Anthocyanins Inhibit Platelet Activation and Attenuate Thrombus Growth In Both Human and Murine Thrombosis Models

Abstract Abstract 3197 Background: Thrombosis and cardiovascular diseases (CVDs) result from blood vessel occlusion by inappropriately activated platelets. They are the leading causes of morbidity and mortality worldwide. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies have demonstrated that anthocyanins are antioxidative and prevent inflammation (J Biol Chem. 2005; 280:36792-01; Arterioscler Thromb Vasc Biol. 2007; 27:519-24), which may indirectly affect platelet function. It has also been reported that anthocyanins affect platelet activities in whole blood and platelet rich plasma (PRP). However, the direct effects of anthocyanins on platelet function and thrombus formation have not been studied. Methods: Here we investigated the effects of anthocyanins on thrombosis using purified platelets as well as several thrombosis models in vitro and in vivo. Cyaniding-3-gulucoside (Cy-3-g) and delphinidin-3-glucoside (Dp-3-g), the two predominantly bioactive compounds of anthocyanin preparations, were prepared from Polyphenol AS Company in Norway. Purified gel-filtered platelets and PRP from healthy human volunteers and C57BL/6J mice were incubated at 37°C for 10 minutes with different concentrations (0.5μM, 5μM and 50μM) of Cy-3-g, Dp-3-g or PBS buffer as a control. Platelet aggregation was assessed by aggregometry using 5μM ADP, 10μg/ml collagen, or 100μM thrombin receptor activating peptide (TRAP; AYPGKF) as agonists. Platelet adhesion and aggregation were assessed in response to an immobilized collagen matrix in an ex vivo perfusion chamber at both high (1800 s-1) and low (600 s-1) shear rates. The expression of activated GPIIbIIIa was determined via PAC-1 monoclonal antibody in flow cytometry. Lastly, the effects of anthocyanins on thrombus formation in C57BL/6J mice were assessed using a FeCl3-induced intravital microscopy thrombosis model. Results: Both Cy-3-g and Dp-3-g significantly inhibited platelet aggregation induced by collagen and TRAP in gel-filtered platelets, and inhibited aggregation induced by ADP, TRAP and collagen in human and mouse PRP. These inhibitory functions were observed at Cy-3-g and Dp-3-g doses as low as 0.5μM. Cy-3-g and Dp-3-g also reduced the surface expression of activated GPIIbIIIa on resting human platelets in a dose-dependent manner. These compounds also markedly reduced platelet adhesion and aggregation in perfusion chamber assays at both low and high shear rates. Using intravital microscopy, we further demonstrated that Cy-3-g and Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for thrombus formation and vessel occlusion. Conclusions: our data clearly demonstrated for the first time that anthocyanin compounds directly inhibited platelet activation, adhesion and aggregation, as well as attenuated thrombus growth at both arterial and veinous shear stresses. These effects on platelets likely contribute to the protective effects of anthocyanins against thrombosis and CVDs. Disclosures: No relevant conflicts of interest to declare.

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Genetic Disruption of □pdl and □pdlr Phospholipases in Mice Leads to Impaired Platelet Adhesion and Aggregation

Blood ◽

10.1182/blood.v112.11.412.412 ◽

2008 ◽

Vol 112 (11) ◽

pp. 412-412

Author(s):

DingYan Wang ◽

Adili Reheman ◽

Phil Connelly ◽

Youdong Wang ◽

Graham Maguire ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Lipid Raft ◽

Platelet Adhesion ◽

Membrane Lipid ◽

Phospholipase A1 ◽

Perfusion Chamber ◽

Platelet Adhesion And Aggregation ◽

Chamber Model

Abstract Platelet adhesion and aggregation are critical events in thrombosis. Bioactive phospholipid LPA (lysophosphatidic acid) has been identified as an important agonist for platelet aggregation. Plasma LPA can be generated from phospholipid substrates by phospholipase A1 (PLA1) and phospholipase D (PLD). We previously identified two novel PLA1 enzymes, designated lpdl (lpd lipase) and lpdlr (lpdl related lipase). Together with phosphatidylserine phospholipase A1 (PS-PLA1), these three phospholipases form a unique PLA1 lipase subfamily. Phospholipids are important structural components of cellular membranes. Recent studies demonstrate that lipid raft microdomains on the platelet membrane contribute to platelet activation. The gathering of membrane lipid rafts is necessary for ADP-mediated platelet activation. Disruption of lipid raft results in a reduction of ADP-induced platelet aggregation. In addition, it is reported that alteration of membrane lipid composition also affects the function of platelet β3 integrin (GPIIbIIIa). Thus, phospholipases may play an important role in platelet function. However, the roles of lpdl and lpdlr in platelet activation and thrombosis are unknown. To study the function of lpdl and lpdlr, we have recently knocked out both lpdl and lpdlr genes in mice by deleting their exons I and II, including their translation start codon ATG. RT-PCR confirmed that neither lpdl nor lpdlr gene is expressed in homozygous lpdl−/− or lpdlr−/− mice but expressed in wild-type (WT) tissues, indicating successful knockout of these phospholipases. Next, optical platelet aggregometry was used to assess platelet aggregation in platelet rich plasma (PRP). Platelet aggregation was induced with 5, 10 and 20 μM ADP in pooled PRP (3×108 platelets/mL) from age- and sex-matched WT, heterozygous and homozygous mice. Our results demonstrated that both the lpdl−/− and lpdlr−/− mice have decreased platelet aggregation after ADP stimulation. We further studied thrombus formation in lpdl−/− mice using an ex vivo perfusion chamber model on collagen type I-coated rectangular glass microcapillary tubes. Our preliminary data showed that both platelet adhesion and aggregation were impaired in the lpdl−/− mice. We are repeating these experiments and are examining lpdlr−/− mice using the same perfusion chamber model. In vivo thrombosis models with intravital microscopy and the mechanisms of how lpdl and lpdlr enzymes regulate platelet activation will also be investigated. These studies could potentially identify a novel pathway for regulation of platelet function, which may lead to development of new diagnostic and/or therapeutic methods for atherothrombosis. (Drs. D. Wang and A. Reheman contribute equally to this work)

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Novel 12-LOX Inhibitor ML355 Attenuates Platelet Reactivity and Impairs Thrombus Growth, Stability and Vessel Occlusion In Vivo

Blood ◽

10.1182/blood.v126.23.3442.3442 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3442-3442 ◽

Cited By ~ 1

Author(s):

Reheman Adili ◽

Theodore R Holman ◽

Michael Holinstat

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Adhesion ◽

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Vessel Occlusion ◽

Growth Stability

Abstract Background: Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. While current anti-platelet treatments limit platelet function, they often result in an increased risk of bleeding. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated by our lab and others to regulate PAR4 and GPVI-mediated platelet reactivity suggesting a role of 12-LOX in regulation of vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Aims: To determine how 12-LOX regulates thrombus formation in vivo and whether platelet 12-LOX is an effective target for anti-platelet therapeutics, wild-type (WT) or 12-LOX deficient (12-LOX-/-) mice were treated with or without the 12-LOX inhibitor, ML355, and were assessed for inhibitory effects on platelet activation in vitro, ex-vivo and in vivo. Methods: The effect of the novel 12-LOX inhibitor ML355 on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber. In vivo thrombus formation and vessel occlusion in small and large vessels were studied in 12-LOX-/-, WT mice and mice treated with ML355 using intravital microscopy using the FeCl3 injury models. Results: Using in vitro platelet aggregation assays, ML355 dose dependently inhibited thrombin, PAR1-AP, and PAR4-AP-induced aggregation in washed human platelets. Interestingly, the negative regulatory effects of ML355 inhibition of 12-LOX can be overcome by high concentration of thrombin. Additionally, ML355 was able to attenuate ADP-induced platelet aggregation both in platelet-rich-plasma and whole blood. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX-/- mice was impaired in FeCl3-induced mesenteric or carotid artery thrombosis models. Thrombi in 12-LOX-/- mice were unstable and frequently form emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The highly selective 12-LOX inhibitor ML355 inhibits platelets aggregation induced by various platelet agonists and ML355 inhibition of platelet function is not agonist specific. Platelet function at high shear in ex vivo conditions in both mice and human was attenuated in the presence of ML355. Thrombus growth, stability, and vessel occlusion was impaired in mice deficient for 12-LOX. Finally, the highly selective 12-LOX inhibitor ML355 attenuates thrombus formation and prevents vessel occlusion in vivo. Our data strongly indicates 12- LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics. Disclosures No relevant conflicts of interest to declare.

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Apolipoprotein AIV (ApoA-IV) Is a Novel Ligand of Platelet β3 Integrin That Negatively Regulates Platelet Adhesion, Aggregation, and Thrombosis.

Blood ◽

10.1182/blood.v114.22.156.156 ◽

2009 ◽

Vol 114 (22) ◽

pp. 156-156

Author(s):

Christopher M. Spring ◽

Wuxun Jin ◽

Hong Yang ◽

Adili Reheman ◽

Guangheng Zhu ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Protein S ◽

Vessel Occlusion ◽

Platelet Adhesion And Aggregation ◽

Β3 Integrin

Abstract Abstract 156 Platelet adhesion and aggregation at sites of vascular injury are key events required for haemostasis and thrombosis. It has been documented that von Willebrand factor (VWF) and fibrinogen (Fg) are required for platelet adhesion and aggregation. However, we previously showed that occlusive thrombi still form in mice deficient for both Fg and VWF (Fg/VWF−/−) via a β3 integrin-dependent pathway. Here, we have investigated novel, non-classical ligands of β3 integrin that may regulate platelet adhesion and aggregation. To identify potential ligand(s) of β3 integrin, latex beads were coated with purified human platelet β3 integrin and incubated with human plasma. Protein(s) specifically associated with β3 integrin were electrophoresed and apolipoprotein AIV (ApoA-IV) was identified by mass spectrometry. We found that ApoA-IV binds to the surface of stimulated platelets, but not to quiescent platelets or β3−/− platelets, and ApoA-IV/platelet association was blocked by the addition of a specific anti-β3 integrin monoclonal antibody. It appears that ApoA-IV binds to, but is not internalized by platelet β3 integrins. ApoA-IV-deficient (ApoA-IV−/−) mice exhibited enhanced platelet aggregation induced by ADP, Collagen, and TRAP in plasma (but not PIPES buffer) compared to wild type (WT) littermates. This enhancement was diminished when ApoA-IV−/− plasma was replaced by WT plasma, indicating that the reduction was due to plasma ApoA-IV and not an unrelated platelet effect. When platelets were incubated with FITC-Fg, ApoA-IV was able to reduce platelet/Fg association, indicating that ApoA-IV may act to displace pro-thrombotic β3 integrin ligand(s). In support of this, ApoA-IV reduced the number of adherent platelets on immobilized Fg in perfusion chamber assays and enhanced thrombus formation was observed when ApoA-IV−/− mouse blood was perfused over collagen. We found that addition of recombinant ApoA-IV inhibited platelet aggregation and thrombus formation in vitro, while the control apolipoprotein ApoA-I did not. Using intravital microscopy, we further demonstrated that early platelet deposition was increased, and the time for thrombus formation and vessel occlusion were shorter in ApoA-IV−/− mice, which can be corrected by recombinant ApoA-IV transfusion. Furthermore, recombinant ApoA-IV inhibited WT platelet aggregation, thrombus formation and enhanced thrombus dissolution both in vitro and in vivo. Our data demonstrate for the first time that ApoA-IV is a novel ligand of platelet β3 integrin that negatively regulates thrombosis. These new data are consistent with the reported association between ApoA-IV and reduced cardiovascular diseases, and establish the first link between ApoA-IV and thrombosis. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of Platelet Adhesion to Fibrin(ogen) in Flowing Whole Blood by Arg-Gly-Asp and Fibrinogen γ-Chain Carboxy Terminal Peptides

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646346 ◽

1992 ◽

Vol 68 (06) ◽

pp. 694-700 ◽

Cited By ~ 25

Author(s):

Roy R Hantgan ◽

Silvia C Endenburg ◽

I Cavero ◽

Gérard Marguerie ◽

André Uzan ◽

...

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Platelet Adhesion ◽

Integrin Receptor ◽

Shear Rates ◽

Perfusion Chamber ◽

Receptor Recognition ◽

Adhesive Interactions ◽

Coated Surfaces ◽

Carboxy Terminal

SummaryWe have employed synthetic peptides with sequences corresponding to the integrin receptor-recognition regions of fibrinogen as inhibitors of platelet aggregation and adhesion to fibrinogen-and fibrin-coated surfaces in flowing whole blood, using a rectangular perfusion chamber at wall shear rates of 300 s–1 and 1,300 s–1. D-RGDW caused substantial inhibition of platelet aggregation and adhesion to fibrinogen and fibrin at both shear rates, although it was least effective at blocking platelet adhesion to fibrin at 300 s–1. RGDS was a weaker inhibitor, and produced a biphasic dose-response curve; SDRG was inactive. HHLGGAK-QAGDV partially inhibited platelet aggregation and adhesion to fibrin(ogen) at both shear rates. These results support the identification of an RGD-specific receptor, most likely the platelet integrin glycoprotein IIb: III a, as the primary receptor responsible for platelet: fibrin(ogen) adhesive interactions under flow conditions, and indicate that platelet adhesion to surface bound fibrin(ogen) is stabilized by multivalent receptor-ligand contacts.

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The Influence of Shear Rate and Red Cell Concentration on Platelet Adhesion and Thrombus Formation in Human Blood

10.1055/s-0039-1687367 ◽

1979 ◽

Author(s):

V.T. Turitto ◽

H.J. Weiss ◽

H. R. Baumgartner

Keyword(s):

Platelet Adhesion ◽

Cell Concentration ◽

Thrombus Formation ◽

Size Variation ◽

Arrival Rate ◽

Red Cells ◽

Steady Increase ◽

Shear Rates ◽

Perfusion Chamber ◽

Diffusion Controlled

The interaction of platelets with subendothelium requires the transport of platelets to the vicinity of the surface, as well as the basic cell-surface reaction. Exposure of subendothelium to human citrated blood flowing in an annular perfusion chamber at wall shear rates (α) of 50-10,000 sec-1 indicates that a diffusion controlled (DC) transport regime exists below 650 sec- l in which platelet adhesion (C+S) was strongly dependent on α, and thrombus formation (T) was absent . Above 800 sec-1, an apparently reaction controlled (RC) regime predominates in which C+S was independent of α, and T increased in both extent and size. Variation of hematocrit (H) from 0-701. in the RC regime (2600 sec-1) lead to a steady increase of C+S with H, and an exponential increase in T as H increased from 30 to 70%. In the DC regime (200 sec-1) virtually no thrombi were formed for all H, and C+S increased as H increased to 40%; above 40%, C+S became independent of H. Thus, at low α (venous), the platelet-subendothelial reaction is controlled primarily by the arrival rate of platelets at the surface and the red cells increase this transport for H up to 40%. At high α (microcirculatory) , the platelet-vessel wall reactivity becomes more dominant and red cells increase the ability of platelets to attach to the subendothelium.

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C57BL/6JOlaHsd Mice with Tandem Deletion of the Multimerin 1 and Alpha-Synuclein Genes Have Impaired Platelet Function in Vivo and in Vitro That Can Be Corrected by Multimerin 1

Blood ◽

10.1182/blood.v112.11.3926.3926 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3926-3926 ◽

Cited By ~ 1

Author(s):

Subia Tasneem ◽

Adili Reheman ◽

Heyu Ni ◽

Catherine P.M. Hayward

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Knockout Mice ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Alpha Synuclein ◽

Type I ◽

Significant Difference

Abstract Studies of mice with genetic deficiencies have provided important insights on the functions of many proteins in thrombosis and hemostasis. Recently, a strain of mice (C57BL/6JOlaHsd, an inbred strain of C57BL/6J) has been identified to have a spontaneous, tandem deletion of the multimerin 1 and α-synuclein genes, which are also adjacent genes on human chromosome 4q22. Multimerin 1 is an adhesive protein found in platelets and endothelial cells while α-synuclein is a protein found in the brain and in blood that is implicated in neurodegenerative diseases and exocytosis. In vitro, multimerin 1 supports platelet adhesion while α-synuclein inhibits α-granule release. We postulated that the loss of multimerin 1 and α-synuclein would alter platelet function and that recombinant human multimerin 1 might correct some of these abnormalities. We compared platelet adhesion, aggregation and thrombus formation in vitro and in vivo in C57BL/6JOlaHsd and C57BL/6 mice. Thrombus formation was studied by using the ferric-chloride injured mesenteric arteriole thrombosis model under intravital microscopy. We found that platelet adhesion, aggregation and thrombus formation in C57BL/6JOlaHsd were significantly impaired in comparison to control, C57BL/6 mice. The number of single platelets, deposited 3–5 minutes after injury, was significantly decreased in C57BL/6JOlaHsd mice (P <0.05, platelets/min: C57BL/6 = 157 ± 15, n=16; C57BL/6JOlaHsd = 77 ± 13, n=17). Moreover, thrombus formation in these mice was significantly delayed. Thrombi in C57BL/6JOlaHsd were unstable and easily dissolved, which resulted in significant delays (P<0.001) in vessel occlusion (mean occlusion times: C57BL/6 = 15.6 ± 1.2 min, n=16; C57BL/6JOlaHsd = 31.9 ± 2.1 min, n=17). We further tested platelet function in these mice by ADP and thrombin induced platelet aggregation using platelet rich plasma and gel-filtered platelets, respectively. Although no significant differences were seen with ADP aggregation, thrombin-induced platelet aggregation was significantly impaired in C57BL/6JOlaHsd mice. Platelet adhesion to type I collagen (evaluated using microcapillary chambers, perfused at 1500 s−1 with whole blood) was also impaired in C57BL/6JOlaHsd mice. However, platelets from C57BL/6JOlaHsd mice showed a normal pattern of agonist-induced release of α-granule P-selectin. Multimerin 1 corrected the in vitro aggregation and adhesion defects of C57BL/6JOlaHsd platelets. Furthermore, the transfusion of multimerin 1 into C57BL/6JOlaHsd mice corrected the impaired platelet deposition and thrombus formation in vivo. No significant difference was found in tail bleeding time between the two groups of mice. As α-synuclein knockout mice have a shortened time to thrombus formation (Circulation2007;116:II_76), the effects of multimerin 1 on impaired platelet function in C57BL/6JOlaHsd mice provide supportive evidence that multimerin 1 contributes to platelet adhesion and thrombus formation at the site of vessel injury. The findings suggest multimerin 1 knockout mice will be useful to explore platelet function. The first two authors and participating laboratories contributed equally to this study.

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Revised Model for Platelet Adhesion to Collagen.

Blood ◽

10.1182/blood.v114.22.2999.2999 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2999-2999

Author(s):

Lucia Stefanini ◽

Moritz Stolla ◽

Sean F Maloney ◽

Timothy Daniel Ouellette ◽

Claire Roden ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Mapk Signaling ◽

Low Flow ◽

Flow Conditions ◽

Integrin Activation ◽

Shear Rates ◽

Slow Kinetics ◽

Rap1 Activation

Abstract Abstract 2999 Poster Board II-968 The Gi-coupled ADP receptor, P2Y12, is the target of clopidogrel bisulfate (Plavix), currently the most successful anti-platelet strategy used in the clinic. In a recent study, we have shown that the Ca2+-sensing nucleotide exchange factor, CalDAG-GEFI, and P2Y12 represent the major signaling pathways leading to Rap1 and integrin activation in platelets (Cifuni et al., 2008, Blood). In the present study, we have further evaluated the importance of CalDAG-GEFI signaling and Rap1 activation for various aspects of platelet activation, and we have compared thrombus formation of CalDAG-GEFI−/− and WT/clopidogrel platelets under static and flow conditions in vitro. Our studies establish a revised model for platelet activation by collagen. In platelets activated with threshold concentrations of GPVI agonists, CalDAG-GEFI serves as a highly sensitive response element to Ca2+ that allows for the rapid activation of Rap1. CalDAG-GEFI-mediated Rap1 activation triggers a first wave of integrin activation and ERK (MAPK) signaling, followed by TxA2 release. TxA2 provides crucial feedback for the activation of PKC and granule/ADP release. ADP in turn triggers the second, P2Y12-dependent wave of Rap1-mediated signaling events, leading to the sustained activation of integrins and further release of TxA2. Higher concentrations of GPVI agonists lead to the concomitant activation of CalDAG-GEFI and PKC, facilitating platelet aggregation independent of feedback by endogenous TxA2. Under physiological flow conditions, CalDAG-GEFI-dependent platelet activation (clopidogrel-treated WT platelets) allowed for the formation of small but unstable thrombi, which rapidly disintegrated at high shear rates. In contrast, CalDAG-GEFI−/− platelets (P2Y12-dependent platelet activation) in anticoagulated blood firmly adhered to the thrombogenic surface but failed to form thrombi, even at high concentrations of collagen. Addition of exogenous TxA2 to anticoagulated CalDAG-GEFI−/− blood did not restore thrombus formation under flow. However, small thrombi were observed with non-anticoagulated CalDAG-GEFI−/− blood perfused at venous but not arterial shear rates, suggesting that a) locally generated thrombin facilitates the recruitment of free flowing CalDAG-GEFI−/− platelets to already adherent platelets, and b) the slow kinetics of P2Y12-dependent Rap1 activation only supports thrombin-induced platelet-platelet cohesion at low shear conditions. In conclusion, our studies demonstrate that CalDAG-GEFI/Rap1 signaling plays a critical role for the first wave of integrin activation and TxA2 generation important for platelet adhesion to a thrombogenic surface. Signaling by P2Y12/Rap1 is essential for sustained platelet activation/thrombus stabilization and partially compensates for CalDAG-GEFI/Rap1-mediated platelet adhesion under low flow conditions. Disclosures: No relevant conflicts of interest to declare.

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CRGT Peptide In Cytoplasm Regulates Thrombus Formation Via Dissociating c-Src-β3 Interaction

Blood ◽

10.1182/blood.v122.21.3501.3501 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3501-3501

Author(s):

Jiansong Huang ◽

Xiaofeng Shi ◽

Wenda Xi ◽

Ping Liu ◽

Xiaodong Xi

Keyword(s):

Molecular Mechanisms ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Human Platelets ◽

Flow Conditions ◽

Cho Cell ◽

Integrin Αiibβ3 ◽

Shear Rates ◽

Platelet Adhesion And Aggregation

Abstract The RGT sequences of the integrin β3 tail directly and constitutively bind the inactive c-Src, regulating integrin αIIbβ3 signaling and platelet function. Previous work has shown that disrupting the interaction of c-Src with β3 via myristoylated RGT peptide or deletion of the RGT sequences in β3 selectively inhibits integrin αIIbβ3 outside-in signaling in platelets. However, the precise molecular mechanisms by which the Src-β3 association regulates integrin αIIbβ3 signaling need to be clarified. We found that active c-Src phosphoylated the Y747 and Y759 residues of β3 directly at the in vitro protein/protein level or in CHO cell models bearing Tac-β3 chimeras, which were devoid of the intact β3 signal transduction. Furthermore, data from mass spectrometry, [γ-32P] ATP incorporation assays and CHO cell/Tac-β3 chimeras demonstrated that the direct phosphorylation of Y747 and Y759 by active c-Src did not depend on the binding of c-Src to the RGT sequences of the β3 tail. To further investigate the biological functions of Src-β3 association in signal transduction we employed a cell-permeable and reduction-sensitive peptide (myr-AC∼CRGT), which disrupted the Src-β3 association in platelets independent of membrane-anchorage, and found that when platelets were stimulated by thrombin the c-Src activation and the phosphorylation of the tyrosine residues of the β3 tail were substantially inhibited by the presence of the peptide. These results suggest that one of the crucial biological functions of Src-β3 association is to serve as a “bridge” linking integrin signaling with the c-Src full activation and phosphorylation of the tyrosines of the β3 tail. To answer whether the RGT peptide binding to Src is able to alter the enzymatic activity of c-Src, we examined the Src-Csk association, the phosphorylation status of Y416 and Y527 of c-Src and the c-Src kinase catalytic activity. Results showed that myr-AC∼CRGT did not dissociate Csk from c-Src in resting platelets and the phosphorylation level of Y416 and Y527 of c-Src remained unaltered. Consistent data were also obtained from in vitro analysis of the c-Src kinase catalytic activity in the presence of CRGT peptide. These results suggest that myr-AC∼CRGT peptide per se does not fully activate c-Src. Myr-AC∼CRGT was also found to inhibit integrin αIIbβ3 outside-in signaling in human platelets. To examine the effect of the myr-AC∼CRGT on platelet adhesion and aggregation under flow conditions, we measured the platelet thrombus formation under different shear rates. Myr-AC∼CRGT did not affect the platelet adhesion at a wall shear rate of 125 s-1. The inability of myr-AC∼CRGT to affect platelet adhesion and aggregation remained at 500 s-1 shear rates. At 1,500 s-1, or 5,000 s-1 rates, myr-AC∼CRGT partially inhibited platelet adhesion and aggregation. These observations indicate that the Src-regulated outside-in signaling plays a pivotal role in the stable thrombus formation and the thrombus growth under flow conditions. The present study reveals novel insights into the molecular mechanisms by which c-Src regulates integrin αIIbβ3 signaling, particularly the phorsphorylation of the β3 cytoplasmic tyrosines, and provides first evidence in human platelets that the RGT peptide or derivatives regulate thrombus formation through dissociating the Src-β3 interaction. The data of this work allow us to anticipate that intracellular delivery of the RGT peptide or its analogues may have potential in the development of a new antithrombotic strategy where only the Src-β3 interaction is specifically interrupted so as to provide an effective inhibition on thrombosis together with a decent hemostasis. Disclosures: No relevant conflicts of interest to declare.

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Glycoprotein Ib, von Willebrand factor, and glycoprotein IIb:IIIa are all involved in platelet adhesion to fibrin in flowing whole blood

Blood ◽

10.1182/blood.v76.2.345.bloodjournal762345 ◽

1990 ◽

Vol 76 (2) ◽

pp. 345-353 ◽

Cited By ~ 1

Author(s):

RR Hantgan ◽

G Hindriks ◽

RG Taylor ◽

JJ Sixma ◽

PG de Groot

Keyword(s):

Platelet Aggregation ◽

Shear Rate ◽

Von Willebrand Factor ◽

Whole Blood ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Shear Rates ◽

Wall Shear ◽

Von Willebrand ◽

Willebrand Factor

We have investigated the molecular basis of thrombus formation by measuring the extent of platelet deposition from flowing whole blood onto fibrin-coated glass coverslips under well-defined shear conditions in a rectangular perfusion chamber. Platelets readily and specifically adhered to fibrin-coated coverslips in 5 minute perfusion experiments done at either low (300 s-1) or high (1,300 s-1) wall shear rates. Scanning electron microscopic examination of fibrin-coated coverslips after perfusions showed surface coverage by a monolayer of adherent, partly spread platelets. Platelet adhesion to fibrin was effectively inhibited by a monoclonal antibody (MoAb) specific for glycoprotein (GP) IIb:IIIa. The dose-response curve for inhibition of adhesion by anti-GPIIb:IIIa at both shear rates paralleled that for inhibition of platelet aggregation. Platelet aggregation and adhesion to fibrin were also blocked by low concentrations of prostacyclin. In contrast, anti- GPIb reduced adhesion by 40% at 300 s-1 and by 70% at 1,300 s-1. A similar pattern of shear rate-dependent, incomplete inhibition resulted with a MoAb specific for the GPIb-recognition region of von Willebrand factor (vWF). Platelets from an individual with severe von Willebrand's disease, whose plasma and platelets contained essentially no vWF, exhibited defective adhesion to fibrin, especially at the higher shear rate. Addition of purified vWF restored adhesion to normal values. These results are consistent with a two-site model for platelet adhesion to fibrin, in which the GPIIb:IIIa complex is the primary receptor, with GPIb:vWF providing a secondary adhesion pathway that is especially important at high wall shear rates.

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Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

Blood ◽

10.1182/blood.v122.21.3513.3513 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3513-3513

Author(s):

Wenxiu Yi ◽

Wei Li ◽

Lijie Ren ◽

Xinliang Mao ◽

Li Zhu

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Venous Blood ◽

Pi3k Pathway ◽

Dependent Manner ◽

Clot Retraction ◽

Occlusion Time

Abstract The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

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