Immunoblot analysis was performed with nonreduced fibrinogen degradation products. Electrophoresed proteins were transferred onto nitrocellulose sheets and incubated at room temperature with the following antibodies: polyclonal rabbit anti-human fibrinogen (A0080; Dako); polyclonal rabbit anti-human fibrinogen ␥-chain prepared by Hazelton Research Products (Denver, PA) using ␥-chain purified from inclusion bodies expressed in Escherichia coli as the antigen 13 ; monoclonal anti-human serum albumin (clone HAS-11, A6684; Sigma, St Louis, MO); monoclonal antibody E2F8E5 to fragment E, recognizing the sequence GHRPLDK (␤ 15-21) (Immunotech, Marseilles, France); peroxidase-conjugated antimouse or anti-rabbit IgG (Calbiochem, La Jolla, CA).…”

Section: Sds-page and Immunoblot Analysis Of Fibrinogen Degradation Pmentioning

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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“…In the presence of calcium ions or GPRP peptide, plasmin digestion of normal fibrinogen generates the fragments D1 and E, whereas in the presence of EDTA the C-terminal part of the ␥ chain is further degraded with fragment D1 transformed to D2 and D3. 26,27 In the presence of Ca ϩϩ ions or GPRP, plasmin digests of fibrinogen Longmont were similar to normal, except for an additional fragment with a Mol wt of approximately 200 kd ( Figure 3A). Immunoblotting analysis with an antihuman fibrinogen confirmed that this novel fragment was derived from fibrinogen ( Figure 3B).…”

Section: Fibrinogen Longmont Structurementioning

confidence: 81%

The impaired polymerization of fibrinogen Longmont (Bβ166Arg→Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers

Lounes

Lefkowitz

Henschen-Edman

et al. 2001

Blood

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This study identified a new substitution in the B␤ chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Removing the dimeric molecules using gel filtration did not correct the fibrin polymerization defect. Fibrinogen Longmont had normal fibrinopeptide A and B release and a functional polymerization site "a." Thus, the sites "A" and "a" can interact to form protofibrils, as evidenced by dynamic light-scattering measurements. These protofibrils, however, were unable to associate in the normal manner of lateral aggregation, leading to abnormal clot formation, as shown by an impaired increase in turbidity. Therefore, it is concluded that the substitution of Arg1663Cys-Cys alters fibrinogen Longmont polymerization by disrupting interactions that are critical for normal lateral association of protofibrils. IntroductionThe fibrinogen molecule is composed of 2 copies of 3 polypeptide chains called A␣, B␤, and ␥. The molecule consists of a central domain (E) linked to 2 distal domains (D) by a coiled-coil connector. The N-terminal ends of the 6 chains form the E domain, whereas the C-terminal parts of the ␥ and B␤ chains and a short fragment of the C-terminal domain of the A␣ chain constitute the D domain.The fibrinogen-to-fibrin conversion is an ordered reaction. It is initiated by the cleavage of fibrinopeptides A and B (FpA and FpB) by thrombin, which unmasks polymerization sites "A" and "B" in the neo-N-terminal ends of the ␣ and ␤ chains, respectively. 1 Thereafter, the fibrin monomers interact spontaneously and form a fibrin clot. Concomitantly, thrombin-activated factor XIII stabilizes the clot by introducing interchain cross-links between ␥ chains and between ␣ chains. [2][3][4] Fibrin polymerization is a 2-step process. First, half-staggered, end-to-end interactions form double-stranded protofibrils, which result from interactions between site "A" and its complementary site "a," 5 always present in the ␥ chain part of the D domain. 6 In fact, the peptide Gly-Pro-Arg-Pro (GPRP), which resembles the natural Gly-Pro-Arg-Val sequence of the N-terminus extremity of the ␣ chain depleted in FpA, prevents clot formation. 7 In the second step, termed "lateral association," the protofibrils are assembled into thick fibers of fibrin that branch to form a fibrin network. 8,9 The interactions involved in the lateral association are less well characterized. It has been proposed that the release of FpB enhances lateral association. 10,11 Alternatively, the release of FpB may be a consequence of fibrin formation. 12 Moreover, the C-terminal domains of the A␣ chain may also participate in lateral association. 13,14 In this report, we describe fibrinogen Longmont,...

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The synthetic peptide Gly‐Pro‐Arg‐Pro‐amide limits the plasmic digestion of fibrinogen in the same fashion as calcium ion

Cited by 29 publications

References 6 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 impaired polymerization of fibrinogen Longmont (Bβ166Arg→Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers

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