Novel Peptide Motifs Containing Asp-Glu-Gly Target P2Y12 and Thromboxane A2 Receptors to Inhibit Platelet Aggregation and Thrombus Formation

Yijie Yang ◽  
Bo Li
2010 ◽  
Vol 638 (1-3) ◽  
pp. 5-12 ◽  
Fernanda C.F. Brito ◽  
Arthur E. Kummerle ◽  
Claire Lugnier ◽  
Carlos A.M. Fraga ◽  
Eliezer J. Barreiro ◽  

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2762-2762
Yiwen Zhang ◽  
Jian Zhang ◽  
Rong Yan ◽  
Jie Zhang ◽  
Mengxing Chen ◽  

Abstract Objective: Receptor-interacting protein 3 (RIP3) is a member of RIP family with a Ser/Thr protein kinase domain in its amino-terminus which is essential for kinase activity and autophosphorylation. The roles of RIP3 in embryonic development and different disease pathologies, such as inflammation and infections, have been reported in recent years. However, the role of RIP3 in thrombosis and hemostasis remains unknown. Methods: Hematologic analysis was performed and tail bleeding time was monitored. Mouse platelets were isolated from anti-coagulated whole blood. Platelet aggregation and secretion were recorded at real time. Platelet P-selectin exposure and specific fibrinogen binding were detected by flow cytometry. TXA2 generation was measured with enzyme immunoassay (EIA) kit. Protein phosphorylations were detected by western blotting. Result: RIP3-/- mice had tail-bleeding times that were significantly prolonged compared with their wild type littermates. In an in vivo model of mesenteric arteriole thrombosis, mice lacking RIP3 exhibited delayed thrombus formation, fewer accumulated platelets, smaller thrombi, and prolonged occlusion times. RIP3 was expressed in both human and mouse platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 (TXA2) analogue, U46619. The defect in ADP secretion appears responsible for the impaired platelet aggregation, because addition of exogenous ADP rescued the reduced platelet aggregation. Although TXA2 generation and α-granule secretion were not impaired, integrin αIIbβ3 activation was attenuated in RIP3-/- platelets. Moreover, phosphorylation of Akt induced by U46619 or thrombin was markedly reduced in the absence of RIP3. Activation of Akt signaling restored the impaired aggregation of RIP3-/- platelets. ERK and p38 phosphorylation elicited by either U46619 or thrombin was attenuated in RIP3-/- platelets. In contrast, U46619- and thrombin-induced activation of PTEN, PDK1, or Src was not impaired in RIP3-/- platelets. Conclusion: Our data demonstrate a novel role for RIP3 in amplifying U46619- and thrombin-induced platelet activation by mediating Akt-dependent ADP secretion, and in supporting hemostasis and thrombus formation in vivo. RIP3 may represent a novel target to modulate PARs and TP signaling and a potential new target for antithrombotic strategy. Disclosures No relevant conflicts of interest to declare.

2000 ◽  
Vol 84 (S1) ◽  
pp. 99-102 ◽  
Kay J. Rutherfurd ◽  
H. S. Gill

Based on amino acid sequence similarities that exist between the fibrinogen γ-chain and κ-casein, and also functional similarities between milk and blood coagulation, considerable effort has been made to investigate the effects of milk proteins and peptides on platelet function and thrombosis. In particular, a number of peptides derived from the glycomacropeptide segment of κ-casein, have been shown to inhibit platelet aggregation and thrombosis. KRDS, a peptide from lactoferrin has also been shown to inhibit platelet aggregation but to a lesser extent than its fibrinogen analogue RGDS. Despite their functional and structural similarities they do not act in the same way on platelet function and are thought to affect thrombus formation differently. Further investigation is needed to determine if these milk-derived bioactive peptides are released naturally following ingestion and might therefore be useful as the basis for milk-based products with anti-thrombotic properties.

1993 ◽  
Vol 70 (04) ◽  
pp. 672-675 ◽  
H F Kotzé ◽  
S Lamprecht ◽  
P N Badenhorst ◽  
V van Wyk ◽  
J P Roodt ◽  

SummaryBay U3405 is a thromboxane A2 (TxA2)-receptor antagonist that inhibits the binding of TxA2 to its target cells. The aim of this study was to determine if Bay U3405 could be used to inhibit arterial thrombosis. A thrombogenic de vice, consisting of uncrimped Dacron vascular graft material (0.5 cm2) built into the wall of silicone rubber tubing with 4 mm inside diameter, was exposed to native flowing blood under arterial blood flow conditions (100-140 ml/min) by interposing the devices as extension segments into permanent femoral arteriovenous shunts implanted in baboons. Thrombus formation was quantified in vivo by measuring the deposition of 111In-labelled platelets onto the graft material with a scintillation camera. In six baboons, a bolus injection of Bay U3405, calculated to attain an initial plasma concentration of 300 ng/ml, reduced the maximum thrombus formation measured over a 2 h study period. Platelet deposition was reduced by 33 ± 14% (SD) at 2 h as compared to control studies done in the same baboons. The accumulation of additional platelets onto a thrombus that was allowed to form for 1 h, was reduced by 58 ± 28% at 2 h. Ex vivo platelet aggregation in response to ADP, activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were not affected by the treatment. Ex vivo platelet aggregation in response to collagen was markedly inhibited for 2 h after treatment. The results demonstrated that selective blocking of the TxA2-receptor on platelets reduced platelet-dependent thrombus formation and the accumulation of additional platelets in a freshly formed thrombus. This may provide a viable approach for preventing excessive thrombus formation in patients undergoing arterial reconstructive surgery.

Molecules ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2380 ◽  
Dongshuai Shen ◽  
Ning Ma ◽  
Yajun Yang ◽  
Xiwang Liu ◽  
Zhe Qin ◽  

Aspirin eugenol ester (AEE) is a novel compound that is formed from the esterification of aspirin (acetylsalicylic acid (ASA)) and eugenol. This study aimed to investigate the effects of AEE on blood stasis in rats and to characterize the underlying mechanisms using a plasma metabolomic study. The results indicate that AEE and ASA could modulate whole blood viscosity (WBV), plasma viscosity (PV), blood coagulation parameters, platelet count, platelet aggregation, lactate dehydrogenase (LDH), creatinine (CR) and the levels of thromboxane A2 (TXA2) and 6-keto prostaglandin F1α (6-keto-PGF1α). The metabolic profiles of the plasma samples from all groups were clearly separated in the score plots. Nineteen potential metabolites were selected and identified, and disordered levels of these metabolites could be regulated by AEE and ASA. Pathway analysis showed that the mechanism of action of AEE on blood stasis might be principally related to the metabolism of amino acid, fatty acid, energy and glycerophospholipid. The above results indicate that AEE protected the rats against blood stasis, and that this effect might have been caused by the anticoagulation activity of AEE and its abilities to maintain a balance between TXA2 and PGI2, reduce blood viscosity, inhibit platelet aggregation and normalize the plasma metabolic profile.

1973 ◽  
Vol 30 (02) ◽  
pp. 352-362
B. A Spilker ◽  
H van Balken

SummaryCharacteristics of thrombi produced upon electrical stimulation of mesentery and brain vessels were studied in five species. Parameters for measuring drug effects were also evaluated to determine which were sensitive to platelet aggregation inhibitors. The current required to cause vasoconstriction in 50% of the rat mesentery arteries stimulated was increased by vasodilators, but not by inhibitors of platelet aggregation. The threshold of current necessary to cause thrombus formation was increased by Imipramine in both acute and chronic experiments and by aspirin and papaverine in chronic experiments. Since these were the only agents tested which inhibit platelet aggregation there appears to be a relationship between this property and the threshold of current necessary to cause thrombus formation. This parameter may possibly be used to differentiate between platelet aggregation inhibitors and streptokinase-like or heparin-like agents. Changes in mean embolization time under present conditions were not related to activity in inhibiting platelet aggregation.

1981 ◽  
Gundu H R Rao ◽  
James G White

Previous work has shown that platelets irreversibly aggregated by ADP or thrombin (T) can be dissociated by various agents and that the refractory state of disaggregated cells can be reversed immediately by treatment with epinephrine (E). In the present study we have evaluated the influence of drugs which affect different steps in the process of prostaglandin (PG) synthesis on platelet DA-RA. Aspirin and indomethacin did not cause DA of platelets in the process of aggregation nor did they prevent reversal of the refractory state by E and subsequent RA of previously dissociated platelets. Imidazole, which inhibits conversion of endoperoxide to thromboxane A2, also failed to influence DA or restoration of sensitvity and RA of disaggregated platelets. On the other hand, chemicals which interfere with release of AA from the membrane of activated platelets, such as mepacrine, chlorpromazine and trifluoperazine, caused rapid DA. Products of PG synthesis, such as PGE1, PGD2 and PGI2, which usually inhibit platelet aggregation, also caused rapid DA. The refractory state of platelets dissociated from aggregates by most of these agents could be reversed by E treatment. However, trifluoperazine disaggregated platelets could be reaggregated only by the combination of E and AA. Agents which block the a-adrenergic receptors did not cause dissociation of aggregating platelets, but prevented correction of the refractory state of dissociated platelets by E. Thus interference with AA release, even after aggregation, can cause DA of clumped platelets, but blockade of peroxidase, cyclo-oxygenase and thromboxane synthetase do not cause reversal once it is in progress. A membrane linked mechanism associated with AA availability, but not metabolism, regulates DA and restoration of membrane sensitivity for RA.

Neurosurgery ◽  
1981 ◽  
Vol 9 (6) ◽  
pp. 679-685 ◽  
Shigeharu Suzuki ◽  
Eiji Sobata ◽  
Takashi Iwabuchi

Abstract The results of our previous experimental and clinical studies led us to the hypothesis that, in the pathogenesis of cerebral vasospasm, subarachnoid focal acidosis resulting from anaerobic changes of subarachnoid clots may be a factor upsetting the balanced synthesis of both thromboxane A2 and prostaglandin I2 from prostaglandin endoperoxides on the inner surface of cerebral arteries. Thus, there is a higher concentration of thromboxane A2, a prostanoid that causes arterial contraction and platelet aggregation. We tested the administration of trapidil, an antagonist and selective synthesis inhibitor of thromboxane A2, in a series of 20 cases for the prevention of cerebral vasospasm and cerebral ischemia after aneurysmal rupture. Vasospasm was demonstrated by angiography in 9 of these cases, but only 2 of the 9 showed mild signs of cerebral ischemia. Of the 20 patients, 15 were discharged from the hospital as cured and 3 had a neurological deficit at discharge. Our findings suggest the significance in symptomatic vasospasm of thrombus formation by platelet aggregation and the effectiveness of trapidil as a preventive.

2004 ◽  
Vol 91 (06) ◽  
pp. 1168-1176 ◽  
Qiumin Lu ◽  
Alexei Navdaev ◽  
Jeannine Clemetson ◽  
Kenneth Clemetson

SummaryMucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) Ib is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLCγ2. Mucetininduced phosphorylation of the Fcγ chain of platelet was greatly promoted by inhibition of αIIbβ3 by the peptidomimetic EMD 132338, suggesting that phosphatases downstream of αIIbβ3 activation are involved in dephosphorylation of Fcγ. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates αIIbβ3. Inhibition of αIIbβ3 strongly reduced the aggregation response to mucetin, indicating that activation of αIIbβ3 and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxane A2 are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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