Surface morphology of human platelets during in vitro aggregation

The alterations of surface morphology of human platelets during the course of the aggregometer tracing were studied by scanning electron microscopy (SEM). Prior to activation the platelet rich plasma was pre-incubated at 37°C for at least 30 min in order to obtain a sufficient number of discoid platelets. Immediately following the addition of collagen or ADP platelets showed slender pseudopods which thereafter were replaced by bulbous protrusions. The decrease in percent light transmission (%T) in the aggregometer tracing was characterized in SEM by a significant enlargement of bulbous protrusions and by the transformation of platelet shape from disc to sphere. During the increase in %T platelets forming primary aggregates displayed spiny pseudopods at their surface indicating that, during in vitro aggregation induced by collagen or ADP, two generations of pseudopods are formed. Using a low dose of ADP, the return of aggregometer tracing to the base line was regularly accompanied by dissociation of primary aggregates, but platelets remained spheroid and displayed pseudopods for a long time. Our study indicates that the course of aggregometer tracing is closely associated to the surface morphology of platelets. Single morphological changes, however, are not reflected by the aggregometer method.     

Effect of epinephrine on fibrinogen receptor exposure by aspirin-treated platelets and platelets from concentrates in response to ADP and thrombin

Synergistic effects between agonists on platelet aggregation have long been appreciated. Recently epinephrine was reported to induce maximal aggregation of aspirin-treated platelets when combined with ADP or thrombin, and to increase fibrinogen binding of non-aspirin treated platelets stimulated with low doses of ADP. The present study extends these observations to correlate fibrinogen binding in response to various combinations of ADP, epinephrine, and thrombin with platelet aggregation and 14C-serotonin release using aspirin-treated platelets as well as platelets from stored concentrates. When fresh platelets were stimulated with epinephrine (5 microM) together with either ADP (10 microM) or thrombin (150 mU/ml), fibrinogen binding increased by 180% compared to binding observed in response to ADP or thrombin alone. This was accompanied by enhanced platelet aggregation, but no increase in 14C-serotonin release. While both ADP and epinephrine potentiated the aggregation and fibrinogen binding of stored platelets in response to high doses of thrombin (150 mU/ml), maximal aggregation was achieved only with thrombin (150 mU/ml) and epinephrine (5 microM) in combination. The data thus suggest that 1) epinephrine induces maximal aggregation of aspirin-treated platelets stimulated with thrombin or ADP by significantly enhancing fibrinogen receptor exposure independently of the cyclooxygenase-mediated release reaction; 2) epinephrine stimulates platelets by a mechanism different from that of thrombin or ADP; and 3) as demonstrated by others, the ability of platelets from stored concentrates to aggregate and to bind fibrinogen in response to ADP can be enhanced by epinephrine, and, in addition, these platelets can aggregate and bind fibrinogen maximally when stimulated with combinations of epinephrine and thrombin.     

ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Galphaq

Platelets from Galphaq knockout mice are unable to aggregate in response to physiological agonists like adenosine 5'-diphosphate (ADP), thromboxane A(2), thrombin, or collagen, although shape change still occurs in response to all of these agonists except ADP. ADP-induced platelet aggregation results from simultaneous activation of the purinergic P2Y(1) receptor coupled to calcium mobilization and shape change and of a distinct P2 receptor, P2cyc, coupled through Gi to adenylyl cyclase inhibition, which is responsible for completion and amplification of the response. P2cyc could be the molecular target of the antithrombotic drug clopidogrel and the adenosine triphosphate (ATP) analogs AR-C69931MX, AR-C67085, and AR-C66096. The aim of the present study was to determine whether externally added ADP could still act through the Gi pathway in Galphaq-deficient mouse platelets and thereby amplify the residual responses to agonists such as thrombin or collagen. It was found that (1) ADP and adrenaline still inhibited cyclic AMP accumulation in Galphaq-deficient platelets; (2) both agonists restored collagen- but not thrombin-induced aggregation in these platelets; (3) the effects of ADP were selectively inhibited in vitro by the ATP analog AR-C69931MX and ex vivo by clopidogrel and hence were apparently mediated by the P2cyc receptor; and (4) high concentrations of ADP (100 micromol/L) induced aggregation without shape change in Galphaq-deficient platelets through activation of P2cyc. Since adrenaline was not able to induce platelet aggregation even at high concentrations, we conclude that the effects of ADP mediated by P2cyc are not restricted to the inhibition of adenylyl cyclase through Gi(2).     

Human melanoma cell lines differ in their capacity to release ADP and aggregate platelets

Summary. In this study we have investigated, using three different human melanoma cell lines (M₁Do., M₃Da., M₄Be.), the varying capacity of melanoma cells to induce platelet aggregation in the presence or absence of inhibitors of ADP or thrombin. The expression levels of different integrins (α_v, β₃, α_vβ₃, α_IIb, α_vβ₃) were evaluated by immunoprecipitation, binding and flow cytometry studies. The level of ADP in supernatants of melanoma cells were quantified by ADP bioassay and HPLC. Platelets were irreversibly aggregated by M₃Da. as shown by electron microscopy, in contrast to M₁Do, which induced a slow reversible aggregation. M₄Be. did not induce platelet aggregation. In both cases, with M₃Da. or M₁Do., apyrase but not PPACK inhibited platelet induced aggregation. An anti-α_vβ₃ monoclonal antibody (LYP18) or polyclonal antibody inhibited platelet aggregation. A similar number of LYP18 molecules bound to the surface of M₁Do., M₃Da. and M₄Be. cell lines. Biological HPLC assays of ADP present in the supernatant of tumour cell lines showed the highest concentration of ADP to be secreted by M₃Da., followed by M₁Do., and none detected for M₄Be. These results show that differences in in vitro aggregating potential of the three human melanoma cell lines are not related to low integrin expression levels but to their ability to generate ADP. Generation of ADP by human melanoma cells may act as important modulator of melanoma-platelet interactions.     

Differing platelet aggregating effects by two tumor cell lines: Absence of role for platelet-derived ADP

Two different mechanisms of aggregation of heparinized human platelet-rich plasma have been identified with two tumor cell lines: In neither case are these mechanisms dependent on platelet-derived ADP. U87MG cells from a glioblastoma line of human origin caused a single irreversible wave of aggregation simultaneously with the onset of platelet secretion, and this was inhibited by heparin and hirudin but not by apyrase or phospholipase D. In contrast, Hut 20 cells from an undifferentiated tumor cell line of murine origin gave an initial reversible wave followed by a second irreversible wave, which then led to secretion. The first wave of platelet aggregation was unaffected by heparin or hirudin but was inhibited by apyrase, and the second wave was inhibited by phospholipase D. Citrate caused irreversible inhibition with either cell line, and aggregation did not occur with gel filtered platelets. These results suggest that platelet aggregation by the Hut 20 line is initially dependent on ADP released from the tumor cells, whereas aggregation induced by the U87MG line is dependent on a procoagulant activity of the tumor cell surface.     

Transient adhesion refractoriness of circulating platelets under shear stress: the role of partial activation and microaggregate formation by suboptimal ADP concentration

Exposure of whole blood (WB) to subendothelial extracellular matrix (ECM) under shear stress in the cone and plate(let) analyser (CPA) results in platelet adhesion, followed by release reaction and aggregation of circulating platelets on the adherent platelets. The properties of circulating non-adhered platelets in the CPA was studied by exposure of WB to ECM at a high shear rate (1300/s) for 2 min (1st run), followed by transfer of the suspension to a new ECM-coated well for a second run (2nd run) under similar conditions. The results of the 2nd run demonstrated transient adhesion refractoriness associated with platelet microaggregate formation in the suspension. The adhesion refractoriness was dependent on platelet activation during the 1st run and was prevented by addition of apyrase (ADP scavenger) or ADP receptor inhibitor, suggesting a role for ADP in mediating this response. Furthermore, exposure of WB samples to suboptimal concentrations of ADP (0.4-1 micromol/l) or a thrombin receptor activating peptide (TRAP) (5 micromol/l) for 2 min resulted in a similar transient platelet adhesion refractoriness to ECM under flow conditions. The transient platelet refractoriness and microaggregate formation induced by ADP was associated with a transient reduction in glycoprotein (GP)Ib, increased P-selectin expression and increased fibrinogen binding by circulating platelets. These data suggest a role for platelet agonists at suboptimal concentrations in modulating platelet function and limiting the expansion of the thrombus.     

The balance of concurrent aggregation and deaggregation processes in platelets is linked to differential occupancy of ADP receptor subtypes

Deaggregation, the partial reversal of the initial aggregation of platelets is observed following low, but not higher, micromolar ADP concentrations. This study tested the hypothesis that deaggregation results from a balance between concurrent, opposing, aggregation and deaggregation processes which are ADP (adenosine 5'-diphosphate) receptor occupancy-dependent. Aggregation of human platelet-rich plasma (PRP) prepared in r-hirudin was assayed in a 96-well plate reader over 20 min by measurement of the optical density (OD) at 580 nm. Aggregation and the time to reach peak aggregation were directly proportional to ADP receptor occupancy. The magnitude and time course of the response to ADP were comparable to those previously reported with standard aggregometry. The rate constant of platelet deaggregation, as assessed by a fourcompartment kinetic model, was inversely proportional to agonist concentration. The ratio of the rate constants of aggregation and deaggregation was receptor occupancy-dependent and directly proportional to aggregation. Consequently, platelet aggregation was proportional, and deaggregation inversely proportional, to ADP receptor occupancy. We propose that the response of PRP to ADP and to 2-MeS-ADP (2-methylthioadenosinediphosphate), in vitro , consists of at least two active, concurrent processes, aggregation and deaggregation. Incremental occupancy of the P 2T ADP receptor subtype attenuates deaggregation and governs the balance between these two processes.     

ADP causes subsecond changes in protein phosphorylation of platelets

We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched- flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.     

Effect of steroids on the activation status of platelets in patients with Immune thrombocytopenia (ITP)

The activation status of platelets in Immune Thrombocytopenia (ITP) patients – which is still somewhat controversial – is of potential interest, because activated platelets tend to aggregate (leading to excessive clotting or thromboembolic events) but cannot do so when platelet numbers are low, as in ITP. Although corticosteroids are the first line of therapy in ITP, the effect of steroids on activation of platelets has not been evaluated so far. We examined the status of platelet activation (with and without stimulation with ADP) in ITP patients, at the start of therapy (pre-steroid treatment, naive) and post-steroid treatment (classified on the basis of steroid responsiveness). We used flow cytometry to evaluate the levels of expression of P-selectin, and PAC-1 binding to platelets of 55 ITP patients and a similar number of healthy controls, treated with and without ADP. We found that platelets in ITP patients exist in an activated state. In patients who are responsive to steroids, the treatment reverses this situation. Also, the fold activation of platelets upon treatment with ADP is more in healthy controls than in ITP patients; treatment with steroids causes platelets in steroid-responsive patients to become more responsive to ADP-activation, similar to healthy controls. Thus steroids may cause changes in the ability of platelets to get activated with an agonist like ADP. Our results provide new insights into how, and why, steroid therapy helps in the treatment of ITP.     

Radiolabelling of human platelets with 99mTc-HMPAO

The optimum conditions for labelling platelets with lipophilic 99mTc-hexamethyl propylene amine oxime (99mTc-HMPAO) were evaluated. An aseptic closed system was used throughout the procedure in patient studies. 48 ml blood were withdrawn into 12 ml ACD using sterile 60 ml plastic syringes. After mixing, the blood was transferred to sterile 10 ml vacuum tubes and platelets were isolated according to standard centrifugation procedures. The labelling efficiency was not dependent upon incubation temperature (22 degrees C, 37 degrees C) but was greater in saline than in the presence of plasma. The labelling efficiency increased with time up to 60 min in saline. The elution of 99mTc from platelets was about 25% in plasma milieu in vitro but did not increase with time during 160 min. 5 patients with verified fresh deep vein thrombosis in the lower leg were imaged after injection of labelled autologous platelets. All 4 of the patients without anticoagulant therapy showed positive uptake of 99mTc-HMPAO-labelled platelets, but the 5th patient--under heparin therapy--was negative in scintigraphy. Our results are encouraging and 99mTc-HMPAO-labelled platelets offer a promising tool for evaluating various clinical situations.     

Effect of clopidogrel treatment on ADP-induced phosphorylations in rat platelets

Phosphorylations induced by 2-MeS-ADP, a potent agonist of platelet ADP receptors, have been studied in rat platelets, and the effect of clopidogrel, a compound which inhibits platelet aggregation by selectively reducing the binding of ADP to its low affinity receptors on platelets, has been determined. 2-MeS-ADP induced platelet activation (shape change and aggregation) simultaneously with the phosphorylation of myosin light chain (P₂₀) and plekstrin (P₄₇). Phosphorylation of P₂₀ and P₄₇ was transient, a maximum being observed 10 s after addition of the agonist when shape change reached its maximum. P₂₀ and P₄₇ phosphorylations were not strongly affected by clopidogrel treatment. Following stimulation of platelets with 2-MeS-ADP, several proteins were phosphorylated at tyrosine residues. Clopidogrel treatment inhibited the increase in phosphorylation of P₁₄₀ , P₁₀₀ , P_80/85 , P₆₆ and P₅₅ concomitantly with the inhibition of platelet aggregation. However, clopidogrel did not interfere with the early phosphorylation of the P_80/85 kD doublet which occurs at the time of the shape change. P_80/85 , identified by immunodetection as cortactin, could be involved in the reorganization of the cytoskeleton necessary for morphological changes.   Thus, by using clopidogrel-treated rat platelets, we were able to determine some of the phosphorylations coupled either to clopidogrel-resistant high-affinity ADP receptors leading to shape change or to clopidogrel sensitive low-affinity ADP receptors coupled to the aggregation process.     

Platelet-activating factor is a weak platelet agonist: Evidence from normal human platelets and platelets with congenital secretion defects

We have examined the effects of a novel platelet agonist, platelet activating factor (PAF), on human platelets. Irreversible aggregation and ¹⁴C-serotonin secretion in response to PAF (10^?5 M) was found to be dependent on both thromboxane production and secreted adenosine diphosphate (ADP). Liberation of arachidonic acid (AA) from membrane-bound phospholipids is a prerequisite step in platelet thromboxane production. Studies with ³H-AA-labeled platelets revealed that PAF (10^?5 M) was a weak stimulus for the mobilization of AA. In addition, PAF (10^?5M) was found to be a weak inducer of thromboxane synthesis (mean = 6 pmol/10⁸ platelets) as compared to thrombin 5 U/ml (mean = 177 pmol/10⁸ platelets), measured using a radioimmunoassay for thromboxane B₂. Formation of phosphatidic acid is an early step in stimulus-response coupling in platelets. Our studies indicate that PAF is a weak stimulus for phosphatidic acid formation as well. To obtain further insights into its action, we examined the effect of PAF on platelets from three groups of patients with congenital secretion defects: patients with the storage pool deficiency, those with impaired thromboxane synthesis due to impaired liberation of AA from phospholipids, and those with impaired secretion despite normal granule stores and thromboxane production. The response to PAF was impaired in all patients, providing further evidence that PAF-induced platelet activation is dependent on secreted ADP and thromboxane A₂ synthesis, and occurs by mechanisms common to a number of agonists. Overall, these studies indicate that PAF is a weak platelet agonist.     

Primary dysfunction in aggregation and secretion of SHRSP platelets: Not secondary to the circulation of “exhausted” platelets

Summary The thrombin-induced secretion of [¹⁴C]-serotonin and adenine nucleotides from stroke-prone spontaneously hypertensive rats (SHRSP) platelets was markedly reduced with the development of hypertension accompanying hypoaggregability compared with that from age-matched Wistar Kyoto rats (WKY) platelets. Calcium Ionophore A 23187-induced secretion and aggregation were also attenuated in SHRSP platelets. Additionally, an enhancement of platelet secretion as well as aggregation by extracellular Ca²⁺ was less in SHRSP platelets than in WKY platelets. The platelet contents of adenine nucleotides and serotonin were not different between SHRSP and WKY at 5–16 weeks of age whereas they became significantly lower in SHRSP beginning at 22 weeks. The serotonin content in SHRSP platelets at 36 weeks of age was only 55% of that in WKY platelets. It is suggested that the reduced platelet aggregation and secretion observed in SHRSP platelets at ages lower than approximately 20 weeks are not secondary phenomena to the circulation of degranulated platelets, but the primary defect of SHRSP platelets appears to be an impaired function of Ca²⁺.     

Effect of clopidogrel administration to healthy volunteers on platelet phosphorylation events triggered by ADP

The action of clopidogrel on platelet receptors was analysed using platelets obtained from 11 healthy volunteers given 75 mg of clopidogrel daily for 8 d. Samples of blood were taken before treatment and after 8 d of medication. Determination of 2-methylthioadenosine diphosphate trisodium (2MesADP)-induced platelet aggregation, serine/threonine and tyrosine phosphorylations were performed in the absence or presence of the P2Y1-receptor-specific antagonist: adenosine 3'-phosphate 5'-phosphate (A3P5P) or the strong inhibitor of GPIIb/IIIa activation: SR121566. Major conclusions: 1). Serine and threonine phosphorylations of the myosin light chain (P20) and pleckstrin (P47) do not behave similarly, although they are both recognized as the result of phospholipase C pathway stimulation triggered by the P2Y1 receptor. P47 is strongly affected by the A3P5P, and this appears to be highly dependent on P2Y12. However, P20 phosphorylation occurs in the presence of A3P5P, suggesting that the P2Y12 receptor signal contributes to P20 phosphorylation mediated by a calcium-independent pathway. The results suggest that P2Y1 and P2Y12 receptors interact to modulate the phosphorylation of P20 and P47. 2). The inside-out signalling dependent on both P2Y12 and P2Y1 is necessary for GPIIb/IIIa activation. 3). Clopidogrel and SR121566 inhibited the increase in tyrosine phosphorylation induced by 2MesADP and concomitantly inhibited platelet aggregation, indicating that most of the phosphorylations are GPIIb/IIIa dependent. However, neither clopidogrel nor SR121566 inhibited the first wave of 80 kDa substrate (cortactin) which is involved in the reorganization of the cytoskeleton necessary for shape change and which appeared to be essentially P2Y1 dependent.     

Myristoylated alanine-rich C kinase substrate phosphorylation is involved in thrombin-induced serotonin release from platelets

Stimulation of platelets by thrombin induces protein kinase C (PKC) activation, phosphorylation of pleckstrin, aggregation and serotonin release. Here, we demonstrate that, in human platelets, thrombin stimulation also induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) and serotonin release in intact and digitonin-permeabilized platelets. MARCKS is known to bind actin and cross-link actin filaments, and this is inhibited by PKC-evoked MARCKS phosphorylation. MARCKS phosphorylation and serotonin release in response to increasing concentrations of thrombin have a similar EC50 and time course and, in permeabilized platelets, peptide MPSD, with an amino acid sequence corresponding to the phosphorylation site domain of MARCKS, blocked both responses. However, pleckstrin and myosin light chain phosphorylations were not modified. Ala-MPSD, in which the four serine residues of MPSD were substituted by alanines was ineffective. The results suggest a role for MARCKS in platelet secretion. The fact that pleckstrin phosphorylation has a different time course and was not modified in the presence of MPSD when MARCKS phosphorylation and serotonin release were inhibited would suggest either that pleckstrin phosphorylation is unrelated to secretion or that it might only be involved upstream in the events leading to secretion.     

Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors

Summary. Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP-induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP-induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca²⁺ influx. Binding studies, using the non- hydrolysable ligand [³³P]2MeSADP, have shown that human platelets contain about 600 high-affinity binding sites for 2MeSADP (K_d∼ 5 niw). These sites present pharmacological characteristics of a P_2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP-induced platelet aggregation but normal shape change display very low levels of [³³P]2MeSADP binding sites.The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca²⁺ influx and the other a Gi protein-coupled receptor responsible for Ca²⁺ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.     

Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2?×?10³?leukocytes/µl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1–10?µM) and ATP (10–100?µM) was markedly reduced, but to ATP (1000?µM) was enhanced (all p?<?0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t_1/2?=?4?min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4?min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.     

Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists

On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific.

Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70.

Platelet activation triggered the release of 57–79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r² > 0.98; p < 0.0001 for all), and with the microRNA content of the parent platelets (r² > 0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect.

These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.