Identification of a Distinct Platelet Phenotype in the Elderly: ADP Hypersensitivity Coexists With Platelet PAR (Protease-Activated Receptor)-1 and PAR-4-Mediated Thrombin Resistance.

BACKGROUND: Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. METHODS: We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. RESULTS: The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1ß/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). CONCLUSIONS: Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.

Colchicine as a Modulator of Platelet Function: A Systematic Review.

The microtubule inhibitor and anti-inflammatory agent colchicine is used to treat a range of conditions involving inflammasome activation in monocytes and neutrophils, and is now known to prevent coronary and cerebrovascular events. In vitro studies dating back more than 50 years showed a direct effect of colchicine on platelets, but as little contemporary attention has been paid to this area, we have critically reviewed the effects of colchicine on diverse aspects of platelet biology in vitro and in vivo. In this systematic review we searched Embase, Medline, and PubMed for articles testing platelets after incubation with colchicine and/or reporting a clinical effect of colchicine treatment on platelet function, including only papers available in English and excluding reviews and conference abstracts. We identified 98 relevant articles and grouped their findings based on the type of study and platelet function test. In vitro, colchicine inhibits traditional platelet functions, including aggregation, clotting, degranulation, and platelet-derived extracellular vesicle formation, although many of these effects were reported at apparently supraphysiological concentrations. Physiological concentrations of colchicine inhibit collagen- and calcium ionophore-induced platelet aggregation and internal signaling. There have been limited studies of in vivo effects on platelets. The colchicine-platelet interaction has the potential to contribute to colchicine-mediated reduction in cardiovascular events, but there is a pressing need for high quality clinical research in this area.

Cardiac Remote Ischaemic Preconditioning: Mechanistic and Clinical Considerations.

Brief, non-harmful ischaemic insults to an organ remote from the heart, remote ischaemic preconditioning (RIPC), has been proposed to confer protection to the heart against ischaemia-reperfusion injury. While most clinical trials of RIPC during coronary interventions (PCI) suggest benefit, recent large, multicentre trials in coronary artery bypass surgery suggest a lack of efficacy. Mechanistically, RIPC most likely promotes the release of circulating factors which modulate multiple cellular pathways in the heart, promoting cell survival. This review explores potential mechanisms underlying RIPC and includes a contemporary evaluation of clinical studies in PCI and cardiac surgery, highlighting methodological differences which may explain discrepant findings between these two clinical groups.

CD147 in cardiovascular disease and thrombosis.

Thrombotic and inflammatory pathways play a key role in coronary artery disease (CAD) development. Extracellular matrix metalloproteinase (aka CD147) is a member of the immunoglobulin superfamily that is expressed on many cell types including hematopoietic, endothelial cells, leukocytes, keratinocytes, platelets, and others. The binding partners of CD147 are numerous and diverse, and give some indication to the various roles that CD147 can play; these include homophilic interactions, integrins, cyclophilins, glycoprotein VI (GPVI), caveolin 1, and monocarboxylate transporters. Recent evidence suggests a role for CD147 in both thrombosis and inflammation, as well as involvement in CAD and cancer. In this review, we summarize the role of CD147 and its binding partners in platelets, thrombosis, and arterial disease and assess mechanistic aspects of CD147 biology.

Pathologic shear triggers shedding of vascular receptors: a novel mechanism for down-regulation of platelet glycoprotein VI in stenosed coronary vessels.

Ligand-induced ectodomain shedding of glycoprotein VI (GPVI) is a metalloproteinase-dependent event. We examined whether shear force, in the absence of GPVI ligand, was sufficient to induce shedding of GPVI. Human-citrated platelet-rich plasma or washed platelets were subjected to increasing shear rates in a cone-plate viscometer, and levels of intact and cleaved GPVI were examined by Western blot and ELISA. Pathophysiologic shear rates (3000-10 000 seconds(-1)) induced platelet aggregation and metalloproteinase-dependent appearance of soluble GPVI ectodomain, and GPVI platelet remnant. Shedding of GPVI continued after transient exposure to shear. Blockade of α(IIb)ß(3), GPIbα, or intracellular signaling inhibited shear-induced platelet aggregation but minimally affected shear-induced shedding of GPVI. Shear-induced GPVI shedding also occurred in platelet-rich plasma or washed platelets isolated from a von Willebrand disease type 3 patient with no detectable VWF, implying that shear-induced activation of platelet metalloproteinases can occur in the absence of GPVI and GPIbα ligands. Significantly elevated levels of sGPVI were observed in 10 patients with stable angina pectoris, with well-defined single vessel coronary artery disease and mean intracoronary shear estimates at 2935 seconds(-1) (peak shear, 19 224 seconds(-1)). Loss of GPVI in platelets exposed to shear has potential implications for the stability of a forming thrombus at arterial shear rates.

Intravascular blood sampling using the Thrombuster II catheter does not cause artifactual platelet activation.

Platelets and leukocytes play an important role in atherosclerotic plaque progression. Determining the activation state of both the platelet and leukocyte population at the lesion site has become increasingly of interest. A novel thrombus aspiration catheter, the Thrombuster II (Kaneka Medical Products, Osaka, Japan), has recently been introduced into clinical practice, and is finding rapidly increasing use. This catheter is capable of local blood collection within the coronary tree, but to our knowledge has not previously been validated to demonstrate lack of platelet activation. This study therefore aims to establish whether or not blood sampling via the Thrombuster II results in artefactual platelet activation. Duplicate blood samples were obtained from the descending aorta using both the Thrombuster II and a femoral arterial sheath (control). The samples were collected into CTAD (to minimize ex vivo activation) blood collection tubes and processed for flow cytometry analysis. Platelet activation was assessed based on the expression of CD62P, PAC-1 binding and platelet CD147 expression. Platelet-leukocyte aggregates were also examined. No significant difference was noted between the sheath samples and the Thrombuster II catheter. The Thrombuster II catheter is a novel thrombus aspiration device capable of providing the assessment of platelet activation and platelet-leukocyte aggregates in coronary arteries.

Intracoronary shear-related up-regulation of platelet P-selectin and platelet-monocyte aggregation despite the use of aspirin and clopidogrel.

Recent in vitro studies have shown that shear stress can cause platelet activation by agonist-independent pathways. However, no studies have assessed the extent of shear-induced platelet activation within human coronary arteries. We sampled blood from the coronary arteries proximal and distal to coronary lesions and from the coronary sinus in humans with stable coronary disease who were taking both aspirin and clopidogrel. A novel, computationally based technique for estimating shear stress from 3-dimensional coronary angiographic images of these arteries was developed, and the effect of stenosis severity and calculated shear stress on in vivo platelet and related leukocyte activation pathways were determined. We provide evidence of intracoronary up-regulation of platelet P-selectin, platelet-monocyte aggregation, and monocyte CD11b without platelet glycoprotein IIb-IIIa activation or soluble P-selectin up-regulation. This correlates with intracoronary stenosis severity and calculated shear stress and occurs despite the concurrent use of aspirin and clopidogrel. Our results show for the first time shear-related platelet and monocyte activation in human coronary arteries and suggest this as a potential therapeutic target that is resistant to conventional antiplatelet agents.

Perspective: Collagen induced platelet activation via the GPVI receptor as a primary target of colchicine in cardiovascular disease.

Colchicine has been demonstrated to reduce cardiovascular death, myocardial infarction (MI), ischemic stroke, and ischemia-driven coronary revascularization in people with coronary artery disease (CAD). These reductions were observed even in patients already taking antiplatelet therapy. As well as having anti-inflammatory effects, colchicine demonstrates antiplatelet effects. We propose that colchicine's antiplatelet effects primarily target collagen-induced platelet activation via the collagen receptor, glycoprotein (GP)VI, which is critical for arterial thrombosis formation. In settings such as stroke and MI, GPVI signaling is upregulated. We have demonstrated in vitro that therapeutic concentrations of colchicine lead to a decrease in collagen-induced platelet aggregation and alter GPVI signaling. Clinical studies of colchicine given for 6 months lead to a significant reduction in serum GPVI levels in CAD patients, which may ameliorate thrombotic risk. Future evaluation of the effects of colchicine in clinical trials should include assessment of its effects on collagen-mediated platelet activation, and consideration be given to quantifying the contribution of such antiplatelet effects additional to the known anti-inflammatory effects of colchicine.

Rapid Release of Interleukin-1ß from Human Platelets Is Independent of NLRP3 and Caspase.

OBJECTIVE: Platelets are critical in mediating both rapid responses to injury and the development and progression of coronary disease. Several studies have shown that, after prolonged exposure to agonists, they produce and release inflammatory mediators including interleukin-1ß (IL-1ß), via the classical pathway (NLRP3 inflammasome and caspase-1 cleavage to release active IL-1ß) as described for leukocytes. This study aimed to determine whether there is rapid release of IL-1ß in response to soluble platelet agonists and whether such rapid release is NLRP3- and caspase-1-dependent. METHODS AND RESULTS: Using flow cytometry to detect platelet activation (and release of α and dense granule contents) and the combination of Western blotting, enzyme-linked-immunosorbent assay, and immunogold labeling transmission electron and immunofluorescence microscopy, we identified that resting human platelets contain mature IL-1ß. Platelets release IL-1ß within minutes in response to adenosine diphosphate (ADP), collagen, and thrombin receptor agonists, but not in response to conventional NLRP3 inflammasome agonists-lipopolysaccharide and adenosine triphosphate. The rapid release of IL-1ß in response to ADP and thrombin receptor agonists was independent of caspases (including caspase-1) and NLRP3. Immature and mature IL-1ß were identified as low-abundance proteins on transmission electron microscopy of human platelets, and were localized to the platelet cytosol, open canalicular system, and the periphery of α granules. CONCLUSION: Unlike monocytes and neutrophils, human platelets are capable of rapid agonist- and time-dependent release of IL-1ß by a mechanism which is independent of caspase-1 and NLRP3.

Circulating platelet-derived extracellular vesicles are decreased after remote ischemic preconditioning in patients with coronary disease: A randomized controlled trial.

BACKGROUND: Brief nonharmful ischemia, remote ischemic preconditioning (RIPC) has been proposed to confer benefit to patients with coronary artery disease via unknown mechanisms. OBJECTIVES: We aimed to investigate the effect of RIPC on circulating levels of extracellular vesicles (EVs) and global coagulation and fibrinolytic factors in patients with coronary disease. PATIENTS/METHODS: Blood samples were taken from 60 patients presenting for coronary angiography enrolled in a randomized, controlled trial before and after RIPC (3 × 5 min administration of 200 mmHg sphygmomanometer on the arm, n = 31) or sham (n = 29) treatment. Most patients (n = 48) had significant coronary artery disease and all were taking at least one antiplatelet agent. RESULTS: Remote ischemic preconditioning significantly decreased circulating levels of EVs expressing platelet markers CD41 and CD61 detected by flow cytometry in plasma, whereas no such effect was found on EVs expressing phosphatidylserine, CD62P, CD45, CD11b, CD144, CD31+ /CD41- , or CD235a. RIPC had no effect on the overall hemostatic potential assay or circulating antigen levels of tissue plasminogen activator, urokinase, plasminogen activator inhibitor-1, or plasminogen. Sham treatment had no effect on any studied parameter. Statin use inhibited the effect of RIPC on CD61+ EVs, diabetes modified the effect of RIPC on CD45+ and CD11b+ EVs, and hypertension modified the effect of RIPC on CD235a+ EVs. CONCLUSIONS: Remote ischemic preconditioning decreased circulating levels of platelet-derived EVs in patients with coronary disease taking conventional antiplatelet therapy. This may reflect increased EV clearance/uptake or change in production. Clinical variables may alter the effectiveness of RIPC.

Remote ischemic preconditioning inhibits platelet αIIb ß3 activation in coronary artery disease patients receiving dual antiplatelet therapy: A randomized trial.

OBJECTIVES: We investigated whether remote ischemic preconditioning (RIPC) inhibits agonist-induced conformational activation of platelet αIIb ß3 in patients with coronary artery disease already receiving conventional antiplatelet therapy. PATIENTS/METHODS: Consecutive patients with angiographically confirmed coronary artery disease were randomized to RIPC or sham treatment. Venous blood was collected before and immediately after RIPC/sham. Platelet aggregometry (ADP, arachidonic acid) and whole blood platelet flow cytometry was performed for CD62P, CD63, active αIIb ß3 (PAC-1 binding) before and after stimulation with ADP, thrombin ± collagen, or PAR-1 thrombin receptor agonist. RESULTS: Patients (25 RIPC, 23 sham) were well matched, 83% male, age (mean ± standard deviation) 63.3 ± 13.2 years, 95% aspirin, 81% P2Y12 inhibitor. RIPC did not affect platelet aggregation, nor agonist-induced expression of CD62P, but selectively and significantly decreased αIIb ß3 activation after stimulation with either PAR-1 agonist peptide or the combination of thrombin + collagen, but not after ADP nor thrombin alone. The effect of RIPC on platelet αIIb ß3 activation was evident in patients receiving both aspirin and P2Y12 inhibitor, and was not associated with an increase in vasodilator-stimulated phosphoprotein phosphorylation. CONCLUSIONS: Remote ischemic preconditioning inhibits conformational activation of platelet αIIb ß3 in response to exposure to thrombin and collagen in patients with coronary artery disease receiving dual antiplatelet therapy. These findings indicate agonist-specific inhibition of platelet activation by RIPC in coronary artery disease that is not obviated by the prior use of P2Y12 inhibitors.

Procoagulant Effects of Low-Level Platelet Activation and Its Inhibition by Colchicine.

Platelets play an important role in diseases such as cardiovascular disease and cancer, especially through their release of extracellular vesicles (EVs) and role in thrombosis. The effects of the anti-inflammatory drug colchicine on platelets are not well understood. We investigated the effect of colchicine on the release of pro-coagulant EVs from platelets under low-level activation. Citrated platelet-rich plasma (PRP) from healthy donors was incubated with 2 mM colchicine or vehicle at 37°C for 30 minutes with gentle rotation. The incubation conditions caused mild platelet activation (expression of CD62P and increased surface lactadherin binding) and release of EVs expressing phosphatidylserine (PS, measured by binding of lactadherin), CD61 and CD62P, both of which were attenuated by colchicine. The incubation conditions shortened the delay to fibrin generation and this correlated with elevated levels of PS+/CD61+ EVs. Removal of EVs from plasma abrogated clot formation in the overall haemostatic potential (OHP) assay. Colchicine decreased levels of both PS+/CD61+ and CD62P+ EVs and abrogated the shortened delay to fibrin generation achieved by platelet activation. Similar results were observed after incubation of PRP with 200 µM vinblastine, suggesting a microtubular effect. An alternative method of platelet activation using platelet agonists 20 µM ADP or 10 µM epinephrine also increased CD62P+ EV levels, and this too was attenuated by prior incubation with colchicine. Our novel findings demonstrate procoagulant effects of low-level platelet activation and EV formation which are inhibited by colchicine.

Flow Cytometry Protocols for Assessment of Platelet Function in Whole Blood.

Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large numbers of platelets in whole blood. In the following chapter, we provide a number of flow cytometry-based protocols broadly aimed at (1) assessment of constitutively expressed platelet membrane receptors to diagnose inherited platelet bleeding disorders and (2) investigation of basal and agonist-induced platelet functional responses including generation of platelet-leukocyte aggregates, alpha and dense granule release, calcium flux, and phosphatidylserine exposure.

Detection of hypofibrinolysis in stable coronary artery disease using the overall haemostatic potential assay.

INTRODUCTION: Patients with stable coronary artery disease (CAD) are at risk of arterial thrombosis causing myocardial infarction. Detection of global haemostatic markers of hypercoagulability and hypofibrinolysis may be important for risk stratification and individualised treatment. We examined overall haemostatic potential (OHP) and thrombin generation in a group of stable CAD patients. We also sought to investigate associations between fibrinolytic inhibitors and abnormal global fibrinolysis in these patients. MATERIALS AND METHODS: Blood samples were collected from 56 patients defined by coronary anatomy as symptomatically stable CAD. Medications were recorded. Samples were analysed using the global coagulation assays OHP and thrombin generation (calibrated automated thrombogram, CAT), platelet aggregometry measured by Multiplate®, and levels of plasminogen activator inhibitor-1 (PAI-1) antigen measured by ELISA. Results were compared with a reference group of healthy controls. RESULTS: Stable CAD patients displayed increased fibrin and thrombin generation and impaired fibrinolysis (decreased overall fibrinolytic potential, OFP, and increased clot lysis time) compared with healthy controls. No effect of antiplatelet agents or other medications on these parameters was observed using platelet-poor plasma. After multivariate adjustment, OFP of healthy individuals was significantly associated with fibrinogen, but in CAD patients PAI-1 became an important determinant. CONCLUSIONS: Hypercoagulability of plasma is observed in stable CAD, with both increased thrombin generation and reduced fibrinolytic potential making a significant contribution. The OHP assay may provide a simple method of identifying hypercoagulability in individual patients.

Off-pump coronary artery bypass surgery induces prolonged alterations to host neutrophil physiology.

Persistent alteration to host polymorphonuclear cell (PMN) physiology has been demonstrated after cardiac surgery performed with cardiopulmonary bypass (CPB). However, to date, PMN physiology and function beyond the first 24 h have not been investigated after cardiac surgery performed without CPB (off-pump coronary artery bypass grafting [OPCAB]). Blood samples of 15 patients were collected preoperatively and on days 1, 3, and 5 after OPCAB. Expression of CD11b, CD18, CBRM1/5, and CD62L were assessed by flow cytometry under resting conditions and after stimulation with formyl methionyl-leucyl-phenylalanine (fMLF), and respiratory burst activity was also measured. Under resting conditions, PMN CD11b, CBRM1/5, and CD62L expressions were minimally altered by surgery. Compared with the response of preoperative PMNs, PMNs assayed on days 3 and 5 after OPCAB demonstrated a significantly blunted increase in the expression of CD11b and CBRM1/5 after fMLF, significantly diminished shedding of CD62L in response to platelet-activating factor and fMLF, and diminished superoxide production after stimulation on day 3. The alteration of PMN function after OPCAB implies that cardiac surgical trauma without CPB directly modulates host PMN physiology.

Intravascular blood sampling using the export catheter does not induce artifactual platelet activation.

We sought to investigate the feasibility of using the Export catheter to sample blood from the coronary arteries for studies of platelet activation. A pilot comparison between the Export catheter and the established Simmons catheter--currently used to sample blood from the coronary sinus--was performed. Triplicate blood samples were obtained from the descending aorta for each catheter in 10 patients, and analyzed for platelet P-selectin expression, PAC-1 expression, and platelet-leukocyte aggregate formation by flow cytometry. We found no significant increase in levels of platelet activation above that of reference values and no significant differences between the two catheters.