Platelet-targeted pharmacologic treatments as anti-cancer therapy.

Platelets act as multifunctional cells participating in immune response, inflammation, allergy, tissue regeneration, and lymphoangiogenesis. Among the best-established aspects of a role of platelets in non-hemostatic or thrombotic disorders, there is their participation in cancer invasion and metastasis. The interaction of many different cancer cells with platelets leads to platelet activation, and on the other hand platelet activation is strongly instrumental to the pro-carcinogenic and pro-metastatic activities of platelets. It is thus obvious that over the last years a lot of interest has focused on the possible chemopreventive effect of platelet-targeted pharmacologic treatments. This article gives an overview of the platelet-targeted pharmacologic approaches that have been attempted in the prevention of cancer development, progression, and metastasis, including the application of anti-platelet drugs currently used for cardiovascular disease and of new and novel pharmacologic strategies. Despite the fact that very promising results have been obtained with some of these approaches in pre-clinical models, with the exclusion of aspirin, clinical evidence of a beneficial effect of anti-platelet agents in cancer is however still largely missing. Future studies with platelet-targeted drugs in cancer must carefully deal with design issues, and in particular with the careful selection of patients, and/or explore novel platelet targets in order to provide a solution to the critical issue of the risk/benefit profile of long-term anti-platelet therapy in the prevention of cancer progression and dissemination.

Major bleeding in patients undergoing PCI and triple or dual antithrombotic therapy: a parallel-cohort study.

The combination of oral anticoagulants with dual antiplatelet therapy (DAT) in patients undergoing percutaneous coronary intervention with stent implantation (PCI-stenting) is subject to controversy due to the high risk of bleeding. In this multicenter retrospective parallel-group study, we compared the rate of adverse events in chronically anticoagulated patients who underwent PCI-stenting and were discharged on aspirin, clopidogrel and warfarin (triple antithrombotic therapy [TT] group) and were followed in Italian anticoagulation centers, with a parallel cohort of patients who underwent PCI-stenting and were discharged on DAT group. The primary endpoint was the incidence of major bleeding while the patients were in TT and DAT. A secondary endpoint was the occurrence of major ischemic adverse events (MACEs). The final cohort consisted of 229 TT patients and 231 DAT patients followed up for 6 and 7 months, respectively. There were 11 (4.8%; 9.1% patient/years) major bleeding events in the TT group (1 was fatal) as compared to 1 (0.4%; 0.7% patient/years) event in the DAT group (p = 0.003). Of the 28 (6.1%) MACE recorded during the follow-up, 12 (5.2%) occurred in the TT group and 16 (6.9%) in the DAT group. In conclusion, despite close monitoring of anticoagulated patients in dedicated centers, the major bleeding incidence remains high among unselected patients undergoing PCI-stenting and treated with TT. Any efforts to minimize these events should be pursued.

High-on-treatment platelet reactivity predicts adverse outcome after carotid artery stenting: A prospective study.

BACKGROUND AND PURPOSE: High-on-treatment platelet reactivity (HTPR) has been established as a predictor of major adverse cardiovascular events (MACE) in patients undergoing percutaneous coronary interventions on dual antiplatelet therapy (DAPT), but no data are available on its predictive value in patients on DAPT after carotid artery stenting (CAS). We aimed to evaluate the possible association between HTPR in patients on aspirin plus clopidogrel therapy after CAS and subsequent MACE. METHODS: All consecutive patients treated with CAS in a single institution were enrolled in a prospective clinical study. HTPR was evaluated with 5 different laboratory assays carried out just before CAS. MACE incidence (cerebral ischemia, myocardial infarction, stent thrombosis, acute limb ischemia and vascular death) was evaluated at 30 days and thereafter at yearly visits. RESULTS: A total of 300 patients were enrolled in the study, and eight were then excluded because blood samples resulted unsuitable for the laboratory testing or CAS aborted for technical problems. Median follow-up was 5.8 years and during this period 47 MACE occurred. HTPR detected by multiplate electronic aggregometry (MEA) and the VASP phosphorylation assay (VASP) were associated with a significantly enhanced risk of MACE (p = 0.048 and p = 0.038, respectively). However, HTPR to three tests (HTPR3) was more strongly predictive of increased risk of a vascular event at follow up (p = 0.005) at bivariate analysis and also at Cox regression multivariate analysis (p = 0.002). CONCLUSIONS: HTPR to three different assays (mainly to VASP + PFA P2Y+ VerifyNow) in patients on DAPT after CAS has predictive value for subsequent MACE. Prospective studies to assess whether platelet function testing-guided antiplatelet therapy is superior to standard DAPT in patient undergoing CAS should be considered.

In vitro effect of anti-ß2 glycoprotein I antibodies on P-selectin expression, a marker of platelet activation.

OBJECTIVE: Antiphospholipid antibodies (aPL) associated with thrombembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS). Beta2glycoprotein I (ß2GPI) represents the major target antigen for aPL, but the pathogenic role of anti-ß2GPI antibodies (aß2GPI) is still unclear. Some authors assume they play a role in activating platelets. The effects of aß2GPI antibodies on platelet P-selectin expression were evaluated in this study. METHODS: Aß2GPI antibodies in the plasma of a pregnant APS patient were isolated by affinity chromatography during two different stages (catastrophic and quiescent) of the disease. Gel filtered platelets (100,000/µl) from healthy volunteers were incubated with ß2-GPI (20 µg/ml) and with different concentrations (5, 25 e 50 µg/ml) of aß2GPI antibodies. P-selectin surface expression on platelets was assessed by flow cytometry using a specific fluorescent antibody directed against P-selectin. RESULTS: Aß2GPI antibodies induced platelet activation only in the presence of thrombin receptor activator for peptide 6 (TRAP-6), a platelet agonist, at a subthreshold concentration. Aß2GPI antibody enhancement on platelet surface P-selectin expression was stronger in the catastrophic than in the quiescent phase of the disease (47% versus 15%). CONCLUSIONS: TRAP-6 dependent platelet activation by aß2GPI antibodies is consistent with the "two hit" pathogenetic hypothesis for thrombosis. Aß2GPI antibodies induce higher platelet P-selectin expression during the active rather than in the acute phases.

The role of platelets, neutrophils and endothelium in COVID-19 infection.

INTRODUCTION: COVID-19 is associated to an increased risk of thrombosis, as a result of a complex process that involves the activation of vascular and circulating cells, the release of soluble inflammatory and thrombotic mediators and blood clotting activation. AREAS COVERED: This article reviews the pathophysiological role of platelets, neutrophils, and the endothelium, and of their interactions, in the thrombotic complications of COVID-19 patients, and the current and future therapeutic approaches targeting these cell types. EXPERT OPINION: Virus-induced platelet, neutrophil, and endothelial cell changes are crucial triggers of the thrombotic complications and of the adverse evolution of COVID-19. Both the direct interaction with the virus and the associated cytokine storm concur to trigger cell activation in a classical thromboinflammatory vicious circle. Although heparin has proven to be an effective prophylactic and therapeutic weapon for the prevention and treatment of COVID-19-associated thrombosis, it acts downstream of the cascade of events triggered by SARS-CoV-2. The identification of specific molecular targets interrupting the thromboinflammatory cascade upstream, and more specifically acting either on the interaction of SARS-CoV-2 with blood and vascular cells or on the specific signaling mechanisms associated with their COVID-19-associated activation, might theoretically offer greater protection with potentially lesser side effects.

Incomplete inhibition of platelet function as assessed by the platelet function analyzer (PFA-100) identifies a subset of cardiovascular patients with high residual platelet response while on aspirin.

Sixty-six patients with a history of ischemic events (myocardial infarction, unstable angina, or stroke) on chronic aspirin therapy were studied by different platelet function tests: 37 patients had suffered a recurrent event while on aspirin and 29 were without recurrences. Based on results from light transmission aggregometry (LTA) induced by arachidonic acid (AA) and serum TxB(2) both COX-1-dependent methods, only one patient could be identified as aspirin "resistant". However, when methods only partially-dependent on platelet COX-1 activity were considered, the prevalence of aspirin non-responders ranged, according to the different tests, from 0 to 52%. No difference was observed between patients with recurrences and those without. Among patients with recurrent events, those with an incomplete inhibition of platelet function, as assessed by the PFA-100, had significantly higher residual serum TxB(2) (2.4 ± 2.4 ng/mL vs 0.4 ± 0.1 ng/mL, p = 0.03), residual LTA-AA (9.2 ± 10.6% vs 2.0 ± 1.6%, p = 0.008), LTA-Coll (49.3 ± 14.6% vs 10.2 ± 8.3%, p = 0.007) and LTA-ADP (50.9 ± 16.2% vs 34.3 ± 11.0%, p = 0.04). In conclusion, laboratory tests solely exploring the AA-mediated pathway of platelet function, while being the most appropriate to detect the effect of aspirin on its pharmacologic target (platelet COX-1), may fail to reveal the functional interactions between minimal residual TxA(2) and additional stimuli or primers potentially leading to aspirin-insensitive platelet aggregation. High residual platelet response in platelet function tests only partially dependent on COX-1 may reveal a condition of persistent platelet reactivity in a subset of aspirin-treated patients characterizing them as a subgroup at higher vascular risk.

Platelets and Matrix Metalloproteinases: A Bidirectional Interaction with Multiple Pathophysiologic Implications.

Platelets contain and release several matrix metalloproteinases (MMPs), a highly conserved protein family with multiple functions in organism defense and repair. Platelet-released MMPs as well as MMPs generated by other cells within the cardiovascular system modulate platelet function in health and disease. In particular, a normal hemostatic platelet response to vessel wall injury may be transformed into pathological thrombus formation by platelet-released and/or by locally generated MMPs. However, it is becoming increasingly clear that platelets play a role not only in hemostasis but also in immune response, inflammation and allergy, atherosclerosis, and cancer development, and MMPs seem to contribute importantly to this role. A deeper understanding of these mechanisms may open the way to novel therapeutic approaches to the inhibition of their pathogenic effects and lead to significant advances in the treatment of cardiovascular, inflammatory, and neoplastic disorders.

Peripheral arterial disease has a strong impact on cardiovascular outcome in patients with acute coronary syndromes: from the START Antiplatelet registry.

BACKGROUND: Peripheral arterial disease (PAD) was reported to increase the risk of new cardiovascular events in patients with acute coronary syndromes (ACS). However, most of the evidence comes from randomized clinical trials. We aimed to assess the impact of PAD on cardiovascular outcome and treatment decisions in ACS patients in a current real-life setting. METHODS: START-ANTIPLATELET is a multicenter registry enrolling ACS patient. Baseline clinical characteristics and treatment at discharge were recorded and follow-up was repeated at 6-months and 1-year. PAD was defined as intermittent claudication and/or previous revascularization. RESULTS: Among 1442 patients enrolled, 103 (7.1%) had PAD. PAD patients were older (71.8 ± 10.6vs66.2 ± 12.6 yrs., p < 0.0001), more frequently hypertensive (90.3vs68.6%, p< 0.0001), hypercholesterolemic (66vs52%, p= 0.037), diabetic (51.5vs24%, p= 0.0001), obese (28.2vs19.3%, p= 0.029) and with previous TIA (7.8vs2.8%, p= 0.005) or stroke (11.7vs3.1%, p< 0.0001). Clinical presentation and acute treatment were similar in non-PAD and PAD patients, but the latter were discharged significantly less frequently on dual antiplatelet therapy (DAPT) (68.9vs85%, p= 0.005). After a median follow-up time of 11.1 months, major cardio/cerebrovascular event-free survival [MACCE, including cardiovascular death, MI, TIA and stroke, target-vessel revascularization (TVR) and major arterial ischemic events] was significantly shorter (9.0vs11.2 months, p= 0.02; HR 3.2, 2.4-8.4) in PAD patients and net adverse cardiovascular events (NACE = MACCE plus major hemorrhages) were significantly more frequent (19.1%vs10.5%, p = 0.049). CONCLUSIONS: PAD identifies a subgroup of ACS patients at significantly increased cardiovascular risk, but these patients tend to be undertreated. Patients admitted for ACS should be screened for PAD and optimal medical therapy at discharge should be implemented.

Patients with primary antiphospholipid antibody syndrome and without associated vascular risk factors present a normal endothelial function.

INTRODUCTION: Primary antiphospholipid antibody syndrome (PAPS) is characterized by venous or arterial thrombosis and positive antiphospholipid antibodies. It is controversial whether PAPS patients have early atherosclerosis. Endothelial dysfunction is an early event in the natural history of atherosclerosis. Aim of our study was to compare endothelial function of patients with PAPS and no associated risk factors with that of age- and sex-matched controls. MATERIALS AND METHODS: Patients with PAPS, carefully selected to exclude all known risk factors for cardiovascular diseases, estrogen therapy, pregnancy, intake of drugs affecting endothelial function, vitamins or antioxidants, were included in a case-control study. Controls were age- (+/-5 years) and sex-matched subjects with the same exclusion criteria but without PAPS. Flow-mediated dilation of the brachial artery and some plasmatic markers of endothelial and platelet activation were measured. Measures are expressed as mean+/-SEM. RESULTS: Twenty cases (mean age 42+/-4.0 years, 11 females) and 39 controls (mean age 41+/-2.9, 22 females) were studied. FMD was 5.7+/-0.8% in cases (95% CI: 4.1 to 7.3) and 6.8+/-0.5% (5.7 to 7.9) in controls (p=NS). Plasma von Willebrand factor was 128+/-11.3% and 134.2+/-16.1% in cases and controls, respectively (p=NS). Soluble P-selectin and soluble CD40L were 94.1+/-4.9 ng/ml and 0.7+/-0.1 ng/ml in cases and 87.7+/-4.0 ng/ml and 1.0+/-0.2 in controls, respectively (p=NS). In a substudy, circulating progenitor and mature endothelial cells were comparable between the two groups. CONCLUSIONS: Endothelial function in patients with PAPS and no associated risk factors is similar to that of age- and sex- matched controls. These data suggest that the alterations leading to thrombosis in PAPS concern primarily the clotting system.

Nitric oxide-enhancing or -releasing agents as antithrombotic drugs.

Nitric oxide (NO) is a powerful biological mediator provided with a number of activities of relevance for the prevention of thrombosis, like vasodilation, inhibition of platelet adhesion and aggregation, prevention of smooth muscle cell proliferation. Several cells in the circulation release NO, like endothelial cells which are the largest source, red blood cells, platelets and white blood cells, and conditions associated with an impaired production or bioavailability of NO predispose to arterial and venous thrombosis. It seems thus logical to use NO as an antithrombotic agent. However, given the extremely short half-life, limited water solubility and radical nature of this mediator, several chemical strategies to generate drugs releasing NO and/or favouring its endogenous production/bioavailability have been developed. Here we review the pharmacologic approaches to enhance endogenous NO or to induce NO-release developed over the last decades for their effects on platelet activation in vitro and in vivo and on thrombosis, in animal models and in humans. One limitation to the development of NO-releasing agents as antithrombotic drugs is represented by their concomitant vasodilatory action which, by inducing hypotension, limits their applicability. Further pharmacologic and clinical research of novel NO-enhancing and/or -releasing molecules is highly warranted in order to fully exploit the great antithrombotic potential of NO.

Laboratory diagnosis of clinically relevant platelet function disorders.

Inherited platelet function disorders (IPFDs) represent a significant fraction of congenital hemorrhagic disorders, and may be associated with bleeding of considerable severity. IPFDs may be difficult to diagnose and a preliminary accurate clinical examination and an objective evaluation of the severity of the bleeding history are mandatory. The laboratory investigation of IPFDs should follow a rational algorithm based on a streamlined panel of laboratory tests with subsequent steps of increasing levels of complexity. First screening tests include platelet count, peripheral blood smear, light transmission aggregometry, measurement of platelet granule content and release, and the expression of glycoproteins by flow cytometry. Several of these tests have been largely employed, and a few validated, for the diagnosis of IPFDs and some recent developments are discussed. Point-of-care tests may provide the advantage of rapidity and the possibility to study platelet function in whole blood, but further studies are required to clarify their potential diagnostic application. Genotyping is recommended for some conditions (genotype/phenotype correlations, forms associated with a high risk of developing hematologic malignancies) but, especially when carried out by next-generation sequencing (NGS) techniques, needs to be critically evaluated taking into account clinical and laboratory phenotypes.

Role of proaggregatory and antiaggregatory prostaglandins in hemostasis. Studies with combined thromboxane synthase inhibition and thromboxane receptor antagonism.

Thromboxane synthase inhibition can lead to two opposing effects: accumulation of proaggregatory cyclic endoperoxides and increased formation of antiaggregatory PGI2 and PGD2. The elimination of the effects of the cyclic endoperoxides by an endoperoxide-thromboxane A2 receptor antagonist should enhance the inhibition of hemostasis by thromboxane synthase blockers. We have carried out a series of double-blind, placebo-controlled, crossover studies in healthy volunteers to check if this hypothesis may be operative in vivo in man. In a first study, in 10 healthy male volunteers, the combined administration of the thromboxane receptor antagonist BM 13.177 and the thromboxane synthase inhibitor dazoxiben gave stronger inhibition of platelet aggregation and prolonged the bleeding time more than either drug alone. In a second study, in 10 different healthy male volunteers, complete inhibition of cyclooxygenase with indomethacin reduced the prolongation of the bleeding time by the combination BM 13.177 plus dazoxiben. In a third study, in five volunteers, selective cumulative inhibition of platelet TXA2 synthesis by low-dose aspirin inhibited platelet aggregation and prolonged the bleeding time less than the combination BM 13.177 plus dazoxiben. In vitro, in human platelet-rich plasma stimulated with arachidonic acid, the combination of BM 13.177 and dazoxiben increased intraplatelet cAMP while the single drugs did not affect it. Our results indicate that prostaglandin endoperoxides can partly substitute for the activity of TXA2 in vivo in man and that an increased formation of endogenous antiaggregatory and vasodilatory prostaglandins, as obtained with selective thromboxane synthase inhibitors, may contribute to the impairment of hemostasis.

Activated human protein C prevents thrombin-induced thromboembolism in mice. Evidence that activated protein c reduces intravascular fibrin accumulation through the inhibition of additional thrombin generation.

Activated protein C (APC) is a potent physiologic anticoagulant with profibrinolytic properties, and has been shown to prevent thrombosis in different experimental models. We investigated the effect of human APC on thrombin-induced thromboembolism in mice, a model of acute intravascular fibrin deposition leading to death within minutes. APC given intravenously (i.v.) as a bolus 2 min before thrombin challenge (1,250 U/kg) reduced mortality in a dose-dependent manner despite the lack of thrombin inhibitor activity. Significant inhibition of thrombin-induced death was observed at the dose of 0.05 mg/kg, and maximal protection was obtained with 2 mg/kg (> 85% reduction in mortality rate). Histology of lung tissue revealed that APC treatment (2 mg/kg) reduced significantly vascular occlusion rate (from 89.2 to 46.6%, P < 0.01). The protective effect of APC was due to the inhibition of endogenous thrombin formation as indicated by the fact that (a) the injection of human thrombin caused a marked decrease in the coagulation factors of the intrinsic and common pathways (but not of Factor VII), suggesting the activation of blood clotting via the contact system; (b) APC pretreatment reduced markedly prothrombin consumption; (c) the lethal effect of thrombin was almost abolished when the animals were made deficient in vitamin K-dependent factors by warfarin treatment, and could be restored only by doubling the dose of thrombin, indicating that the generation of endogenous thrombin contributes significantly to death; and (d) APC failed to protect warfarin-treated animals, in which mortality is entirely due to injected thrombin, even after protein S supplementation. Other results suggest that APC protects from thrombin-induced thromboembolism by rendering the formed fibrin more susceptible to plasmin degradation rather than by reducing fibrin formation: in thrombin-treated mice, fibrinogen consumption was not inhibited by APC; and inhibition of endogenous fibrinolysis by epsilon-aminocaproic or tranexamic acid resulted in a significant reduction of the protective effect of APC. Since APC did not enhance plasma fibrinolytic activity, as assessed by the measurement of plasminogen activator (PA) or PA inhibitor (PAI) activities, PAI-1 antigen, or 125I-fibrin degrading activity, we speculate that the inhibition of additional (endogenous) thrombin formation by APC interrupts thrombin-dependent mechanisms that make fibrin clots more resistant to lysis, so that the intravascular deposited fibrin can be removed more rapidly by the endogenous fibrinolytic system.

Survey of lupus anticoagulant diagnosis by central evaluation of positive plasma samples.

OBJECTIVE: To determine whether the diagnosis of lupus anticoagulant (LAC) in a large cohort of positive patients was confirmed at a reference laboratory. METHODS: Over a 1-year period, each participating center collected samples from LAC-positive patients. Plasma was filtered and kept deep-frozen until it was sent on dry ice to the reference laboratory by express courier. Centers returned detailed laboratory information and clinical data from each patient. The reference laboratory screened plasma samples by diluted Russell viper venom time (dRVVT) and kaolin clotting time (KCT). When these were prolonged, 1:1 mixing studies were carried out, and confirmatory tests were performed as appropriate. Positive samples were further tested by thrombin time (TT). The presence of heparin was checked by measuring antifactor Xa activity when TT was prolonged. Negative samples were tested by activated partial thromboplastin time using hexagonal phospholipids. RESULTS: Plasma samples from 302 patients from 29 anticoagulation clinics were analyzed. LAC was excluded in 71 samples (24%), because dRVVT and KCT screening test results were normal (34) or reversed to normal by mixing studies (35). The remaining two samples were considered negative because they contained heparin. LAC-negative patients showed different characteristics from those in whom diagnosis was confirmed. They were significantly older (49.7 vs. 45.0 years, P < 0.03), were more often first diagnosed (66% vs. 41%, P < 0.001), and were more frequently judged as mild in LAC potency (60% vs. 25%, P < 0.0001). Moreover, anticardiolipin and anti-beta(2)-glycoprotein I antibody values were more often normal in LAC-negative (82%) than in LAC-positive (42%) samples (P < 0.0001). LAC-positive samples identified by both dRVVT and KCT (146/231, 63%) showed a LAC potency that was significantly stronger than that in samples in which LAC diagnosis was made by a single test. CONCLUSIONS: A false-positive LAC diagnosis is not uncommon across specialized centers. Patients' characteristics and a complete antiphospholipid antibody profile may help to identify these individuals.

Activation of phospholipase A2 and beta-thromboglobulin release in human platelets: comparative effects of thrombin and fluoroaluminate stimulation.

Several reports have suggested that the activity of platelet phospholipase A2 is modulated by GTP-binding protein(s) whose nature and properties need to be defined. Fluoroaluminate is known to activate G-proteins and this leads to a number of cellular responses including the activation of phospholipases. This paper demonstrates that human platelets, prelabelled with [3H]arachidonic acid, produce free arachidonic acid when stimulated with fluoroaluminate and this effect is time- and dose-dependent. The production of arachidonic acid is not inhibited by neomycin, a PI-cycle inhibitor, but is completely abolished by mepacrine, an inhibitor of both phospholipase A2 and C. At low concentration of fluoroaluminate (10 mM NaF) phospholipase A2 but not phospholipase C is activated. In addition, fluoroaluminate treatment releases beta-thromboglobulin (beta-TG) and this effect is not inhibited by acetylsalicylic acid. Under identical conditions both neomycin and mepacrine suppress the release of arachidonic acid and beta-TG induced by thrombin. Sodium nitroprusside, which increases cGMP levels in platelets, inhibits arachidonic acid liberation and beta-TG release in thrombin-stimulated platelets but has no effect in fluoroaluminate-treated platelets; cGMP was reported to suppress phospholipase C activation. These results are consistent with the hypothesis that, in thrombin-stimulated platelets, the liberation of arachidonic acid and beta-TG are strictly dependent on the activation of phospholipase C. We have also provided evidence for the existence of a phospholipase A2 activated by a G-protein which is independent from the degradation of phosphoinositides and, contrary to phospholipase C, it is not down regulated by cGMP.

Thromboxane synthase inhibitors, thromboxane receptor antagonists and dual blockers in thrombotic disorders.

Thromboxane A2 (TXA2) plays a pivotal role in platelet activation and is involved in the development of thrombosis. Thromboxane synthase inhibitors suppress TXA2 formation and increase the synthesis of the antiaggregatory prostaglandins PGI2 and PGD2; however, accumulated PGH2 may interact with the platelet and vessel wall TXA2 receptor, thus reducing the antiplatelet effects of this class of drug. TXA2 receptor antagonists block the activity of both TXA2 and PGH2 on platelets and the vessel wall. Very recently, drugs possessing both thromboxane synthase-inhibitory and thromboxane receptor-antagonist properties have been developed. Paolo Gresele and colleagues explain here why these drugs can be more efficacious than traditional antiplatelet agents and review the available experimental studies involving these drugs.

Intraplatelet signaling mechanisms of the priming effect of matrix metalloproteinase-2 on platelet aggregation.

OBJECTIVE: Platelets contain and release some matrix metalloproteinases (MMPs), enzymes involved in the degradation of extracellular matrix, and one of these (MMP-2) exerts a proaggregatory effect. We explored the signal transduction mechanisms activated by MMP-2 in human blood platelets. METHODS AND RESULTS: Recombinant, human MMP-2, added before stimulation with subthreshold doses of different agonists, potentiated platelet activation, calcium influx, IP3 formation, and pleckstrin phosphorylation. Wortmannin and LY29400, two PI3-K inhibitors, suppressed the potentiating effects of MMP-2 and preincubation with MMP-2 enhanced the thrombin-induced association of the p85alpha PI3-K subunit with the cytoskeleton and increased the phosphorylation of PKB. Protein tyrosine kinase inhibitors, MAP kinase inhibitors, PLA2 inhibitors, cyclooxygenase inhibitors and antagonists of the P2Y1 and P2Y12 receptors did not affect the potentiating activity of MMP-2 on platelets. CONCLUSION: Our data show that MMP-2, at a concentration released by activated platelets, facilitates platelet activation acting at the level of a second messenger system common to different agonists and related to the activation of PI3-K. Platelet-released MMP-2 may contribute to platelet activation in vivo.

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression.

BACKGROUND: NO-releasing statins are new chemical entities, combining HMG-CoA reductase inhibition and slow NO release, that possess stronger anti-inflammatory and antiproliferative activities than the native statins. OBJECTIVE: We evaluated the antithrombotic effects of nitropravastatin (NCX-6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo. METHODS AND RESULTS: In vitro, NCX-6550 inhibited (1) U46619- and collagen-induced platelet aggregation in buffer and plasma; (2) collagen-induced P-selectin expression in whole blood and (3) platelet adhesion to collagen-coated coverslips under high shear stress. These effects were displayed at concentrations of NCX-6550 ranging from 25 to 100 mum, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 microm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS- and PMA-induced TF expression by blood mononuclear cells was also inhibited by NCX-6550 (IC50 13 microm), but not by pravastatin, as assessed by functional and immunological assays and by real-time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX-6550 (24-48 mg kg(-1)) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity. CONCLUSIONS: Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.

Effect of glyceryl trinitrate on distensibility of peripheral muscular arteries in humans is not mediated by prostaglandins.

STUDY OBJECTIVE: Vasodilator prostaglandins have been claimed to be responsible for the coronary haemodynamic and venodilator effects of glyceryl trinitrate, although conflicting results have been reported. The aim of this study was to evaluate whether vasodilator prostaglandins play a role in the effect of glyceryl trinitrate on the distensibility of peripheral muscular arteries in healthy humans. DESIGN: A non-invasive technique, impedance plethysmography, was applied to the assessment of the effects of sublingual glyceryl trinitrate on the compliance of the forearm and digital arteries. The subjects studied received placebo (on two separate occasions), indomethacin (100 mg orally), or ibuprofen (800 mg orally) 1 h before glyceryl trinitrate (0.3 mg sublingual) on four occasions separated from each other by at least 48 h. Blood pressure and heart rate were measured by standard techniques; changes in peripheral arterial compliance were evaluated by impedance plethysmography of the forearm and finger. The study was double blind, cross over, placebo controlled, and randomised. SUBJECTS: 12 healthy male volunteers were enrolled in the study. All subjects were fasting for at least 10 h and had abstained from smoking and from methylxanthine or alcohol containing beverages. MEASUREMENTS AND MAIN RESULTS: Glyceryl trinitrate increased heart rate by 11.6(SEM 1.6) beats.min-1 (p less than 0.0005) and diastolic blood pressure by 8.7(1) mm Hg (p less than 0.01), and decreased systolic blood pressure by 8.7(1.5) mm Hg (p less than 0.01) in placebo treated volunteers; the amplitude of the plethysmograph c wave in the forearm and in the finger was also augmented, by 120(11)% and 78(13)% respectively, indicating an increase in arterial compliance. The results obtained in the two placebo sessions did not differ, indicating good reproducibility of the system. Pretreatment with either indomethacin or ibuprofen did not modify the effects of glyceryl trinitrate on heart rate, blood pressure, and arterial compliance in the forearm and the finger. Both indomethacin and ibuprofen suppressed prostaglandin synthesis, as shown by the striking inhibition of serum TxA2 concentration, by 97.2(1.5)% and 93.7(3.0)%, respectively. CONCLUSIONS: Sublingual glyceryl trinitrate, in doses used clinically, induces a reproducible increase in peripheral arterial compliance in healthy volunteers; prostaglandins do not play any significant role in this effect.