CD44 sensitivity of platelet activation, membrane scrambling and adhesion under high arterial shear rates

2016 ◽  
Vol 115 (01) ◽  
pp. 99-108 ◽  
Author(s):  
Kousi Alzoubi ◽  
Madhumita Chatterjee ◽  
Britta Walker ◽  
Patrick Münzer ◽  
Dong Luo ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
Protein Abundance ◽  
Integrin Activation ◽  
Platelet Surface ◽  
Shear Rates ◽  
Occlusive Vascular Disease ◽  
Phosphatidylserine Exposure

SummaryCD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44-/- ) were compared to platelets from corresponding wild-type mice (cd44+/+ ). In resting platelets, P-selectin abundance, αllbβ3 inte-grin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44-/- and cd44+/+ platelets. Platelet degranulation and αllbβ3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca2+-store depletion with thapsigargin (1 µM), effects more pronounced in cd44-/- than in cd44+/+ platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44-/- than in cd44+/+ platelets. Thrombin further decreased forward scatter in cd44-/- and cd44+/+ platelets, an effect which tended to be again more pronounced in cd44-/- than in cd44+/+ platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s-1) were significantly augmented in cd44-/- mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and prothrombotic potential of platelets.

Medicine Meets Mathematics: Measuring ADAM10 Activity Directly On a Forming Thrombus

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 196-196
Author(s):  
Jane F Arthur ◽  
Isaac Pinar ◽  
Adam Facey ◽  
Andreas Fouras ◽  
Kris Ryan ◽  
...  
Keyword(s):  
Shear Stress ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Maximal Activity ◽  
Confocal Imaging ◽  
Platelet Surface ◽  
Shear Rates ◽  
Platelet Receptor ◽  
Fluorescent Peptide

Abstract Platelets respond rapidly to injury, infection and changes in blood shear stress where they engage subendothelial collagen and von Willebrand Factor via platelet-specific receptors glycoprotein (GP)VI and the GPIb-IX-V complex, respectively. Metalloproteolytic shedding of GPVI is one important consequence of platelet activation and occurs via activation of the receptor sheddase A Disintegrin and Metalloproteinase (ADAM)10. We demonstrated that exposure of platelets to brief, elevated shear was sufficient to activate ADAM10-mediated shedding of GPVI and that this activation did not require platelet receptor engagement, intracellular signalling or release of soluble mediators (Al Tamimi et al., Blood, 2012). A critical global question, however, is how ADAM10 activity towards vascular substrates can be so rapidly upregulated in a high shear environment? To directly examine shear-induced activation of a vascular metalloproteinase, we developed the first sensor capable of visualising ADAM10 activity on a forming thrombus, consisting of a GPVI sequence-based ADAM10-sensitive fluorescent peptide with an ADAM10-releasable quencher (GPVI-Cy3). Rapid recombinant (r) ADAM10 (Km = 24.3 μM, kcat = 0.27 s-1) but not rADAM17 cleavage of GPVI-Cy3 permitted direct ADAM10 monitoring on platelets. First, suspensions of human washed platelets were exposed to variable rates of uniform shear in a cone-plate viscometer, or were treated with 10 μg/ml collagen-related peptide (CRP; a GPVI ligand), or 5 mM NEM (a potent generic activator of ADAMs) then mixed with 5-10 μM GPVI-Cy3. Real-time fluorescence was monitored in a fluorescence plate reader. Untreated platelets displayed basal (37% of maximal) levels of ADAM10 activity (2.3 pmol/min/106 platelets; 100% denoting NEM-induced levels) consistent with active ADAM10, lacking an inhibitory pro-domain by western blot and flow cytometry, being present on the non-activated platelet surface. ADAM10 activity increased to 59% of maximal activity following CRP treatment and 86% of maximal activity after exposure to 10,000 s-1 shear stress for 5 min (5.4 pmol/min/106 platelets). Cleavage of GPVI-Cy3 was completely blocked by inclusion of 100 μM GM6001 or 2 μM GI254023 (specific ADAM10 inhibitor). Second, ADAM10 activity was visualised using fluorescence multi-channel confocal imaging (Nikon A1R Plus si, Piezo z-stage and perfect focus system) of thrombi formed by perfusion of collagen-coated capillaries with hirudinated whole blood (input wall shear rate = 1,800 s-1). The channel profile was reconstructed digitally, and a Computational Fluid Dynamics package (OPEN-Foam) accurately simulated the shear forces acting on the flow throughout the domain as a function of time. Importantly, this technique permits shear rates to be precisely determined at the thrombi surface, and provides time-varying data on shear rates experienced by platelets traversing the domain in vitro. Highest ADAM10 activity was observed 5-10 minutes post thrombus formation and colocalized with areas of high (>6000 s-1) shear on the surface of the thrombus. When GPVI-Cy3 was included for the duration of thrombus formation, serial z-stack thrombus cross section images revealed areas of high and low ADAM10 activity within the thrombus core, consistent with elevated ADAM10 activity on platelets involved in the initial stages of thrombus formation. Together, our findings (i) define a novel ADAM10-selective substrate GPVI-Cy3 which reports on ADAM10 activity in vitro and under hydrodynamic flow ex vivo, (ii) enable simultaneous quantitation of ADAM10 activity and local shear rates, and (iii) demonstrate correlation between ADAM10 activity and regions of elevated shear stress for the first time. Future studies will investigate how shear stress under pulsatile or continuous flow activates ADAM10 on vascular cell membranes, ultimately enabling the design of therapeutic agents that discretely target shear-mediated up-regulation of ADAMs activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Involvement of Ca2+ Activated Cl- Channel Ano6 in Platelet Activation and Apoptosis

2015 ◽  
Vol 37 (5) ◽  
pp. 1934-1944 ◽  
Author(s):  
Guoxing Liu ◽  
Guilai Liu ◽  
Hong Chen ◽  
Oliver Borst ◽  
Meinrad Gawaz ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Blood Platelets ◽  
Annexin V ◽  
Bleeding Disorder ◽  
Integrin Activation ◽  
Cell Shrinkage ◽  
Platelet Surface ◽  
Cl Channel ◽  
Phosphatidylserine Exposure ◽  
Αiibβ3 Integrin

Background/Aims: The ubiquitously expressed Ca2+ Activated Cl- Channel Ano6 participates in the stimulation of cell membrane scrambling. Defective Ano6 underlies the Scott syndrome, an inherited bleeding disorder with impaired scrambling of plasma membrane phospholipids. At least in theory, the bleeding disorder of Scott syndrome may result from impaired platelet function. Activators of platelets include thrombin and collagen related peptide (CRP), which trigger increase of cytosolic Ca2+-activity ([Ca2+]i), production of reactive oxygen species (ROS), degranulation, integrin activation, as well as cell shrinkage and phospholipid scrambling of the cell membrane. The present study thus explored whether Ano6 modifies activation-induced alterations of cytosolic Ca2+-activity ([Ca2+]i), degranulation (P-selectin exposure), integrin activation, phosphatidylserine exposure on the platelet surface and platelet volume. Methods: Platelets from mice lacking Ano6 (ano6-/-) were compared to platelets from corresponding wild-type mice (ano6+/+). [Ca2+]i was estimated from Fluo-3 fluorescence, ROS from DCFDA fluorescence, degranulation from P-selectin abundance, integrin activation from αIIbβ3-integrin abundance, phosphatidylserine abundance from annexin-V-binding, and cell volume from forward scatter. Results: Platelet number in blood was slightly higher in ano6-/- mice than in ano6+/+ mice. Without activation [Ca2+]i and volume were similar in ano6-/- and ano6+/+ platelets as well as ROS abundance, P-selectin abundance, αIIbβ3 integrin activation, and phosphatidylserine exposure were negligible in both genotypes. Thrombin (0.01 U/ml) and CRP (2 or 5 µg/ml) increased [Ca2+]i, ROS abundance, platelet degranulation, αIIbβ3 integrin activation, and triggered annexin-V-binding as well as cell shrinkage, all effects less pronounced in ano6-/- than in ano6+/+ platelets. Conclusions: Genetic knockout of Ano6 blunts thrombin- and CRP-induced activation and apoptosis of blood platelets.

scholarly journals Rapid Upregulation of Orai1 Abundance in the Plasma Membrane of Platelets Following Activation with Thrombin and Collagen Related Peptide

2015 ◽  
Vol 37 (5) ◽  
pp. 1759-1766 ◽  
Author(s):  
Guilai Liu ◽  
Guoxing Liu ◽  
Hong Chen ◽  
Kousi Alzoubi ◽  
Anja T. Umbach ◽  
...  
Keyword(s):  
Blood Platelets ◽  
Protein Abundance ◽  
Wild Type ◽  
Atpase Inhibitor ◽  
Platelet Surface ◽  
Related Peptide ◽  
Primary Hemostasis ◽  
Occlusive Vascular Disease ◽  
Small G Protein ◽  
Store Depletion

Background: Blood platelets accomplish primary hemostasis following vascular injury and contribute to the orchestration of occlusive vascular disease. Platelets are activated by an increase of cytosolic Ca2+-activity ([Ca2+]i), which is accomplished by Ca2+-release from intracellular stores and subsequent store operated Ca2+ entry (SOCE) through Ca2+ release activated Ca2+ channel moiety Orai1. Powerful activators of platelets include thrombin and collagen related peptide (CRP), which are in part effective by activation of small G- protein Rac1. The present study explored the influence of thrombin and CRP on Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i) in platelets drawn from wild type mice. Methods: Orai1 protein surface abundance was quantified utilizing CF™488A conjugated antibodies, and [Ca2+]i was determined with Fluo3-fluorescence. Results: In resting platelets, Orai1 protein abundance and [Ca2+]i were low. Thrombin (0.02 U/ml) and CRP (5ug/ml) within 2 min increased [Ca2+]i and Orai1 protein abundance at the platelet surface. [Ca2+]i was further increased by Ca2+ ionophore ionomycin (1 µM) and by store depletion with the sarcoendoplasmatic Ca2+ ATPase inhibitor thapsigargin (1 µM). However, Orai1 protein abundance at the platelet surface was not significantly affected by ionomycin and only slightly increased by thapsigargin. The effect of thrombin and CRP on Orai1 abundance and [Ca2+]i was significantly blunted by Rac1 inhibitor NSC23766 (50 µM). Conclusion: The increase of [Ca2+]i following stimulation of platelets with thrombin and collagen related peptide is potentiated by ultrarapid Rac1 sensitive translocation of Orai1 into the cell membrane.

Action of Lysolecithin on Blood Platelets

1963 ◽  
Vol 09 (03) ◽  
pp. 512-524 ◽  
Author(s):  
Chava Kirschmann ◽  
Sara Aloof ◽  
Andre de Vries
Keyword(s):  
Blood Platelets ◽  
Protective Action ◽  
Platelet Surface ◽  
Washed Platelet ◽  
Sufficient Concentration ◽  
Saline Medium

SummaryLysolecithin is adsorbed to washed blood platelets and, at sufficient concentration, lyses them, inhibits their clot-retracting activity and promotes their thromboplastin-generating activity. Lysolecithin adsorption to the platelet was studied by using P32-labelled lysolecithin obtained from the liver of rats injected with labelled orthophosphate. The amount of lysolecithin adsorbed to the surface of the washed platelet in saline medium is dependent on the concentration of lysolecithin in solution and reaches saturation — 5 × 10-8 jig per platelet — at a concentration of 9—10 µg per ml. Platelet lysis in saline medium begins at a lysolecithin concentration higher than 18 jig per ml. Plasma and albumin prevent adsorption of lysolecithin to the platelet and protect the platelet from damage by lysolecithin. Albumin is able to remove previously adsorbed lysolecithin from the platelet surface. The protective action of plasma explains the lack of platelet damage in blood, the plasma lecithin of which has been converted to lysolecithin by the action of Vipera palestinae venom phosphatidase, in vitro and in vivo.

Protocatechuic Acid Protects Platelets from Apoptosis via Inhibiting Oxidative Stress-Mediated PI3K/Akt/GSK3β Signaling

2021 ◽  
Author(s):  
Fuli Ya ◽  
Kongyao Li ◽  
Hong Chen ◽  
Zezhong Tian ◽  
Die Fan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caspase 3 ◽  
Protocatechuic Acid ◽  
Human Platelets ◽  
Ros Generation ◽  
Inhibitory Effects ◽  
Platelet Apoptosis ◽  
Phosphatidylserine Exposure ◽  
Underlying Mechanisms

AbstractOxidative stress plays crucial roles in initiating platelet apoptosis that facilitates the progression of cardiovascular diseases (CVDs). Protocatechuic acid (PCA), a major metabolite of anthocyanin cyanidin-3-O-β-glucoside (Cy-3-g), exerts cardioprotective effects. However, underlying mechanisms responsible for such effects remain unclear. Here, we investigate the effect of PCA on platelet apoptosis and the underlying mechanisms in vitro. Isolated human platelets were treated with hydrogen peroxide (H2O2) to induce apoptosis with or without pretreatment with PCA. We found that PCA dose-dependently inhibited H2O2-induced platelet apoptosis by decreasing the dissipation of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and decreasing phosphatidylserine exposure. Additionally, the distributions of Bax, Bcl-xL, and cytochrome c mediated by H2O2 in the mitochondria and the cytosol were also modulated by PCA treatment. Moreover, the inhibitory effects of PCA on platelet caspase-3 cleavage and phosphatidylserine exposure were mainly mediated by downregulating PI3K/Akt/GSK3β signaling. Furthermore, PCA dose-dependently decreased reactive oxygen species (ROS) generation and the intracellular Ca2+ concentration in platelets in response to H2O2. N-Acetyl cysteine (NAC), a ROS scavenger, markedly abolished H2O2-stimulated PI3K/Akt/GSK3β signaling, caspase-3 activation, and phosphatidylserine exposure. The combination of NAC and PCA did not show significant additive inhibitory effects on PI3K/Akt/GSK3β signaling and platelet apoptosis. Thus, our results suggest that PCA protects platelets from oxidative stress-induced apoptosis through downregulating ROS-mediated PI3K/Akt/GSK3β signaling, which may be responsible for cardioprotective roles of PCA in CVDs.

A Role for Bile Salt-Dependent Lipase in Platelet Activation and in Thrombus Formation in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3526-3526 ◽  
Author(s):  
Laurence Panicot-Dubois ◽  
Christophe Dubois ◽  
Barbara C. Furie ◽  
Bruce Furie ◽  
Dominique Lombardo
Keyword(s):  
Bile Salt ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Pancreatic Acinar Cells ◽  
Cremaster Muscle ◽  
Terminal Domain ◽  
Laser Injury ◽  
Phosphatidylserine Exposure

Abstract Bile Salt Dependent Lipase (BSDL) is an enzyme secreted by pancreatic acinar cells. BSDL, in the presence of primary bile salts, participates in the hydrolysis of dietary lipid esters in the duodenum lumen. This 105 kDa N and O-glycosylated protein has been detected in the plasma of normal subjects. Recent in vitro and in vivo studies demonstrated that pancreatic BSDL reaches the blood via transcytosis through enterocytes. Other studies showed that pancreatic human BSDL is captured by human umbilical vein endothelial cells and induces the proliferation of smooth muscle cells in vitro at BSDL concentrations found in blood, suggesting that this enzyme may play a role in hemostasis and thrombosis. However the specific role of circulating BSDL is unknown. The goal of this study was to determine the possible involvement of circulating BSDL in thrombus formation. We investigated the participation of circulating mouse BSDL in thrombus formation using widefield intravital microscopy in the cremaster muscle of living mice. Thrombi were formed following laser injury of the vessel wall of an arteriole in the cremaster muscle. Pancreatic mouse BSDL, a 74 kDa glycoprotein, was detected using several antibodies directed against either the whole human BSDL (pAbL64, pAbL32) or a peptide based on a sequence in the N-terminal domain of BSDL (Ser326-Thr350; pAbAntipeptide). Mouse and human BSDL share about 80% sequence homology, the main difference localized in the C-terminal domain, which is truncated to the mouse BSDL compared with the human enzyme. All the antibodies are able to specifically recognize the mouse pancreatic BSDL. Using antibodies pAbL64, pAbL32, or pAbAntipeptide we observed specific accumulation of circulating mouse BSDL into the growing thrombus. The circulating BSDL co-localized with platelets present in the thrombus. These results suggest that circulating BSDL is involved in thrombus formation in vivo. In order to determine if BSDL plays a role in platelet activation and aggregation, we performed in vitro studies on human washed platelets. BSDL increased both the amount of phosphatidylserine exposure on the surface of platelets and the activation of αIIbβ3 induced by thrombin. These results indicate that this enzyme can amplify the activation of platelets in vitro. While BSDL alone cannot induce the aggregation of platelets, this enzyme significantly increases the amount of platelet aggregation induced by SFLLRN peptide or thrombin. Altogether, these data suggeste that circulating BSDL participates in the thrombus formation after laser injury of the arterial wall and can amplify both the activation of platelets and the phosphatidylserine exposure, increasing the thrombotic response after vessel injury. This mechanism may be operative in the development of venous thromboembolic disease in pancreatic cancer.

Evaluation of Platelet Thrombus Formation under Flow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3964-3964
Author(s):  
Volker R. Stoldt ◽  
Jan Peveling ◽  
Robert Loncar ◽  
Andreas Beck ◽  
Volker Aurich ◽  
...  
Keyword(s):  
Shear Rate ◽  
Laser Scanning ◽  
Type I Collagen ◽  
Thrombus Formation ◽  
Confocal Laser ◽  
Lag Phase ◽  
Type I ◽  
Shear Rates ◽  
Platelet Aggregate

Abstract We have used confocal laser microscopy and a novel “voxel”-based imaging software to study the dynamics of platelet aggregation and thrombus formation when anticoagulated blood was perfused over collagen-coated surfaces at shear rates simulating arterial flow. The objective was to evaluate the three-dimensional growth of platelet thrombi over time (“4-D” imaging). Blood from healthy donors, anticoagulated with either PPACK (80 μM) or, depending on the type of experiment, with trisodium citrate (11 mM), was incubated with mepacrine (10 μM) to render platelets fluorescent. Blood was aspirated with a syringe pump through a rectangular perfusion chamber (flow path height of 80 μm) at a flow rate of 160 or 480 μl per min to provide initial shear rates of 500 or 1,500 sec−1, respectively. Prior to perfusion, glass coverslips were coated with fibrillar type I collagen (Roche Diagnostics, Mannheim, Germany) prepared in 0.5 M acedic acid, pH 2.8, and blocked with 2 % BSA. The chamber was mounted on a Zeiss Axiovert 100M/LSM 510 invert laser scanning confocal microscope (Carl Zeiss, Oberkochem, Germany). Upon perfusion, a series of stacks, i.e. 30 confocal optical sections, from the bottom to the apex of the forming platelet aggregate or thrombus, were obtained every 25 sec with a 488-nm laser and a scanning time of < 500 msec on an area of 26,450 μm2. Images corresponding to an area of 0.202 μm2 were analyzed by a “voxel”-based procedure, whereby a voxel is defined by a volume of 0.202 μm3 (0.45 μm x 0.45 μm x 1 μm). For calibration, fluorescent beads (Invitrogen, Carlsbad, CA, USA) were used, and the volume coresponding to a 1.0 μm thick stack was calculated pursuant to the voxel technique. A threshold was applied to distinguish adherent platelets from the background. Using these procedures, a uniform profile of thrombus formation and volume was observed (n=7). With citrate anticoagulated blood at an initial shear rate of 500 sec−1, thrombus growth begun after a lag phase of 220 sec, and, after 420 sec, thrombus volume reached a maximum (mean ± SD, 5x104 ± 4.9x103 μm3). Thrombus progression occurred in a two-step way with an apical growth (height extension) at the interval of 220 and 300 sec, and a further growth in the plane section at the interval of 300 and 420 sec after perfusion. Prolonged perfusion resulted in markedly abnormal flow pattern due to thrombus growth and increased shear rates. Again at an initial shear rate of 500 sec−1, platelet aggregate formation and thrombus progression were completely suppressed in the presence of anti-αIIbβ3 antibody (abciximab, 4 μg/ml). Interestingly, the polymorphism (HPA-1, PlA) of the β subunit of αIIbβ3 had a dramatic effect on thrombus growth. Thus, when comparing blood from homozygous carriers of HPA-1b (n=8) and HPA-1a (n=8), thrombus formation and progression occurred more rapidly with HPA-1b than with HPA-1a platelets, resulting in significantly larger thrombi from HPA-1b than from HPA-1a individuals (p=0.001). In conclusion, the voxel-based determination of thrombus formation and progression in vitro provides an appropriate technique to assess volumina of thrombi. Moreover, this technique can detect phenotypic differences related to an αIIbβ3 polymorphism which is postulated to modulate platelet thrombogenicity.

Revised Model for Platelet Adhesion to Collagen.

Blood ◽  
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.

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

Blood ◽  
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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