KRDS--a tetrapeptide derived from lactotransferrin--inhibits binding of monoclonal antibody against glycoprotein IIb-IIIa on ADP-stimulated platelets and megakaryocytes

Abstract Short peptides isolated from fibrinogen and K-casein have been shown to inhibit platelet aggregation and fibrinogen binding to stimulated platelets. We studied the effects of synthetic peptides occurring in milk proteins (bovine K-casein, KNQDK, and human lactotransferrin, KRDS) and in fibrinogen (RGDS and L10) on subsequent binding of monoclonal antibodies (MoAb) against the glycoprotein (GP) IIb-IIIa complex (AP2 and P2) on adenosine diphosphate (ADP)-stimulated and unstimulated human platelets and megakaryocytes (MKs) by using an immunoperoxidase method to visualize antibody binding. Only KRDS (900 mumol/L) inhibited the binding of AP2 and P2 on ADP (5 mumol/L)- stimulated platelets, but not on unstimulated platelets. However, the binding of P2 was considerably more inhibited than that of AP2 as judged by immunoperoxidase intensity. Radiolabeled AP2 binding was inhibited by 30% with KRDS on ADP-stimulated platelets as compared with platelets incubated in the absence of ADP. KRDS did not inhibit the binding of MoAbs against GP IIIa (SZ 21), GP IIb (SZ 22), and GP Ib (SZ 2) on ADP-stimulated human platelets. Inhibition of P2 binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 micron ADP. A lower concentration of ADP (5 or 10 mumol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2 binding inhibition was observed in a larger (P less than .001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonist-induced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.

Download Full-text

KRDS--a tetrapeptide derived from lactotransferrin--inhibits binding of monoclonal antibody against glycoprotein IIb-IIIa on ADP-stimulated platelets and megakaryocytes

Blood ◽

10.1182/blood.v72.1.172.bloodjournal721172 ◽

1988 ◽

Vol 72 (1) ◽

pp. 172-178

Author(s):

S Raha ◽

C Dosquet ◽

JF Abgrall ◽

P Jolles ◽

AM Fiat ◽

...

Keyword(s):

Functional Ability ◽

Synthetic Peptides ◽

Milk Proteins ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Short Peptides ◽

Immunoperoxidase Method ◽

Inhibit Platelet Aggregation ◽

Fibrinogen Binding ◽

Binding Inhibition

Short peptides isolated from fibrinogen and K-casein have been shown to inhibit platelet aggregation and fibrinogen binding to stimulated platelets. We studied the effects of synthetic peptides occurring in milk proteins (bovine K-casein, KNQDK, and human lactotransferrin, KRDS) and in fibrinogen (RGDS and L10) on subsequent binding of monoclonal antibodies (MoAb) against the glycoprotein (GP) IIb-IIIa complex (AP2 and P2) on adenosine diphosphate (ADP)-stimulated and unstimulated human platelets and megakaryocytes (MKs) by using an immunoperoxidase method to visualize antibody binding. Only KRDS (900 mumol/L) inhibited the binding of AP2 and P2 on ADP (5 mumol/L)- stimulated platelets, but not on unstimulated platelets. However, the binding of P2 was considerably more inhibited than that of AP2 as judged by immunoperoxidase intensity. Radiolabeled AP2 binding was inhibited by 30% with KRDS on ADP-stimulated platelets as compared with platelets incubated in the absence of ADP. KRDS did not inhibit the binding of MoAbs against GP IIIa (SZ 21), GP IIb (SZ 22), and GP Ib (SZ 2) on ADP-stimulated human platelets. Inhibition of P2 binding by KRDS was also observed in a section of MKs isolated from human bone marrow and stimulated by 15 or 20 micron ADP. A lower concentration of ADP (5 or 10 mumol/L) failed to produce any inhibition of binding. This indicates that MKs may not be equally responsive to agonists as platelets. Moreover, P2 binding inhibition was observed in a larger (P less than .001) percentage of mature MKs (29%) as compared with younger, maturing MKs (11%). The observations suggested that a functional ability possessed by platelets, namely, agonist-induced exposure of the site of interaction of KRDS, may occur at a late stage of MK development.

Download Full-text

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽

10.1182/blood.v97.9.2648 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2648-2656 ◽

Cited By ~ 22

Author(s):

Juan A. Rosado ◽

Else M. Y. Meijer ◽

Karly Hamulyak ◽

Irena Novakova ◽

Johan W. M. Heemskerk ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Actin Polymerization ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Human Platelets ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Integrin Αiibβ3 ◽

Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

Download Full-text

Studies on the mechanism of expression of secreted fibrinogen on the surface of activated human platelets

Blood ◽

10.1182/blood.v73.5.1226.1226 ◽

1989 ◽

Vol 73 (5) ◽

pp. 1226-1234 ◽

Cited By ~ 39

Author(s):

C Legrand ◽

V Dubernard ◽

AT Nurden

Keyword(s):

Monoclonal Antibody ◽

Plasma Membrane ◽

Synthetic Peptides ◽

Divalent Cations ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Plasma Fibrinogen ◽

Fab Fragments ◽

Polymer Formation ◽

Activated Platelets

Abstract Affinity purified anti-fibrinogen (anti-Fg) Fab fragments were used to study the mechanism of expression of alpha-granule fibrinogen on activated platelets. Low amounts of the radiolabeled anti-Fg Fab bound to unstimulated or adenosine diphosphate (ADP)-stimulated cells. They readily bound to platelets stimulated with collagen, alpha-thrombin or gamma-thrombin in the presence of divalent cations. At 1 n mol/L alpha- thrombin or 25 nmol/L gamma-thrombin, platelet fibrinogen was expressed on the surface of the cells notwithstanding the presence of AP-2, a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, or the synthetic peptides Arg-Gly-Asp-Ser and gamma 400–411, all substances that prevented the binding of plasma fibrinogen to platelets. These results suggest that platelet fibrinogen may interact with its receptors during its translocation from the alpha-granules to the plasma membrane and, thus, not occupy the same sites as those available for plasma fibrinogen on the surface of the cell. Furthermore, we found that platelet fibrinogen was expressed on the thrombin-stimulated platelets of a Glanzmann's thrombasthenia variant that failed to bind plasma fibrinogen. Normal platelets stimulated with 5 nmol/L alpha- thrombin bound increased amounts of the anti-fg Fab, the additional expression being inhibited by the anti-GP IIb-IIIa monoclonal antibody or by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymer formation. This suggests that rebinding to externally located GP IIb-IIIa complexes becomes important once fibrin is formed.

Download Full-text

Ticlopidine Selectively Inhibits Human Platelet Responses to Adenosine Diphosphate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646487 ◽

1991 ◽

Vol 66 (06) ◽

pp. 694-699 ◽

Cited By ~ 49

Author(s):

Marco Cattaneo ◽

Benjaporn Akkawat ◽

Anna Lecchi ◽

Claudio Cimminiello ◽

Anna M Capitanio ◽

...

Keyword(s):

Platelet Aggregation ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Clot Retraction ◽

Fibrinogen Binding ◽

Low Concentrations ◽

Binding Inhibition ◽

High Concentrations ◽

Formalin Fixed

SummaryPlatelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrihogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPIIb/IIIa monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their K d were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.

Download Full-text

Participation of ADP in the binding of fibrinogen to thrombin- stimulated platelets

Blood ◽

10.1182/blood.v56.3.553.553 ◽

1980 ◽

Vol 56 (3) ◽

pp. 553-555 ◽

Cited By ~ 46

Author(s):

EF Plow ◽

GA Marguerie

Keyword(s):

Creatine Phosphokinase ◽

Adenosine Diphosphate ◽

Creatine Phosphate ◽

Human Platelets ◽

Serotonin Release ◽

Fibrinogen Binding ◽

Platelet Secretion

Abstract Thrombin and adenosine diphosphate (ADP) supported the binding of 125I- fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.

Download Full-text

Correlation between fibrinogen binding to human platelets and platelet aggregability

Blood ◽

10.1182/blood.v55.5.841.bloodjournal555841 ◽

1980 ◽

Vol 55 (5) ◽

pp. 841-847 ◽

Cited By ~ 3

Author(s):

EI Peerschke ◽

MB Zucker ◽

RA Grant ◽

JJ Egan ◽

MM Johnson

Keyword(s):

Adenosine Diphosphate ◽

Human Platelets ◽

Fibrinogen Concentration ◽

Platelet Aggregability ◽

Single Receptor ◽

Fibrinogen Binding ◽

Fibrinogen Molecule ◽

Bernard Soulier Syndrome

Fibrinogen is essential for aggregating platelets with adenosine diphosphate (ADP) and was recently shown to bind to platelets stimulated with ADP. The present work confirms the specific and saturable nature of the platelet-fibrinogen interaction. Binding of 125iodine-labeled fibrinogen to human gel-filtered platelts was maximal at 1 min, and the receptors were saturated when the fibrinogen concentration in the suspending medium approached 0.8 mg/ml. Assuming that one fibrinogen molecule interacts with a single receptor, experiments with 9 normal donors revealed the presence of 12,896 +/- 2456 receptors per platelet. Much of the bound material dissociated from platelets after incubation with apyrase or EDTA. Binding was markedly inhibited at pH 6.5, in the presence of EDTA, and with platelets from 3 thrombasthenic patients but not with those from a patient with the Bernard-Soulier syndrome. Fibrinogen binding was also virtually absent with platelets that had been incubated with EDTA for 8 min at 37 degrees C and pH 7.8. These platelets could not aggregate when mixed with ADP and adequate CaCl2 and fibrinogen, although they could still change their shape. Thus, ADP-induced binding of fibrinogen correlates with platelet aggregability.

Download Full-text

Immunocytochemical localization of fibrinogen on washed human platelets. Lack of requirement for fibrinogen during adenosine diphosphate-induced responses and enhanced fibrinogen binding in a medium with low calcium levels

Blood ◽

10.1182/blood.v71.4.850.850 ◽

1988 ◽

Vol 71 (4) ◽

pp. 850-860 ◽

Cited By ~ 16

Author(s):

H Suzuki ◽

RL Kinlough-Rathbone ◽

MA Packham ◽

K Tanoue ◽

H Yamazaki ◽

...

Keyword(s):

Electron Microscopy ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Induced Responses ◽

Immunocytochemical Localization ◽

Platelet Surface ◽

Gold Particles ◽

Fibrinogen Binding ◽

Large Numbers ◽

Lowicryl K4m

Abstract The association of fibrinogen with washed human platelets was examined by immunocytochemistry during aggregation induced by adenosine diphosphate (ADP) and during deaggregation. The platelets were suspended either in a medium containing 2 mmol/L Ca2+ or in a medium containing no added Ca2+ (20 mumol/L Ca2+). Platelets were fixed at several times during aggregation and deaggregation, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. To determine whether the method detected fibrinogen associated with the platelets, the platelets were pretreated with chymotrypsin (10 U/mL) and aggregated by fibrinogen; gold particles were apparent not only in the alpha granules but on the platelet surface and between adherent platelets as well. In the medium with 2 mmol/L Ca2+, ADP caused extensive aggregation of normal platelets in the presence of fibrinogen (0.4 mg/mL), and gold particles were evident between the adherent platelets and on the platelet surface; when the platelets deaggregated, gold was no longer present on the surface. In a medium without added Ca2+, ADP caused extensive aggregation in the presence of fibrinogen, and large numbers of gold particles were on the platelet surface and even more between adherent platelets. In this medium, the platelets did not deaggregate, and by five minutes, the granules appeared to be swollen or fused. In the absence of external fibrinogen, ADP caused the formation of small aggregates, and fibrinogen was not detected between adherent platelets. Thus, the association of fibrinogen with the platelet surface enhances platelet aggregation but is not essential for the ADP-induced formation of small aggregates. The association of fibrinogen with platelets is greater under conditions in which platelets release their granule contents and do not deaggregate because both endogenous and exogenous fibrinogen take part in aggregation.

Download Full-text

Species differences in small molecule binding to αIIbβ3 are the result of sequence differences in 2 loops of the αIIb β propeller

Blood ◽

10.1182/blood-2008-09-177337 ◽

2009 ◽

Vol 113 (4) ◽

pp. 902-910 ◽

Cited By ~ 10

Author(s):

Ramesh B. Basani ◽

Hua Zhu ◽

Michael A. Thornton ◽

Cinque S. Soto ◽

William F. DeGrado ◽

...

Keyword(s):

Point Mutations ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Human Platelets ◽

Rgd Motif ◽

Fibrinogen Binding ◽

Evolutionary Response ◽

Arginine Glycine Aspartic Acid ◽

Von Willebrand ◽

Willebrand Factor

Abstract Compared with human platelets, rodent platelets are less responsive to peptides and peptidomimetics containing an arginine-glycine-aspartic acid (RGD) motif. Using chimeric human-rat αIIbβ3 molecules, we found that this difference in Arg-Gly-Asp-Ser (RGDS) sensitivity was the result of amino acid substitutions at residues 157, 159, and 162 in the W3:4-1 loop and an Asp-His replacement at residue 232 in the W4:4-1 loop of the αIIb β propeller. Introducing the entire rat W3:4-1 and W4:4-1 loops into human αIIbβ3 also decreased the inhibitory effect of the disintegrins, echistatin and eristostatin, and the αIIbβ3 antagonists, tirofiban and eptifibatide, on fibrinogen binding, whereas the specific point mutations did not. This suggests that RGDS interacts with αIIb in a different manner than with these small molecules. None of these species-based substitutions affected the ability of αIIbβ3 to interact with RGD-containing macromolecules. Thus, human von Willebrand factor contains an RGD motif and binds equally well to adenosine diphosphate-stimulated human and rodent platelets, implying that other motifs are responsible for maintaining ligand binding affinity. Many venoms contain RGD-based toxins. Our data suggest that these species amino acids differences in the αIIb β-propeller represent an evolutionary response by rodents to maintain hemostasis while concurrently protecting against RGD-containing toxins.

Download Full-text

Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates

Blood ◽

10.1182/blood.v75.5.1081.bloodjournal7551081 ◽

1990 ◽

Vol 75 (5) ◽

pp. 1081-1086 ◽

Cited By ~ 1

Author(s):

M Cattaneo ◽

MT Canciani ◽

A Lecchi ◽

RL Kinlough-Rathbone ◽

MA Packham ◽

...

Keyword(s):

Binding Sites ◽

Creatine Phosphokinase ◽

Adenosine Diphosphate ◽

Creatine Phosphate ◽

Human Platelets ◽

Fibrinogen Binding ◽

Normal Human ◽

Platelet Aggregates ◽

Induced Aggregation ◽

Selective Impairment

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta- thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.

Download Full-text

Platelet binding to polymerizing fibrin is avidity driven and requires activated αIIbβ3 but not fibrin cross-linking

Blood Advances ◽

10.1182/bloodadvances.2021005142 ◽

2021 ◽

Vol 5 (20) ◽

pp. 3986-4002

Author(s):

Lorena Buitrago ◽

Samuel Lefkowitz ◽

Ohad Bentur ◽

Julio Padovan ◽

Barry Coller

Keyword(s):

Platelet Aggregation ◽

Light Transmission ◽

Potent Inhibitor ◽

Thrombin Receptor ◽

Cross Linking ◽

Inhibit Platelet Aggregation ◽

Clot Retraction ◽

Fibrinogen Binding ◽

Platelet Binding ◽

Binding Inhibition

Abstract The molecular basis of platelet-fibrin interactions remains poorly understood despite the predominance of fibrin in thrombi. We have studied the interaction of platelets with polymerizing fibrin by adding thrombin to washed platelets in the presence of the peptide RGDW, which inhibits the initial platelet aggregation mediated by fibrinogen binding to αIIbβ3 but leaves intact a delayed increase in light transmission (delayed wave; DW) as platelets interact with the polymerizing fibrin. The DW was absent in platelets from a patient with Glanzmann thrombasthenia, indicating a requirement for αIIbβ3. The DW required αIIbb3 activation and it was inhibited by the αIIbβ3 antagonists eptifibatide and the monoclonal antibody (mAb) 7E3, but only at much higher concentrations than needed to inhibit platelet aggregation initiated by a thrombin receptor activating peptide (T6). Surface plasmon resonance and scanning electron microscopy studies both supported fibrin having greater avidity for αIIbβ3 than fibrinogen rather than greater affinity, consistent with fibrin’s multivalency. mAb 10E5, a potent inhibitor of T6-induced platelet aggregation, did not inhibit the DW, suggesting that fibrin differs from fibrinogen in its mechanism of binding. Inhibition of factor XIII–mediated fibrin cross-linking by >95% reduced the DW by only 32%. Clot retraction showed a pattern of inhibition similar to that of the DW. We conclude that activated αIIbβ3 is the primary mediator of platelet-fibrin interactions leading to clot retraction, and that the interaction is avidity driven, does not require fibrin cross-linking, and is mediated by a mechanism that differs subtly from that of the interaction of αIIbβ3 with fibrinogen.

Download Full-text