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

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.

Ticlopidine Selectively Inhibits Human Platelet Responses to Adenosine Diphosphate

1991 ◽  
Vol 66 (06) ◽  
pp. 694-699 ◽  
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.

Thiosulfinates Inhibit Platelet Aggregation and Microparticle Shedding at a Calpain-dependent Step

2001 ◽  
Vol 86 (11) ◽  
pp. 1284-1291 ◽  
Author(s):  
Brigitte Brohard-Bohn ◽  
Sabine Pain ◽  
Christilla Bachelot-Loza ◽  
Jacques Auger ◽  
Francine Rendu
Keyword(s):  
Platelet Aggregation ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Inhibit Platelet Aggregation ◽  
Clot Retraction ◽  
Allium Species ◽  
Calpain Activation ◽  
Fibrinogen Binding ◽  
Ic50 Values ◽  
Dose Dependent

SummaryThiosulfinates (TSs) are sulfur compounds generated through the processing of different Allium species with antiplatelet property. To further define this platelet inhibitory effect we studied diallyl-TS (Al2TS), dipropyl-TS (Pr2TS), and dimethyl-TS (Me2TS) on platelet responses. The three TSs inhibited dose-dependent platelet aggregation, with IC50 values of 15 ± 2, 19 ± 2, and 9 ± 1 μM for Al2TS, Pr2TS and Me2TS, respectively. TSs had no effect on the expression of a platelet procoagulant surface, measured by flow cytometry as the binding of annexin V-FITC. They inhibited the microparticle shedding and clot retraction. Since the microparticle shedding is a calpain-activation dependent step, we assessed calpain activation by analysis of autoproteolysis in shorter active forms and by talin proteolysis in the presence of TSs. Calpain activation was inhibited by TSs independently of fibrinogen binding. Thus, TSs represent a new category of platelet inhibitors, acting on cal-pain-induced events.

Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus binds to platelets and inhibits platelet aggregation

2004 ◽  
Vol 91 (04) ◽  
pp. 779-789 ◽  
Author(s):  
Oonagh Shannon ◽  
Jan-Ingmar Flock
Keyword(s):  
Platelet Aggregation ◽  
Potent Inhibitor ◽  
Specific Binding ◽  
Binding Protein ◽  
Dependent Manner ◽  
Platelet Surface ◽  
Putative Receptor ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dose Dependent

Summary S. aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have shown here that Efb is a potent inhibitor of platelet aggregation. Efb can bind specifically to platelets by two mechanisms; 1) to fibrinogen naturally bound to the surface of activated platelets and 2) also directly to a surface localized component on the platelets. This latter binding of Efb is independent of fibrinogen. The specific binding of Efb to the putative receptor on the platelet surface results in a stimulated, non-functional binding of fibrinogen in a dose dependent manner, distinct from natural binding of fibrinogen to platelets. The natural binding of fibrinogen to GPIIb/IIIa on activated platelets could be blocked by a monoclonal antibody against this integrin, whereas the Efb-mediated fibrinogen binding could not be blocked. The enhanced Efb-dependent fibrinogen binding to platelets is of a nature that does not promote aggregation of the platelets; instead it inhibits aggregation. The anti-thrombotic action of Efb may explain the effect of Efb on wound healing, which is delayed in the presence of Efb.

scholarly journals Antiplatelet and Antithrombotic Effects of Epimedium koreanum Nakai

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Muhammad Irfan ◽  
Tae-Hyung Kwon ◽  
Dong-Ha Lee ◽  
Seung-Bok Hong ◽  
Jae-Wook Oh ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Light Transmission ◽  
Thrombus Formation ◽  
Calcium Mobilization ◽  
Potential Candidate ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Atp Secretion ◽  
Antithrombotic Effects ◽  
Epimedium Koreanum Nakai

Background and Objective. Epimedium koreanum Nakai is a medicinal plant known for its health beneficial effects on impotence, arrhythmia, oxidation, aging, osteoporosis, and cardiovascular diseases. However, there is no report available that shows its effects on platelet functions. Here, we elucidated antiplatelet and antithrombotic effects of ethyl acetate fraction of E. koreanum. Methodology. We analyzed the antiplatelet properties using standard in vitro and in vivo techniques, such as light transmission aggregometry, scanning electron microscopy, intracellular calcium mobilization measurement, dense granule secretion, and flow cytometry to assess integrin αIIbβ3 activation, clot retraction, and Western blot, on washed platelets. The antithrombotic effects of E. koreanum were assessed by arteriovenous- (AV-) shunt model in rats, and its effects on hemostasis were analyzed by tail bleeding assay in mice. Key Results. E. koreanum inhibited platelet aggregation in agonist-stimulated human and rat washed platelets, and it also reduced calcium mobilization, ATP secretion, and TXB2 formation. Fibrinogen binding, fibronectin adhesion, and clot retraction by attenuated integrin αIIbβ3-mediated inside-out and outside-in signaling were also decreased. Reduced phosphorylation of extracellular signal-regulated kinases (ERK), Akt, PLCγ2, and Src was observed. Moreover, the fraction inhibited thrombosis. HPLC results revealed that the fraction predominantly contained icariin. Conclusion and Implications. E. koreanum inhibited platelet aggregation and thrombus formation by attenuating calcium mobilization, ATP secretion, TXB2 formation, and integrin αIIbβ3 activation. Therefore, it may be considered as a potential candidate to treat and prevent platelet-related cardiovascular disorders.

Diatrizoate-containing contrast media inhibit platelet aggregation by disrupting fibrinogen binding

1991 ◽  
Vol 2 (3) ◽  
pp. 389-396 ◽  
Author(s):  
David C. Sane ◽  
Tammy L. Moser ◽  
Thomas M. Bashore ◽  
Carrie L. Wagner ◽  
Charles S. Greenberg
Keyword(s):  
Platelet Aggregation ◽  
Contrast Media ◽  
Inhibit Platelet Aggregation ◽  
Fibrinogen Binding

Abstract 3010: Thrombin Receptor Antagonist (TRA;SCH530348) is a Selective, Potent Inhibitor of PAR1 Activity with Predictable Pharmacokinetics

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Lisa K Jennings ◽  
Angela Earhart ◽  
Richard C Becker ◽  
Larissa Reyderman ◽  
Enrico Veltri ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Receptor Antagonist ◽  
Light Transmission ◽  
Controlled Study ◽  
Thrombin Receptor ◽  
Loading Dose ◽  
Post Dose ◽  
Inhibition Of Platelet Aggregation ◽  
Thrombin Receptor Antagonist ◽  
Sustained Inhibition

Background: TRA-PCI was a multicenter, randomized, double-blind, placebo-controlled study demonstrating the safety of SCH 530348, a potent thrombin receptor antagonist (TRA), in patients undergoing non-urgent percutaneous coronary intervention (PCI). A secondary endpoint in a sub-study within the primary evaluable patient cohort was inhibition of platelet aggregation (IPA) induced by appropriate agonists relative to baseline. We hypothesized that TRA therapy would selectively inhibit platelet aggregation induced by the thrombin receptor agonist peptide (TRAP) and that sustained inhibition would be observed. Pharmacokinetic (PK) studies were also carried out. Methods: Platelet aggregation responses to TRAP, adenosine diphosphate (ADP), collagen and arachidonic acid (AA) were measured using light transmission aggregometry (LTA) at baseline, 30, 60, 90, 120 min following a loading dose (10, 20 or 40 mg vs placebo) and after a maintenance dose (0.5, 1.0 or 2.5 mg/day) at 30 and 60 days. PK was assessed at 30 and 60 min and 2 hr after loading dose administration. Results: TRA was active in inhibiting 15 μM TRAP-induced platelet aggregation with onset of inhibition directly related to dose. Loading doses of 10, 20 or 40 mg achieved >90% inhibition of platelet aggregation (IPA) 60 –120 min post dose with the 40 mg dose achieving >90% inhibition between 60 and 90 min. Patients receiving maintenance doses of 0.5, 1.0 or 2.5 mg exhibited >90% IPA at 30 and 60 days. Placebo treated patients had on average ≥10% IPA to TRAP. TRA had no significant effects on ADP, collagen or AA -induced platelet aggregation compared with placebo controls. TRA PK was characterized by fast distribution and slow elimination (t1/2 = 165–311 hr) with clear evidence of hysteresis. Conclusion: Among PCI patients treated with TRA, there was a specific, dose-related, sustained inhibition of TRAP-induced platelet aggregation, without an impact on other aggregation related pathways. These data suggest that TRA is a potent, selective inhibitor of PAR-1 activity induced by thrombin activation of platelets, and, in view of this, is a promising therapeutic utility for treatment of thrombosis.

Platelet Aggregation and Fibrinogen Binding in Human, Rhesus Monkey, Guinea-Pig, Hamster and Rat Blood: Activation by ADP and a Thrombin Receptor Peptide and Inhibition by Glycoprotein IIb/IIIa Antagonists

1993 ◽  
Vol 70 (03) ◽  
pp. 531-539 ◽  
Author(s):  
Nigel S Cook ◽  
Hans-Günter Zerwes ◽  
Carlo Tapparelli ◽  
Max Powling ◽  
Jagjit Singh ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Guinea Pig ◽  
Rhesus Monkey ◽  
Whole Blood ◽  
Flow Cytometric Analysis ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Important Species ◽  
Rat Blood ◽  
Fibrinogen Binding

SummaryPlatelet aggregation and fibrinogen binding in whole blood, induced either by ADP or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin receptor (TRP, thrombin receptor activating peptide), have been studied with blood from different species. Aggregation was assessed by counting the number of single platelets in blood before und after addition of the agonist with an automated cell counter. Both ADP (0.02-0.5 μM) and TRP (1-10 μM) were found to be potent agonists of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal aggregatory response within 2 min of agonist addition. In contrast, hamster and rat platelets were much less responsive to both ADP and TRP in terms of the whole blood aggregation.Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857, inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50’s of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50’s (as determined by flow cytometric analysis of platelet fibrinogen binding) were 4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around 100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.These results highlight important species differences in the response of platelets to activation by two different agonists and also in their inhibition by GP-IIb/IIIa antagonists. In particular, platelets from the rat and hamster were insensitive to agonists and antagonists, whereas guinea-pig and rhesus monkey platelets responded with an affinity similar to human platelets. Since these studies were performed in whole blood, the results should be representative of those expected in animal experiments. These recently developed methods for studying platelet responses in small aliquots of whole blood are simple to perform and provide important information concerning the optimal choice of species for subsequent in vivo studies with these compounds.

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.

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

Blood ◽  
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

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.

Caffeic Acid Phenyl Ester, a Component of the Chinese Herb Propolis, Inhibits Platelet Aggregation Via Competition with Fibrinogen for Binding to aIIbb3

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5367-5367
Author(s):  
Ya Ping Wu ◽  
Albert Huisman ◽  
Cees Weeterings ◽  
Philip G. de Groot ◽  
Yun Xiang Zhang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Caffeic Acid ◽  
Chinese Herb ◽  
Affinity Constant ◽  
Inhibit Platelet Aggregation ◽  
Activation Markers ◽  
Fibrinogen Binding ◽  
History Of ◽  
Set Up ◽  
Plant Sources

Abstract Background: Propolis is a resinous substance collected by honeybees from various plant sources. Honeybees collect resin from cracks in the bark of trees and leaf buds, masticate it and the partially digested material is mixed with beeswax. The precise composition of propolis varies with the source. There is a long history of the use of propolis, at least to 300BC and its use continues till today. Propolis has a reputation to have a large number of healthy properties among others anti-thrombotic properties. Over 180 different compounds have been identified in propolis, among others caffeic acid phenyl esters (CAPE). We have investigated the effects of CAPE on platelet aggregation. Results: Platelet aggregation was induced with ADP, collagen or the thrombin analogue TRAP. Different flavoid components present in propolis (CAPE, galangin, acacetin, pinostrobin) were able to inhibit platelet aggregation, CAPE was the most potent one, especially for the collagen induced platelet aggregation. We examined the effect of 100 mM CAPE on the expression of a number of platelet membrane activation markers after addition of collagen and we observed that CAPE inhibited fibrinogen binding and the binding of LIBS antibody against aIIbb3 for 93% and 82%, respectively, while the expression of P-selectin was only inhibited for 37%. To explain this discrepancy, we studied the binding of CAPE to purified aIIbb3 with a Biacore. CAPE binds to aIIbb3 with an affinity constant of about 400 μM. In an ELISA set-up CAPE was able to inhibit the binding of fibrinogen to purified aIIbb3 (figure). Figure Figure Conclusion: Propolis contains components such as CAPE that inhibit platelet aggregation via competition with fibrinogen for the binding to aIIbb3.

Sign in / Sign up

Export Citation Format

Share Document