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 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 Megakaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of gamma chain-variant polypeptides

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 743-750 ◽  
Author(s):  
PJ Haidaris ◽  
CW Francis ◽  
LA Sporn ◽  
DS Arvan ◽  
FA Collichio ◽  
...  
Keyword(s):  
Rna Processing ◽  
Plasma Fibrinogen ◽  
Chain Gene ◽  
Post Translational Modification ◽  
Gamma Chain ◽  
Specific Expression ◽  
Human Fibrinogen ◽  
Alternative Processing ◽  
C Terminus ◽  
Processing Event

Abstract The gamma chain of human fibrinogen is heterogeneous in length at the C- terminus due to differential RNA processing of the gamma chain-gene primary transcript. We have produced two specific monoclonal antibodies (MoAbs) against the gamma-chain epitopes generated by this alternative processing event: anti-gamma 57.5(408–416) (L2B), which reacts with gamma 57.5 and gamma 55 chains, and anti-gamma 50(337–411) (H9B7), which reacts preferentially with gamma 50 chains. Using these MoAbs we have studied the expression of gamma-chain polypeptides by immunofluorescence microscopy in the tissues of fibrinogen biosynthesis and have determined that gamma 57.5 polypeptide is expressed in hepatocytes but is absent or present in significantly reduced amounts in megakaryocytes. Therefore the gamma 50 chain is found in plasma, platelet, and megakaryocyte fibrinogens, but the gamma 57.5 chain is found only in plasma fibrinogen. The C-terminal amino acid sequence of gamma 55 includes the L2B epitope 57.5(408–416). Using MoAb L2B we have determined that gamma 55, which is a post-translationally modified gamma 57.5 chain, is found only in plasma fibrinogen and is absent or present in markedly reduced amounts in platelet or megakaryocyte fibrinogen. In addition, the conformation of the L2B epitope is preserved in gamma 55, as determined by Western blot analysis. The hepatocyte-specific expression of the gamma 57.5-chain polypeptide and the post-translational modification to gamma 55 result in a compartmentalization of gamma-chain polypeptide expression. This is suggestive of different mechanisms regulating human fibrinogen gamma- chain gene expression in hepatocytes v megakaryocytes that may operate in a tissue-specific manner at the level of 3′ RNA processing events.

scholarly journals Megakaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of gamma chain-variant polypeptides

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 743-750
Author(s):  
PJ Haidaris ◽  
CW Francis ◽  
LA Sporn ◽  
DS Arvan ◽  
FA Collichio ◽  
...  
Keyword(s):  
Rna Processing ◽  
Plasma Fibrinogen ◽  
Chain Gene ◽  
Post Translational Modification ◽  
Gamma Chain ◽  
Specific Expression ◽  
Human Fibrinogen ◽  
Alternative Processing ◽  
C Terminus ◽  
Processing Event

The gamma chain of human fibrinogen is heterogeneous in length at the C- terminus due to differential RNA processing of the gamma chain-gene primary transcript. We have produced two specific monoclonal antibodies (MoAbs) against the gamma-chain epitopes generated by this alternative processing event: anti-gamma 57.5(408–416) (L2B), which reacts with gamma 57.5 and gamma 55 chains, and anti-gamma 50(337–411) (H9B7), which reacts preferentially with gamma 50 chains. Using these MoAbs we have studied the expression of gamma-chain polypeptides by immunofluorescence microscopy in the tissues of fibrinogen biosynthesis and have determined that gamma 57.5 polypeptide is expressed in hepatocytes but is absent or present in significantly reduced amounts in megakaryocytes. Therefore the gamma 50 chain is found in plasma, platelet, and megakaryocyte fibrinogens, but the gamma 57.5 chain is found only in plasma fibrinogen. The C-terminal amino acid sequence of gamma 55 includes the L2B epitope 57.5(408–416). Using MoAb L2B we have determined that gamma 55, which is a post-translationally modified gamma 57.5 chain, is found only in plasma fibrinogen and is absent or present in markedly reduced amounts in platelet or megakaryocyte fibrinogen. In addition, the conformation of the L2B epitope is preserved in gamma 55, as determined by Western blot analysis. The hepatocyte-specific expression of the gamma 57.5-chain polypeptide and the post-translational modification to gamma 55 result in a compartmentalization of gamma-chain polypeptide expression. This is suggestive of different mechanisms regulating human fibrinogen gamma- chain gene expression in hepatocytes v megakaryocytes that may operate in a tissue-specific manner at the level of 3′ RNA processing events.

Interaction of the Integrin αIIbβ3 with Monomeric Fibrin at the Single-Molecule Level.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4018-4018
Author(s):  
Rustem I. Litvinov ◽  
Henry Shuman ◽  
David H. Farrell ◽  
Joel S. Bennett ◽  
John W. Weisel
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Competitive Inhibition ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Rupture Force ◽  
Fab Fragment ◽  
Human Fibrinogen ◽  
Integrin Αiibβ3 ◽  
Binding Strength

Abstract Abstract 4018 Poster Board III-954 Although platelets aggregated with fibrin constitute major components of in vivo blood clots and thrombi, heretofore most research has focused on fibrinogen's role in platelet aggregation. The integrin αIIbβ3 has been shown to primarily mediate platelet-fibrin interactions, but the mechanism of αIIbβ3 binding to fibrin is largely unknown and may be significantly different from binding to fibrinogen. To elucidate mechanisms of platelet interactions with fibrin, we have compared the overall reactivity, binding strength, and specificity towards αIIbβ3 of human fibrinogen vs. monomeric fibrin, using single-molecule optical trap-based rupture force spectroscopy of the surface-bound proteins. In this system, a ligand-coated bead is trapped by the laser and repeatedly brought into contact with a receptor-coated surface so that the forces required to separate the two can be measured and displayed as rupture force histograms. We have applied this technique to the interaction of fibrin(ogen) and αIIbβ3 by measuring the force required to separate a laser-trapped bead coated with either fibrinogen or monomeric fibrin from an immobilized pedestal coated with purified αIIbβ3. Experiments were performed with plasma-purified or recombinant homodimeric γA/γA and γ′/γ′ fibrin(ogen)s. The latter protein represents a splicing variant in which the γ chain has a substitution in the C-terminal four amino acids in 400-411 dodecapeptide, the major αIIbβ3-binding site in fibrinogen. Surface-bound monomeric fibrin was obtained by treating fibrinogen-coated surfaces with thrombin. Control integrin-fibrinogen interactions manifested as a characteristic bimodal rupture force histogram with rupture forces ranging from 20 pN to 140 pN, similar to what we had observed previously (PNAS, 2002, 99, 7426). The interactions were highly sensitive to inhibitory effects of abciximab and eptifibatide, specific αIIbβ3 antagonists, and to Mn2+-induced activation, indicating that they were mediated by the functional integrin. Monomeric fibrin γA/γA was at least as reactive towards αIIbβ3 as the parental fibrinogen γA/γA, sometimes exhibiting even higher binding probabilities and slightly stronger rupture forces. Fibrin-integrin interactions were less sensitive to the inhibitory effect of abciximab compared to fibrinogen, suggesting that some additional structures in αIIbβ3, not completely blocked by this Fab fragment, might be involved in binding to fibrin. Similar to fibrinogen, fibrin-integrin interactions were partially sensitive to eptifibatide, cRGD peptide and γC-dodecapeptide, indicating that their specificity is akin to fibrinogen but may not be identical. Fibrinogen γ′/γ′, lacking the established binding site for the integrin, was reactive with αIIbβ3, but the binding strength was somewhat smaller. The effect of replacing the γA with the γ′ chains in fibrinogen was qualitatively similar to competitive inhibition by the γC-dodecapeptide and resulted in complete cutoff of the larger force peak as well as in partial reduction of weaker interactions. That fibrinogen γ′/γ′ maintained its ability to bind αIIbβ3 suggests that the γ400-411 motif is not the only structure involved in the binding of immobilized fibrinogen to the integrin. Fibrin γ′/γ′ was as reactive with αIIbβ3 as fibrinogen γ′/γ′. Both of them interacted with αIIbβ3 in an RGD-sensitive manner. The data show that both surface-bound fibrinogen and monomeric fibrin are highly reactive with the integrin αIIbβ3. Fibrin is somewhat more reactive than fibrinogen in terms of binding probability and strength and is less sensitive to a specific inhibitor, abciximab, suggesting that αIIbβ3-fibrin interactions have distinct specificity. Susceptibility to competitive inhibitors, such as cRGD and γC-dodecapeptide, along with maintenance of integrin-binding activity of fibrinogen γ′/γ′ suggest that the αIIbβ3-binding sites in fibrin(ogen) are complex and include, but are not confined to, the γC-terminal 400-411 motif. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 732-736 ◽  
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Plasma Fibrinogen ◽  
Platelet Glycoprotein ◽  
Surface Binding ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.

Interaction of platelets with liposomes containing dodecapeptide sequence from fibrinogen

2004 ◽  
Vol 91 (06) ◽  
pp. 1158-1167 ◽  
Author(s):  
Chikaho Toma ◽  
Takako Nishiya
Keyword(s):  
Platelet Aggregation ◽  
Binding Site ◽  
Synthetic Peptide ◽  
Surface Density ◽  
The Other ◽  
Interaction Site ◽  
Ligand Affinity ◽  
Rgd Sequence ◽  
Activated Platelets ◽  
Other Hand

SummaryLiposomes with a covalently bound synthetic peptide containing the dodecapeptide sequence HHLGGAKQAGDV, the putative platelet interaction site at γ400-411 of fibrinogen (dodecapeptide-liposomes), were prepared. These liposomes enhanced platelet aggregation and specifically adhered to platelets activated on the collagen surface. Dodecapeptide-liposomes released encapsulated materials upon interacting with platelets activated on the collagen surface.The rate of content release was dependent on the peptide surface density, indicating that the interaction between the dodecapeptide-liposomes and platelets activated on the collagen surface induces clustering of the surfacecoupled ligands at the binding site on the receptor matrix to facilitate release of the internal contents through the liposome membranes. The level of lipid mixing between the dodecapeptide-liposomes and platelets activated on the collagen surface was relatively low, however it was increased in liposome preparations containing octa-arginine, the (R)8GDV sequence, while content release was maintained at the same level as that of the dodecapeptide-liposomes. The level of content release and lipid mixing for liposome preparations containing the RGD sequence as a ligand (RGD-liposomes) upon interacting with platelets activated on the collagen surface was extremely low. Both the level of the content release and lipid mixing, however, were enhanced in liposome preparations containing octa-arginine, the (R)8RGD sequence. Dodecapeptide-liposomes and RGD-liposomes were not internalized by activated platelets. On the other hand, liposomes containing (R)8PPQ, (R)8RGD, or (R)8GDV were internalized by activated platelets, and the extent of internalization was inversely related to ligand affinity to the target.

scholarly journals Characterization of the gamma chain platelet binding site on fibrinogen fragment D

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2643-2648 ◽  
Author(s):  
NE Kirschbaum ◽  
MW Mosesson ◽  
DL Amrani
Keyword(s):  
Platelet Aggregation ◽  
Binding Site ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Gamma Chain ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Chain Sequence ◽  
Carboxy Terminal

Abstract Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400–411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400–411 peptide in inhibiting ADP- induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.

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

scholarly journals Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 732-736
Author(s):  
RI Parker ◽  
HR Gralnick
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Von Willebrand Factor ◽  
Plasma Fibrinogen ◽  
Platelet Glycoprotein ◽  
Surface Binding ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.

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.

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