Lipid rafts are essential for release of phosphatidylserine-exposing extracellular vesicles from platelets.

Platelets protect the vascular system during damage or inflammation, but platelet activation can result in pathological thrombosis. Activated platelets release a variety of extracellular vesicles (EVs). EVs shed from the plasma membrane often expose phosphatidylserine (PS). These EVs are pro-thrombotic and increased in number in many cardiovascular and metabolic diseases. The mechanisms by which PS-exposing EVs are shed from activated platelets are not well characterised. Cholesterol-rich lipid rafts provide a platform for coordinating signalling through receptors and Ca2+ channels in platelets. We show that cholesterol depletion with methyl-β-cyclodextrin or sequestration with filipin prevented the Ca2+-triggered release of PS-exposing EVs. Although calpain activity was required for release of PS-exposing, calpain-dependent cleavage of talin was not affected by cholesterol depletion. P2Y12 and TPα, receptors for ADP and thromboxane A2, respectively, have been reported to be in platelet lipid rafts. However, the P2Y12 antagonist, AR-C69931MX, or the cyclooxygenase inhibitor, aspirin, had no effect on A23187-induced release of PS-exposing EVs. Together, these data show that lipid rafts are required for release of PS-exposing EVs from platelets.Isaac Newton Trust/ Wellcome Trust ISSF/University of Cambridge Joint Research Grant British Heart Foundation grant SP/15/7/3156

Synergistic effect of peptide inhibitors derived from the extracellular and intracellular domain of αIIb subunit of integrin αIIbβ3 on platelet activation and aggregation

αIIbβ3, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of αIIbβ3 changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence 1000LEEDDEEGE1008 of αIIb (pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313–320 of αIIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of αIIb and β3. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets’ surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin αIIbβ3 activation

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding

Effect of octapeptides on platelet activation.

<p>Washed platelets (2.5x10⁸ platelets/ml) were preincubated with vehicle (NaCl 0.9%) or various peptides (A, B and D: 500 μM) for 5 min. Platelets were then treated with 0.1 U/ml thrombin (activated) or vehicle (resting) as described in “Experimental Procedures”. <b>(A)</b> LIBS (AP5 mAb) expression induced by vehicle, RGDS, ^pYMESRADR, RGDS + ^pYMESRADR, 317-substituted octapeptide (^pY³¹³MESAADR³²⁰), 319-substituted octapeptide (^pY³¹³MESRAAR³²⁰) or 320-substituted octapeptide (^pY³¹³MESRADA³²⁰) on resting or thrombin-activated platelets. AP5 binding was quantified by determining the fluorescence intensity (Geomean UA) of anti-mAb-phycoerythrin binding (n = 6; mean ± SEM). Isotype control binding (NI, first column) was performed in each experiment; <b>(B)</b> Flow cytometry analysis of anti-fibrinogen-FITC, PAC1-FITC, anti-CD62P-PE or isotype control (dashed black lines) antibody binding on resting (black lines) or activated platelets preincubated with vehicle (green lines), ^pYMESRADR octapeptide (red lines), or RGDS (blue lines); <b>(C)</b> Inhibitory effects of octapeptides (^pY³¹³MESRADR³²⁰: black; substituted ^pY³¹³MESRAAR³²⁰: grey or ^pM³¹⁴ESRADRK³²¹: dashed) on human washed platelet aggregation induced by thrombin (0.1 U/ml). Platelets were preincubated with vehicle (peptide 0 μM) or various concentrations of octapeptides and stimulated with thrombin for 5 min. Aggregation is expressed as a percentage of maximal light transmission measured at 5 min. Each point represents the mean (± SEM) of at least 4 experiments, *P<0.05, **P<0.01, ***P< 0,001 versus untreated platelets (peptide 0 μM); <b>(D)</b> Mean fluorescence intensity of PAC1-FITC binding on thrombin-activated platelets preincubated with vehicle, RGDS, ^pYMESRADR or with 317 substituted octapeptide (^pYMESAADR), 319 substituted octapeptide (^pYMESRAAR) or 320 substituted octapeptide (^pYMESRADA).</p