Platelet receptor recognition domain on the .gamma. chain of human fibrinogen and its synthetic peptide analogs [Erratum to document cited in CA110(17):151810E]

Biochemistry ◽  
1989 ◽  
Vol 28 (19) ◽  
pp. 7974-7974
Author(s):  
Marek Kloczewiak ◽  
Sheila Timmons ◽  
Maria A. Bednarek ◽  
Masato Sakon ◽  
Jacek Hawiger
Keyword(s):  
Synthetic Peptide ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
Peptide Analogs ◽  
Platelet Receptor ◽  
Receptor Recognition

Platelet receptor recognition domain on the .gamma. chain of human fibrinogen and its synthetic peptide analogues

Biochemistry ◽  
1989 ◽  
Vol 28 (7) ◽  
pp. 2915-2919 ◽  
Author(s):  
Marek Kloczewiak ◽  
Sheila Timmons ◽  
Maria A. Bednarek ◽  
Masato Sakon ◽  
Jacek Hawiger
Keyword(s):  
Synthetic Peptide ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
Platelet Receptor ◽  
Receptor Recognition ◽  
Peptide Analogues

PLATELET RECEPTOR RECOGNITION DOMAINS AND THEIR SYNTHETIC PEPTIDE ANALOGS

1987 ◽  
Author(s):  
J Hawiger
Keyword(s):  
Adhesion Molecules ◽  
Synthetic Peptide ◽  
Synthetic Peptides ◽  
Gamma Chain ◽  
Platelet Receptors ◽  
Adhesive Protein ◽  
Peptide Analogs ◽  
Platelet Receptor ◽  
Receptor Recognition ◽  
Adhesive Molecules

Adhesive molecules and their receptorsplay an essential role in hemostasis and thrombosis. Platelet thrombi are formed through the interaction of cell adhesion molecules (CAMs) with intercellular adhesion molecules (IAMs)and substrate adhesion molecules (SAMs). Platelet CAMs encompass membrane glycoproteins lb, lib, Ilia,and possibly la and IV, which constitutemembrane receptors for IAMs(e.g., fibrinogen) and for SAMs encompassingvon Willebrand Factor (vWF), fibronectin, vitronectin, collagen, and thrcmbospondin. Receptorfunction of platelet CAMs can be specific,i.e., only one adhesive protein among IAMs and SAMs is selected forbinding as exemplified by GPIb and vWF. Alternatively,more than one adhesive protein can interact with platelet CAMs comprising the GPIIb/IIIa complex.This common adhesive receptor mechanism switched on by thrombin, ADP, phorbol ester or ionophore A23187 is turned off by a rise in intraplatelet cyclic AMP which provides a negative control.Fibrinogen, the most abundant adhesiveprotein in plasma, interacts with platelet CAMs via receptor recognition domains on gamma and alpha chains. Pinpointing platelet receptor recognition domain to a carboxy-terminal segment of the gamma chain encompassing residues 400-411gave rise to a series of synthetic peptide analogs which do not interfere with themetabolic pathways of platelets but blockbinding of I fibrinogen to its receptors on stimulated platelets, inhibit their aggregation in vitro, and formation of a platelet thrombus in vivo. The alpha chain of human fibrinogen contains the sequenceRGD (residues 95-97 and 572-574). Synthetpeptide analogs of the RGD sequence, which constitute the "cell adhesion site" of fibronectin, also inhibit binding of 125I-fibrinogen to stimulated platelets. However, these synthetic peptides are not "specific" for fibrinogen chains because thealpha chain of human fibrinogen which hasnosequence homology with gamma 400-411 is prevented by a peptide gamma 400-411 from interaction with platelet receptors. Viceversa, the human gamma chain is blocked by tetrapeptide RGDS not expressed in the human gamma chain. Interaction of human vWF with human platelets is blocked by synthetic peptide analogs of gamma 400-411 (not present in vWF)and of RGD sequence (present in vWF).These synthetic peptides inhibite "common" receptor pathwaystimulated with ADP, thrombin, or phorbolester, but they do not interfere with binding of 125I-vWF via a "specific" pathvoy induced with ristocetin and involving GPIb.The design of synthetic peptide analogs which inhibit platelet receptors for adhesive molecules includes the following considerations: ligand specificity (is thepeptide inhibitory toward binding of one or more adhesive molecules?),cell speciicity (is the peptide specific for platelets or does it perturb the adhesive properties of other cells, e.g.,endothelium?);the hydrophilic character; protection against degradation by peptidases; and a sufficiently long half-life to achieve platelet inhibitory potency in vivo without overloading the blood with excessive amounts of peptide.This is accomplished by constructing a peptide-albumin conjugate with ahalf-life extended at least 30 times.Whenpeptides are modeled with predominantly hydrophilic or hydrophobic residues, only the hydrophilic peptide remained active to block the platelet receptor. This agreed with the general observation that sequences on adhesive molecules that are knownto interact with cellular receptors have a hydrophilic rather than a hydrophobic character. Furthermore, changing the charge of synthetic peptides toward the negative reduced the reactivity, whereas introducing additional arginine residues enhanced the reactivity toward platelet receptors. Localization of the functionally important binding domain in the flexible segment of an adhesive protein increases the likelihood that the synthetic peptide will assume the conformation mimicking such a domain in the native adhesive protein. Structure-function studies of the receptor recognition domains on adhesive molecules led to development of a new class of platelet inhibitors acting at the membranereceptors responsible for anchoring of platelets to the vessel wall and linking them to each other.

Platelet receptor recognition domains on the .alpha. chain of human fibrinogen: structure-function analysis

Biochemistry ◽  
1989 ◽  
Vol 28 (7) ◽  
pp. 2909-2914 ◽  
Author(s):  
Jacek Hawiger ◽  
Marek Kloczewiak ◽  
Maria A. Bednarek ◽  
Sheila Timmons
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Human Fibrinogen ◽  
Alpha Chain ◽  
Platelet Receptor ◽  
Receptor Recognition

scholarly journals The C-terminal sequences of the gamma 57.5 chain of human fibrinogen constitute a plasmin sensitive epitope that is exposed in crosslinked fibrin

Blood ◽  
1989 ◽  
Vol 74 (7) ◽  
pp. 2437-2444
Author(s):  
PJ Haidaris ◽  
EI Peerschke ◽  
VJ Marder ◽  
CW Francis
Keyword(s):  
Platelet Aggregation ◽  
Binding Site ◽  
Synthetic Peptide ◽  
Synthetic Peptides ◽  
Competitive Inhibition ◽  
Plasma Fibrinogen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Post Translational Modification ◽  
Gamma Chain ◽  
Human Fibrinogen

The gamma chain of human plasma fibrinogen is heterogeneous with three forms differing in length at the C-terminus. Alternative RNA splicing produces two gamma chain mRNAs encoding gamma 50 and gamma 57.5 polypeptides, while fibrinogen gamma 55 is produced by post- translational modification of the gamma 57.5 chain. The composition of purified variant gamma chain fibrinogens, which comprise 10% to 13% total plasma fibrinogen, is predominantly heterodimeric (A alpha, B beta, gamma 50/gamma 55 or A alpha, B beta, gamma 50/gamma 57.5), whereas the composition of purified fibrinogen with the major form of the gamma chain is homodimeric (A alpha, B beta, gamma 50/gamma 50). These gamma chain variations interrupt sequences that mediate platelet- fibrinogen interactions. Therefore, the structure and function of gamma 57.5 C-terminal sequences were investigated using synthetic peptides and a specific monoclonal antibody (MoAb), L2B. The L2B epitope was localized and included gamma 57.5 chain residues 409–412 (Arg-Pro-Glu- His), as determined by differential enzyme-linked immunosorbent assay (ELISA) reactivity with a His-412 deleted synthetic peptide and by Western blot analysis of plasmin cleaved fibrinogen gamma 57.5. L2B had no effect on adenosine diphosphate (ADP)-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. High concentrations (0.5 to 1 mmol/L) of synthetic peptide gamma 57.5 405– 416 only weakly inhibited ADP-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. Binding of fibrinogen gamma 50 (IC50 = 780 mumol/L) or gamma 57.5 (IC50 = 650 mumol/L) to ADP- stimulated platelets was weakly inhibited, and MoAb L2B failed to inhibit fibrinogen gamma 57.5 binding. Peptide gamma 57.5 408–416 failed to dissociate platelet-bound fibrinogens. These data indicate that the gamma 408–416 sequence of fibrinogen gamma 55 or gamma 57.5 alone is unlikely to bind to the platelet fibrinogen receptor, glycoprotein llb-llla (GPllb-llla), in support of platelet aggregation under physiologic conditions. The sequence recognized by L2B does not resemble known GPllb-llla binding site peptide sequences [Arg-Gly-Asp- Ser (RGDS) or gamma 50 400–411] as determined by competitive inhibition ELISA comparing these binding site synthetic peptides with gamma 57.5 408–416. This epitope is available for binding MoAb L2B in gamma 55 or gamma 57.5 chain dimers and binds to all gamma 57.5 408–416 epitopes equally in non-crosslinked and factor Xllla crosslinked fibrin clots.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals The C-terminal sequences of the gamma 57.5 chain of human fibrinogen constitute a plasmin sensitive epitope that is exposed in crosslinked fibrin

Blood ◽  
1989 ◽  
Vol 74 (7) ◽  
pp. 2437-2444 ◽  
Author(s):  
PJ Haidaris ◽  
EI Peerschke ◽  
VJ Marder ◽  
CW Francis
Keyword(s):  
Platelet Aggregation ◽  
Binding Site ◽  
Synthetic Peptide ◽  
Synthetic Peptides ◽  
Competitive Inhibition ◽  
Plasma Fibrinogen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Post Translational Modification ◽  
Gamma Chain ◽  
Human Fibrinogen

Abstract The gamma chain of human plasma fibrinogen is heterogeneous with three forms differing in length at the C-terminus. Alternative RNA splicing produces two gamma chain mRNAs encoding gamma 50 and gamma 57.5 polypeptides, while fibrinogen gamma 55 is produced by post- translational modification of the gamma 57.5 chain. The composition of purified variant gamma chain fibrinogens, which comprise 10% to 13% total plasma fibrinogen, is predominantly heterodimeric (A alpha, B beta, gamma 50/gamma 55 or A alpha, B beta, gamma 50/gamma 57.5), whereas the composition of purified fibrinogen with the major form of the gamma chain is homodimeric (A alpha, B beta, gamma 50/gamma 50). These gamma chain variations interrupt sequences that mediate platelet- fibrinogen interactions. Therefore, the structure and function of gamma 57.5 C-terminal sequences were investigated using synthetic peptides and a specific monoclonal antibody (MoAb), L2B. The L2B epitope was localized and included gamma 57.5 chain residues 409–412 (Arg-Pro-Glu- His), as determined by differential enzyme-linked immunosorbent assay (ELISA) reactivity with a His-412 deleted synthetic peptide and by Western blot analysis of plasmin cleaved fibrinogen gamma 57.5. L2B had no effect on adenosine diphosphate (ADP)-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. High concentrations (0.5 to 1 mmol/L) of synthetic peptide gamma 57.5 405– 416 only weakly inhibited ADP-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. Binding of fibrinogen gamma 50 (IC50 = 780 mumol/L) or gamma 57.5 (IC50 = 650 mumol/L) to ADP- stimulated platelets was weakly inhibited, and MoAb L2B failed to inhibit fibrinogen gamma 57.5 binding. Peptide gamma 57.5 408–416 failed to dissociate platelet-bound fibrinogens. These data indicate that the gamma 408–416 sequence of fibrinogen gamma 55 or gamma 57.5 alone is unlikely to bind to the platelet fibrinogen receptor, glycoprotein llb-llla (GPllb-llla), in support of platelet aggregation under physiologic conditions. The sequence recognized by L2B does not resemble known GPllb-llla binding site peptide sequences [Arg-Gly-Asp- Ser (RGDS) or gamma 50 400–411] as determined by competitive inhibition ELISA comparing these binding site synthetic peptides with gamma 57.5 408–416. This epitope is available for binding MoAb L2B in gamma 55 or gamma 57.5 chain dimers and binds to all gamma 57.5 408–416 epitopes equally in non-crosslinked and factor Xllla crosslinked fibrin clots.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals Reactivity of synthetic peptide analogs of adhesive proteins in regard to the interaction of human endothelial cells with extracellular matrix

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2200-2206 ◽  
Author(s):  
CS Chen ◽  
J Hawiger
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Synthetic Peptide ◽  
Cell Monolayer ◽  
Human Fibrinogen ◽  
Hybrid Peptides ◽  
Peptide Analogs ◽  
Extracellular Matrix Components ◽  
Adhesive Proteins

Abstract Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to “cell adhesion site” of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400–411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). “Hybrid” peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys- Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His- His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.

Effects of Hybrid Peptide Analogs to Receptor Recognition Domains on α- and γ-Chains of Human Fibrinogen on Fibrinogen Binding to Platelets

1993 ◽  
Vol 69 (05) ◽  
pp. 490-495 ◽  
Author(s):  
Hiroshi Mohri ◽  
Takao Ohkubo
Keyword(s):  
Binding Affinity ◽  
Platelet Rich Plasma ◽  
Membrane Receptors ◽  
Hybrid Peptide ◽  
Human Fibrinogen ◽  
Hybrid Peptides ◽  
Peptide Analogs ◽  
Receptor Recognition ◽  
Adhesive Proteins ◽  
Induced Aggregation

SummaryWe synthesized a series of hybrid peptides that correspond to the γ-chain dodecapeptide (400-411), variable numbers of glycine residues, and the RGDS peptide [Y-HHLGGAK-QAGDV(G) n RGDS] to investigate the relationship of these receptor recognition domains of fibrinogen to platelet membrane glycoprotein IIb/IIIa. The tetrapeptide RGDS, the GRGDSPA peptide and the dodecapeptide inhibited binding of fibrinogen to GPIIb/IIIa by 50% (IC50) at concentrations of 17 ± 1.6 μM, 15 ± 2.1 μM, and 87 ± 6.8 μM, respectively. The inhibitory effect of hybrid peptides increased as the number of glycine residues increased, plateauing with 9-11 glycine residues in hybrid peptide analogs, which had an IC50 of 0.68 ± 0.14 μM. These hybrid peptides completely inhibited the binding of fibrinogen to activated platelets when used in sufficient concentrations. The peptide Y-HHLGGAKQAGDV(G)9RGDS blocked ADP-induced aggregation in citrated platelet-rich plasma with IC50 of 3.5 ± 0.6 μM. When the peptide Y-HHLGGAK-QAGDV(G)9RGDS was labeled with 125I to quantify its binding to platelets, maximal binding was observed within 30 min. The binding sites of the hybrid peptide were 43,600 molecules/platelet (K d = 3.1 ± 0.5 × 10-7 M) to stimulated platelets and 12,500 molecules/platelet (K d = 1.4 ± 0.2 × 10-7 M) to nonstimulated platelets. The hybrid peptides had the same binding affinity to platelets as fibrinogen and inhibited platelet function. Moreover, anti-GPIIb/IIIa antibody inhibited the binding of the labeled hybrid peptide to stimulated platelets. These results indicate that in the native fibrinogen molecule the presence of both RGD sequence or γ-chain domain at optimal distances increased the binding affinity to GPIIb/IIIa. These domains may be the source of hybrid peptide, expanding a new class of platelet inhibitors that act at membrane receptors for adhesive proteins.

scholarly journals Reactivity of synthetic peptide analogs of adhesive proteins in regard to the interaction of human endothelial cells with extracellular matrix

Blood ◽  
1991 ◽  
Vol 77 (10) ◽  
pp. 2200-2206
Author(s):  
CS Chen ◽  
J Hawiger
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Synthetic Peptide ◽  
Cell Monolayer ◽  
Human Fibrinogen ◽  
Hybrid Peptides ◽  
Peptide Analogs ◽  
Extracellular Matrix Components ◽  
Adhesive Proteins

Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to “cell adhesion site” of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400–411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). “Hybrid” peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys- Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His- His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.

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