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.

Elastin in asthma.

Extracellular matrix is generally increased in asthma, causing thickening of the airways which may either increase or decrease airway responsiveness, depending on the mechanical requirements of the deposited matrix. However, in vitro studies have shown that the altered extracellular matrix produced by asthmatic airway smooth muscle cells is able to induce increased proliferation of non-asthmatic smooth muscle cells, which is a process believed to contribute to airway hyper-responsiveness in asthma. Elastin is an extracellular matrix protein that is altered in asthmatic airways, but there has been no systematic investigation of the functional effect of these changes. This review reveals divergent reports of the state of elastin in the airway wall in asthma. In some layers of the airway it has been described as increased, decreased and/or fragmented, or unchanged. There is also considerable evidence for an imbalance of matrix metalloproteinases, which degrade elastin, and their respective inhibitors the tissue inhibitors of metalloproteinases, which collectively help to explain observations of both increased elastin and elastin fragments. A loss of lung elastic recoil in asthma suggests a mechanical role for disordered elastin in the aetiology of the disease, but extensive studies of elastin in other tissues show that elastin fragments elicit cellular effects such as increased proliferation and inflammation. This review summarises the current understanding of the role of elastin in the asthmatic airway.

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.

Thrombin Generation and Cancer: Contributors and Consequences.

The high occurrence of cancer-associated thrombosis is associated with elevated thrombin generation. Tumour cells increase the potential for thrombin generation both directly, through the expression and release of procoagulant factors, and indirectly, through signals that activate other cell types (including platelets, leukocytes and erythrocytes). Furthermore, cancer treatments can worsen these effects. Coagulation factors, including tissue factor, and inhibitors of coagulation are altered and extracellular vesicles (EVs), which can promote and support thrombin generation, are released by tumour and other cells. Some phosphatidylserine-expressing platelet subsets and platelet-derived EVs provide the surface required for the assembly of coagulation factors essential for thrombin generation in vivo. This review will explore the causes of increased thrombin production in cancer, and the availability and utility of tests and biomarkers. Increased thrombin production not only increases blood coagulation, but also promotes tumour growth and metastasis and as a consequence, thrombin and its contributors present opportunities for treatment of cancer-associated thrombosis and cancer itself.

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.

Natural history of hypercoagulability in patients undergoing coronary revascularization and effect of preoperative myocardial infarction.

OBJECTIVES: The balance between hyper- and hypocoagulable states is critical after coronary artery surgery both with (coronary artery bypass grafting [CABG]) and without (off-pump coronary artery bypass [OPCAB]) cardiopulmonary bypass to prevent thrombotic or bleeding complications. We aimed to quantify novel parameters of coagulation, fibrinolysis, and overall hemostasis ≤6 months after CABG and OPCAB and to determine the influences on these parameters. METHODS: A total of 63 patients (30 CABG, 33 OPCAB) had blood collected before and at various points ≤6 months after surgery. Fibrin and fibrinolysis time curves were generated by measuring the absorption of 405 nm each minute for 100 minutes after the addition of tissue factor and tissue plasminogen activator to cell-free plasma. The parameters were compared with those from a group of healthy controls. RESULTS: The patients' preoperative prothrombotic assay parameters were compared with those from healthy controls. Both CABG and OPCAB patients were hypercoagulable until at least day 10 after surgery, with elevation of fibrin generation (CABG, peak day 3, +28.9%; OPCAB, peak day 1, +16.3% vs preoperative baseline) and impairment of fibrinolysis capacity (CABG, day 1, -58.4%; OPCAB, day 1, -22.6%). Surgical revascularization resulted in resolution of preoperative hypercoagulability by 6 months postoperatively. Patients with preoperative myocardial infarction (MI) had prolonged hypercoagulability after surgery that was most exaggerated after CABG (overall hemostatic potential day 5, no MI, +64.1% vs with MI, +128.9% compared with baseline; P = .013). CONCLUSIONS: Patients will be vulnerable to thrombotic events for ≤6 weeks after coronary surgery yet will have resolution of hypercoagulability by 6 months. Preoperative factors, such as MI, could require individualized management of thrombosis prophylaxis in the postoperative period.

Tropoelastin modulates TGF-ß1-induced expression of VEGF and CTGF in airway smooth muscle cells.

Elastin is predominantly comprised of crosslinked tropoelastin. For many years elastin was considered to serve a solely structural role but is now being increasingly identified as causal in cell signaling, development and repair. We introduced tropoelastin into an in vitro model in which airway smooth muscle cells (ASMCs) were stimulated with transforming growth factor (TGF)-ß1 to examine the modulatory effect of this modular elastin sequence on release of angiogenic factors and matrix metalloproteinases (MMPs). Human ASMCs were presented to surfaces coated with tropoelastin or collagen and controls, then stimulated with TGF-ß1. Transcript levels of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) were quantified 4 and 24 h after TGF-ß1 stimulation. Protein VEGF release from cells and CTGF sequestered at cell surfaces were measured by ELISA at 24 and 48 h. TGF-ß1 increased VEGF mRNA 2.4 fold at 4 h and 5 fold at 24 h, accompanied by elevated cognate protein release 3 fold at 24 h and 2.5 fold at 48 h. TGF-ß1 stimulation increased CTGF mRNA 6.9 fold at 4 h and 11.8 fold at 24 h, accompanied by increased sequestering of its protein counterpart 1.2 fold at 24 h and 1.4 fold at 48 h. Pre-incubation of cells with tropoelastin did not modulate VEGF or CTGF mRNA expression, but combined with TGF-ß1 stimulation it led to enhanced VEGF release 5.1-fold at 24h and 4.4-fold at 48 h. Pre-incubation with tropoelastin decreased CTGF sequestering 0.6-fold at 24 and 48 h, and increased MMP-2 production. Collagen pre-incubation under the same conditions displayed no effect on TGF-ß1 stimulation apart from a slightly decreased (0.9 fold) sequestered CTGF at 48 h. As CTGF is known to anchor VEGF to the matrix and inhibit its angiogenic activity, a process which can be reversed by digestion with MMP-2, these findings reveal that elastin sequences can disrupt the balance of angiogenic factors, with implications for aberrant angiogenesis. The results suggest a model of molecular crosstalk and support an active role for elastin in vascular remodeling.

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.