Regulation Of Platelet Reactivity Research Articles

Abstract 11291: Ectopic Olfactory Receptor Expression in Platelets as Novel Antithrombotic Targets

Introduction: Platelets are enriched in orphan G-protein-Coupled Receptors (GPCRs) that could potentially regulate platelet reactivity. A remote study in patients with hereditary anosmia reported dysfunctional platelet responses suggesting the presence of platelet olfactory receptor (ORs) that could regulate thrombosis. Hypothesis: Platelet ORs serve as an alternative pharmacological strategy to generate platelet cyclic adenosine monophosphate (cAMP) to prevent thrombosis. Methods: Ribonucleic Acid sequencing (mRNAseq) of human megakaryocytes (MKs) was conducted. Various ORs were validated in platelets by RT-qPCR and immunoblotting (IB). Reporter cell lines with stable integration of cloned ORs identified OR ligands and later validated in adult platelets. Fluorophores for OR second messengers (Fura2 for Ca 2+ and MQAE for Cl - ) and an ELISA for cAMP were used for adult platelets. Platelet reactivity in response to OR agonists was evaluated by FACS and aggregometry, and thrombosis by biomechanical platelet activation was evaluated using ex vivo microfluidics and flow cones to simulate disturbed flow (D-flow). OR agonist injection into mice and CRISPR-Cas9 OR knockdown was conducted, with platelet reactivity and thrombosis tested as proof-of-concept. Results: Only 15 of >400 ORs in the genome were identified in MKs, and only 3 in all adult platelets (highest expression was OR2L13). A terpene family was identified as OR2L13 agonists, generating cAMP in the platelet, and structural modifications were made with IC 50 values in the low-high μM range for inhibiting agonist-induced platelet activation, ex vivo human blood thrombosis by microfluidics, and in vivo platelet activation in mice with prolonged tail bleeding. Deletion of OR2L13 (olfr168 -/- ) in mice augmented platelet reactivity with loss of OR agonist-mediated prolonged tail bleeding in olfr168 -/- . OR2L13 in human platelets is stored and trafficked by alpha granules following surface agonist stimulation and biomechanical activation. Conclusions: Like neurons, platelets have a fully functional OR signal transduction cascade which operates as a negative regulator of platelet reactivity and thrombosis in response to both chemical as well as biomechanical platelet activation.

MiR-204-5p and Platelet Function Regulation: Insight into a Mechanism Mediated by CDC42 and GPIIbIIIa.

Background Several platelet-derived microRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking. Aims To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues. Methods Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor, and a fibrinogen-binding assay. Quantitative polymerase chain reaction and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy. Results miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet–fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation. Conclusion We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.

Functional Validation of microRNA-126-3p as a Platelet Reactivity Regulator Using Human Haematopoietic Stem Cells.

Platelets are an abundant source of micro-ribonucleic acids (miRNAs) that may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, have been noted as potential biomarkers of platelet reactivity and the recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain, and the functional validation of candidate miRNAs on human-derived cells is lacking. This article functionally validates miR-126-3p as a regulator of platelet reactivity in platelet-like structures (PLS) derived from human haematopoietic stem cells. CD34+-derived megakaryocytes were transfected with miR-126-3p and differentiated in PLS. PLS reactivity was assessed using perfusion in a fibrinogen-coated flow chamber. miR-126-3p's selected gene targets were validated using quantitative polymerase chain reaction, protein quantification and a reporter gene assay. CD34+-derived megakaryocytes transfected with miR-126-3p generated PLS exhibiting 156% more reactivity than the control. These functional data were in line with those obtained analysing CD62P expression. Moreover, miR-126-3p transfection was associated with the down-regulation of a disintegrin and metalloproteinase-9 (ADAM9) messenger RNA (mRNA), a validated target of miR-126-3p, and of Plexin B2 (PLXNB2) mRNA and protein, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results to miR-126-3p transfection. Finally, using a reporter gene assay, we validated PLXNB2 as a direct target of miR-126-3p. We functionally validated miR-126-3p as a regulator of platelet reactivity in PLS derived from human haematopoietic stem cells. Moreover, PLXNB2 was validated as a new gene target of miR-126-3p in human cells, suggesting that miR-126-3p mediates its effect on platelets, at least in part, through actin dynamics regulation.

Functional validation of microRNA-126-3p as a platelet reactivity regulator using human hematopoietic stem cells

Platelets are an abundant source of microRNAs (miRNAs), which may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, are pointed out as potential biomarkers of platelet reactivity and recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain and functional validation of these candidate miRNAs on human-derived cells is lacking. To functionally validate miR-126-3p as regulator of platelet reactivity in platelet-like structures derived from human hematopoietic stem cells. The functional impact of miRNAs on platelet-like structures derived from human hematopoietic stem cells was evaluated using a flow-based assay. CD34+ derived megakaryocytes were transfected with selected miRNA or siRNA and were differentiated in platelet-like structures. Platelet adhesion phenotype was assessed by perfusion of differentiated cells in a microfluidic system under a constant shear rate. Overexpression of miR-126-3p increased platelet-like structures adhesion compared to control. Moreover, miR-126-3p transfection was associated with downregulation of ADAM9, a validated target of miR-126-3p, and of PLXNB2, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results than miR-126-3p transfection. Reporter gene assay confirm that miR-126-3p directly targets PLXNB2 3′UTR sequence. Taken together, using a flow-based assay, we functionally validate miR-126-3p as a regulator of platelet reactivity on platelet-like structures derived from human hematopoietic stem cells. Moreover, PLXNB2 was identified as a platelet reactivity regulator supporting the hypotheses that miR-126-3p modulates platelet reactivity at least in part by downregulation of PLXNB2.

Aerobic exercise training lowers platelet reactivity and improves platelet sensitivity to prostacyclin in pre‐ and postmenopausal women

Background The risk of atherothrombotic events increases after the menopause. Regular physical activity has been shown to reduce platelet reactivity in younger women, but it is unknown how regular exercise affects platelet function after the menopause. Objectives To examine the effects of regular aerobic exercise in late premenopausal and recent postmenopausal women by testing basal platelet reactivity and platelet sensitivity to prostacyclin and nitric oxide. Methods Twenty-five sedentary, but healthy, late premenopausal and 24 matched recently postmenopausal women, mean (95% confidence interval) 49.1 (48.2-49.9) and 53.7 (52.5-55.0) years old, participated in an intervention study: 3-month high-intensity supervised aerobic spinning-cycle training (1 h, × 3/week). Basal platelet reactivity was analyzed in platelet-rich plasma from venous blood as agonist-induced % aggregation. In a subgroup of 13 premenopausal and 14 postmenopausal women, platelet reactivity was tested ex vivo after femoral arterial infusion of prostacyclin, acetylcholine, a cyclooxygenase inhibitor, and after acute one-leg knee extensor exercise. Results Basal platelet reactivity (%aggregation) to TRAP-6 (1 μm) was higher in the postmenopausal, 59% (50-68), than the premenopausal women, 45% (35-55). Exercise training reduced basal platelet reactivity to collagen (1 μg mL-1 ) in the premenopausal women only: from 63% (55-71%) to 51% (41-62%). After the training intervention, platelet aggregation was more inhibited by the arterial prostacyclin infusion and the acute exercise in both premenopausal and postmenopausal women. Conclusions These results highlight previously unknown cardioprotective aspects of regular aerobic exercise in premenopausal and postmenopausal women, improving their regulation of platelet reactivity through an increased platelet sensitivity to prostacyclin, which may counterbalance the increased atherothrombotic risk associated with the menopause.

An ω-6 Fatty Acid, Dgla, Prevents Platelet Activation and Thrombosis in Vivo.

AbstractAbstract 2169Platelets play a pivotal role in thrombotic events leading to clot formation and clot stability in vivo. Uncontrolled signaling events in the platelet can result in unwanted thrombosis, which may eventually lead to the development of myocardial infarction or stroke. Previously, we have shown that ex vivo treatment of human platelets with the ω-6 fatty acid dihomo-γ-linolenic acid (DGLA) or its eicosanoid derived from 12-LOX oxidation, 12-hydroxyeicosatrienoic acid (12(S)-HETrE), inhibits PAR and collagen-induced platelet aggregation, clot retraction, and GPIIbIIIa activation. Since early studies have shown dietary supplementation of fatty acids increase fatty acid incorporation into the platelet lipid membrane (Barre, DE Lipids 1992; 27(5): 315–320; Marry, MJ Prostaglandins Leukot Essent Fatty Acids 1997; 56(3):223–223), we postulated that altering fatty acid composition in the platelet through dietary supplementation in vivo may be a viable approach to inhibiting platelet function. Therefore, a longitudinal study of wild-type mice on normal chow compared to mice supplemented with high (.5 g/kg) or low (.13 g/kg) DGLA diets was conducted. Each set of mice (7–8 mice) was given the designated diet for a period of 1, 2, or 3 months. At each time point, tail bleeding times and ex vivo platelet function in PRP were performed. Tail bleeding times from mice on the high DGLA diet were significantly prolonged by more than 15 minutes. Further, a smaller but statistically significant delay in clotting time was observed in mice on the low DGLA diet compared with control mice. Additionally, ex vivo aggregation response to collagen (1 μg/mL to 20 μg/mL) and PAR4-AP (50 μM to 500 μM) in platelets from mice on the high DGLA diet showed significant shifts to the right in their ability to induce platelet aggregation compared with control mice suggesting these mice were protected against thrombosis. JON/A and P-selectin binding to the PRP of high and low DGLA were also significantly attenuated in response to PAR4-AP. This study, which evaluated the in vivo and ex vivo effects of DGLA on regulation of platelet reactivity, supports DGLA as a potent, endogenous anti-thrombotic agent. Understanding the mechanistic details by which DGLA protects against thrombosis and maintains hemostasis through its COX-1 and 12-LOX-dependent bioactive metabolites will help to identify the potential viability of this target for anti-platelet intervention. Disclosures:No relevant conflicts of interest to declare.

12-Lipoxygenase: A Potential Target for Novel Anti-Platelet Therapeutics

Platelets play an essential role in the regulation of hemostasis and thrombosis and controlling their level of activation is central to prevention of occlusive clot formation and stroke. Although a number of anti-platelet targets have been identified to address this issue including COX-1, the P2Y(12) receptor, the integrin αIIbβ3, and more recently the protease-activated receptor-1, these targets often result in a significant increased risk of bleeding which may lead to pathologies as serious as the thrombosis they were meant to treat including intracranial hemorrhage and gastrointestinal bleeding. Therefore, alternative approaches to treat uncontrolled platelet activation are warranted. Platelet-type 12-lipoxygenase is an enzyme which oxidizes the free fatty acid in the platelet resulting in the production of the stable metabolite 12-hydroxyeicosatetraenoic acid (12-HETE). The role of 12-HETE in the platelet has been controversial with reports associating its function as being both anti- and pro-thrombotic. In this review, the role of 12-lipoxygenase and its bioactive metabolites in regulation of platelet reactivity, clot formation, and hemostasis is described. Understanding the mechanisms by which 12-lipoxygenase and its metabolites modulate platelet function may lead to the development of a novel class of anti-platelet therapies targeting the enzyme in order to attenuate injury-induced clot formation, vessel occlusion and pathophysiological shifts in hemostasis.

Interaction between ATP and catecholamines in stimulation of platelet aggregation.

Platelets, on activation by endothelial damage, release ADP, ATP, serotonin, epinephrine, and norepinephrine. Although ATP is known to augment the action of norepinephrine in cardiovascular and endocrine systems, the possible interaction between ATP and catecholamines in regulation of platelet reactivity has not been reported. The addition of ATP (1-5 microM) to human platelet-rich plasma did not induce platelet aggregation; however, it selectively augmented the aggregatory response to norepinephrine and epinephrine, but not to serotonin. This potentiating action of ATP was dose dependent and was not due to contamination by, or hydrolysis to, ADP. The action of ATP was blocked by 10 microM of adenosine 3'-phosphate 5'-phosphosulfate, a selective P(2)Y(1) receptor antagonist. ATP alone did not cause release of intracellular Ca(2+), but produced a significant Ca(2+) response in the presence of norepinephrine. In contrast, the P(2)X(1) receptor agonists P(1),P(6)-diadenosine-5' hexophosphate and alpha,beta-methylene-ATP had no effect on norepinephrine-induced platelet aggregation even when added at 100 microM. This synergistic interaction between ATP and norepinephrine in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress.

Role of extracellular ATP metabolism in regulation of platelet reactivity

Extracellular adenosine triphosphate (ATP) regulates platelet reactivity by way of direct action on platelet purinergic receptors or by hydrolysis to adenosine diphosphate (ADP). Subsequent metabolism of ATP and ADP to adenosine monophosphate (AMP) and adenosine inhibits platelet aggregation. Endothelial cell membrane-bound ecto-ATP/ADPase (CD39, E-NTPDase1) is thought to be the main regulator of platelet responsiveness. However, the findings in studies of CD39-knockout mice imply that nucleotidase(s) in plasma regulates circulating adenine nucleotides levels. Understanding extracellular ATP metabolism by CD39 and plasma nucleotidases is therefore important. In this study, α-phosphorus 32- and γ-phosphorus 32-labeled ATP were rapidly metabolized directly to AMP and pyrophosphate in human plasma at pH 7.4, suggesting the presence of pyrophosphatase/phosphodiesterase-like activity. A specific phosphodiesterase substrate, p-nitrophenol-5′-TMP (p-Nph-5′-TMP), was readily hydrolyzed in human plasma. The antiaggregatory action of β,γ-methylene-ATP (AMPPCP) (5 μmol/L) was blocked by DMPX, an adenosine-receptor antagonist, suggesting that in plasma, AMPPCP was metabolized to AMP and adenosine. Recombinant soluble CD39 (solCD39) was used to assess the role of CD39 in ATP metabolism. As little as 0.25 μg/mL of solCD39 inhibited ADP-induced platelet aggregation. However, in the presence of ADP-free ATP (10 μmol/L), solCD39 induced platelet aggregation in a dose-dependent manner. Because AMPPCP could not substitute for ATP in solCD39-stimulated platelet aggregation, it is likely that ADP formation from ATP was required. Endogenous CD39 may thus have a hemostatic function by promoting ADP formation from released ATP, in addition to its antiaggregatory properties. A plasma nucleotidase hydrolyzes ATP directly to AMP. This prevents ADP accumulation and generates adenosine, a potent, locally acting inhibitor of platelet reactivity. The presence of both endothelial CD39 and plasma nucleotidase appears to be important in the maintenance of normal hemostasis and prevention of excessive platelet responsiveness. (J Lab Clin Med 2002;140:166-75)

Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase

1. Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 microM) promoted a cell number-related inhibition of platelet aggregation induced by thrombin (40 mu ml-1). This inhibition was not attributable to products of the cyclo-oxygenase pathway for the SMC were also treated with indomethacin (10 microM). 2. The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 micrograms ml-1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 micrograms ml-1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non-treated cells. 3. The inhibition of platelet aggregation obtained with non-treated or LPS-treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml-1) and ablated by oxyhaemoglobin (OxyHb, 10 microM). Preincubation of the SMC with NG-monomethyl-L-arginine (L-NMMA, 30-300 microM) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with L-arginine (L-Arg, 100 microM) but not with D-arginine (D-Arg, 100 microM). 4. Exposure of the non-treated SMC (5 x 10(5) cells) to stirring (1000 r.p.m., 37 degrees C) for 10 min led to a significant increase in their levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) but not adenosine 3':5'-cyclic monophosphate (cyclic AMP). L-NMMA (300 microM) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells.5. These findings support the idea that non-treated or LPS-treated cultured SMC can produce an NO-like factor. Production by the latter requires protein synthesis as evidenced by blockade with cycloheximide. This NO-like factor may play a role in the auto-regulation of smooth muscle cell reactivity through a cyclic GMP-dependent mechanism.

Ouabain affects platelet reactivity as measured in vitro

Ouabain, a digitalis glycoside and an inhibitor of the Mg 2+ -dependent Na +-K + ATPase was used to probe the role of intracellular Na + levels in the regulation of platelet reactivity. Platelets preincubated with 10 to 150 uM ouabain exhibited a potentiated aggregation response to collagen (14.4 to 180 μg/mL), ADP (4 to 12 μM) and thrombin (0.03 to 0.10 unit/mL). Ouabain markedly decreased the time interval between addition of collagen and the onset of shape change. At submaximal concentrations of collagen, thrombin and ADP, preincubation with ouabain increased the rate and amplitude of the aggregation response. Irreversible aggregation was achieved in ouabain-treated platelets by using concentrations of ADP which induced only reversible aggregation in the absence of ouabain. In addition, chelation of extracellular calcium with EGTA or EDTA (2 mM) failed to block reactivity to collagen, ADP or thrombin in ouabain-treated platelets. These results suggest that ouabain induces a “preactivation state” in platelets, perhaps via modulation of intra-cellular Na + levels.