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.

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.

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.

Platelet and endothelial activation in catastrophic and quiescent antiphospholipid syndrome.

Antiphospholipid antibodies (aPL) seem to induce a prothrombotic state by activating endothelium and platelets, but no studies have evaluated systematically the effects of aPL from patients with the antiphospholipid syndrome (APS) in quiescent versus catastrophic phase. Our aims were to evaluate the in vitro effects on platelet activation of anti-ß2 glycoprotein I (anti-ß2GPI) antibodiesisolated from APS patientin either quiescent or catastrophic phase and to investigate ex vivo platelet and endothelial activation in patients with quiescent or catastrophic APS. Anti-ß2GPI antibodies were isolated from plasma of a pregnant woman in two different stages of APS (quiescent and catastrophic, respectively). They were co-incubated with washed platelets from healthy controls that were then challenged with TRAP-6 (thrombin receptor activating peptide 6) and the expression of P- selectin (P-sel) on platelets was assessed by flow cytometry. Moreover, plasma samples from six patients with quiescent, four with catastrophic APS and 10 controls were assessed for several markers of platelet and endothelial activation. The results showed that purified anti-ß2GPI antibodies co-incubated with platelets enhanced TRAP-6- induced platelet P-sel expression. Notably, anti-ß2GPI antibodies isolated during the catastrophic phase enhanced platelet P-sel expression more than antibodies isolated from the same patient in the quiescent stage of disease. Moreover, APS patients had significantly higher plasma levels of soluble (s) Psel, sCD40 ligand, soluble vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1 than control subjects. In addition, sP-sel and von Willebrand factor activity were significantly higher during catastrophic than in quiescent phase.

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.

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.

NCX4016: a novel antithrombotic agent.

Despite great advantages in antithrombotic treatments, important limitations of the presently available drugs encourage the search of more effective agents. Within the cardiovascular system, nitric oxide exerts several activities which may have an antithrombotic potential. Nitroaspirin in vitro inhibits platelet aggregation and adhesion under shear conditions and smooth muscle cell proliferation--all activities not exerted by aspirin. In vivo nitroaspirin exerts antithrombotic properties and prevents restenosis in hypercholesterolemic mice while aspirin is inactive. Nitroaspirin has shown a number of significant advantages over the presently available antiplatelet agents; however, only clinical studies will say whether nitroaspirin represents a step forward in antithrombotic treatment.

Low molecular weight heparins prevent thrombin-induced thrombo-embolism in mice despite low anti-thrombin activity. Evidence that the inhibition of feed-back activation of thrombin generation confers safety advantages over direct thrombin inhibition.

BACKGROUND AND OBJECTIVES: Thrombin-induced thromboembolism in mice is a model in which the feed-back clotting activation produced by the injected enzyme greatly contributes to fibrin accumulation in lungs and to mortality. Using this model we have previously shown that activated human protein C (aPC), by interrupting endogenous clotting activation at a high level (factors Va and VIIIa), prevents mortality inducing only a minor hemostatic impairment. With the same model we have now compared the antithrombotic and prohemorrhagic effects of two low molecular weight heparins (LMWHs), reviparin and tinzaparin, which are expected to inhibit preferentially the positive feed-back triggered by thrombin (anti Xa activity), with those of unfractionated heparin (UFH) and PEG-hirudin, which inhibit mainly or exclusively thrombin activity (anti IIa activity). DESIGN AND METHODS: Pulmonary thromboembolism was induced in mice by i.v. injection of bovine thrombin (1,000U/kg). Drugs (from 0.12 to 1.2 mg/kg) were given as bolus injection 2 min prior to thrombin challenge and mortality was assessed within 15 min. The bleeding time was assessed by a tail tip transection model. Activated partial thromboplastin time (aPTT), thrombin clotting time (TcT), fibrinogen assay and anti Xa activity determination were performed in citrated plasma from saline- or drug-treated animals. RESULTS: All drugs protected mice from thrombin-induced mortality in a dose-dependent way. At comparable antithrombotic dosages, the anti IIa activity generated in plasma (assessed by TcT) was highest with UFH, intermediate with tinzaparin and very low with reviparin. Accordingly, the fibrinogen drop, which is caused mainly by the injected thrombin, was prevented by the heparins to an extent that was fairly well related to their anti IIa activity. aPTT and bleeding time, used as measures of hemorrhagic risk, were markedly more prolonged by UFH than by reviparin. Tinzaparin, instead, had an intermediate effect. Interestingly, PEG-hirudin, at equipotent antithrombotic dosages, caused a prolongation of bleeding time comparable to that observed with UFH. INTERPRETATIONS AND CONCLUSIONS: Our data show that, in our model, drugs acting at a high level of the blood clotting cascade, like LMWHs with a high anti Xa/anti IIa ratio, display a better antithrombotic/prohemorrhagic profile than drugs acting prevalently on thrombin.

Involvement of platelets in experimental mouse trypanosomiasis: evidence of mouse platelet cytotoxicity against Trypanosoma equiperdum.

Platelets play an important role in the human response to parasites. Trypanosoma equiperdum, a parasite that has the horse as its natural host, is able to induce infection in mice and thus it may represent a simple model for studying the role of platelets in the development of a parasitosis. Although several aspects of the murine response to T. equiperdum infection have been clarified, the precise mechanism of killing of the parasite is still unclear. We have studied the involvement of blood platelets in experimental murine infection with T. equiperdum. Infected mice show a progressive decrease of the number of circulating platelets. The production of thromboxane A2 (TxA2) by platelets stimulated with collagen decreases progressively with the progression of T. equiperdum infection, compatible with in vivo platelet activation or with a possible antagonistic effect by trypanosomes on the production of TxA2. Finally, mouse platelets exert in vitro a direct parasitocidal activity on T. equiperdum at ratios >/=20:1. Complement fractions do not enhance platelet trypanocidal activity, whereas IgM fractions do, at least in short-term coincubation experiments. Our data show that platelets are involved in experimental murine T. equiperdum infection and confirm that platelet parasitocidal activity is a generalized phenomenon in mammals.

Prevention of pulmonary thromboembolism by NCX 4016, a nitric oxide-releasing aspirin.

We studied the antithrombotic activity of 2-acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), a novel nitric oxide (NO)-releasing aspirin derivative, in vivo in different animal models of platelet-dependent and independent pulmonary thromboembolism and compared it with that of aspirin. NCX 4016 protected mice from death induced by the intravenous (i.v.) injection of collagen plus epinephrine, of 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619) and of thrombin while aspirin was only active against collagen plus epinephrine. The drop in platelet count and number of lung emboli were reduced by NCX 4016 more effectively than aspirin. NCX 4016 protected mice also from mechanical pulmonary embolism (i.v. injection of hardened rat red blood cells) while aspirin was ineffective. In rabbits, NCX 4016 significantly reduced the accumulation of [111In]oxine-labeled platelets in the pulmonary vasculature induced by collagen and by thrombin while aspirin produced reductions which were significant only versus collagen. In conclusion, NCX 4016 exerts a more pronounced antithrombotic activity than aspirin in vivo in two different animal species, largely due to a deeper inhibitory effect on platelets. NCX 4016 may represent a better antithrombotic agent than aspirin.

Endogenous nitric oxide acts as a natural antithrombotic agent in vivo by inhibiting platelet aggregation in the pulmonary vasculature.

Nitric oxide (NO) is a powerful vasodilator and an inhibitor of platelet aggregation in vitro. While the ability of NO to modulate vascular tone in vivo has been proven, only a few studies have assessed its platelet inhibitory activity in vivo. We have employed two complementary animal models of pulmonary platelet thromboembolism to assess the antithrombotic activity of endogenous NO in vivo. The inhibition of nitric oxide synthase (NOS) by L-NAME significantly potentiated while the administration of the NOS substrate L-arginine significantly reduced the accumulation of 111In-labelled platelets in the pulmonary vasculature of rabbits induced by intravenous collagen plus epinephrine. L-NAME or L-arginine did not, however, modify 111In-labelled erythrocyte distribution in lungs and phenylephrine had no effect on platelet accumulation following collagen + adrenaline, suggesting that the effects of L-NAME were not due to vasoconstriction but rather to a direct modification of platelet function. In mice, L-NAME significantly reduced the dose of collagen + adrenaline required to induce thromboembolic mortality, increased the fall in circulating platelets and increased the % of pulmonary vessels occluded by platelet thrombi. The effects of L-NAME were reversed by L-arginine but not by a dose of nicardipine exerting maximal vasodilatation. Phenylephrine did not potentiate collagen + adrenaline-induced mortality. In the pulmonary vasculature in vivo, endogenous NO inhibits collagen + adrenaline-induced aggregation and enhances platelet disaggregation. This natural modulator function of NO is exerted via a direct effect on platelets and not as a result of haemodynamic changes.

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.

The effect of defibrotide on thromboembolism in the pulmonary vasculature of mice and rabbits and in the cerebral vasculature of rabbits.

1. Administration of bovine thrombin (100 u kg-1) into the carotid artery of rabbits induces a sustained accumulation of 111 Indium-labelled platelets within the cranial vasculature over the subsequent 3 h. 2. Intracarotid (i.c.) administration of defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h) prior to i.c. thrombin (100 u kg-1) significantly reduces the ability of thrombin to induce cranial thromboembolism in rabbits. 3. Intravenous (i.v.) administration of thrombin (20 u kg-1) in rabbits induces a reversible accumulation of radiolabelled platelets into the thoracic circulation which is significantly reduced by i.v. administration of defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h) prior to i.v. thrombin. In contrast, platelet accumulation in response to adenosine diphosphate (ADP; 20 micrograms kg-1, i.v.) or platelet activating factor (PAF; 50 ng kg-1, i.v.) is not significantly affected by this treatment. 4. Intravenous administration of the nitric oxide (NO)-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 mg kg-1) potentiates platelet accumulation induced by low dose thrombin (10 u kg-1, i.v.) within the pulmonary vasculature of rabbits. The potentiated response is significantly abrogated following pretreatment with defibrotide (64 mg kg-1 bolus plus 64 mg kg-1 h-1 for 1 h, i.v.). 5. Intravenous injection of human thrombin (1250 u kg-1) to mice induces death within the majority of animals which is significantly reduced by pretreatment with defibrotide (150-175 mg kg-1, i.v.). In contrast, death induced by i.v. collagen (1.25 mg kg-1) plus adrenaline (75 microg kg-1) is not significantly affected by defibrotide pretreatment.6. The inhibitory effect of defibrotide in mice is abolished following concomitant treatment with the inhibitor of fribrinolysis, tranexamic acid (100 mg kg-1, i.v.), but is unaffected following treatment with the cyclo-oxygenase inhibitor, aspirin (300 mg kg-1, i.p.).7. The protective effect of defibrotide against thrombin-induced thromboembolism in the mouse is potentiated by recombinant tissue-plasminogen activator (rt-PA; 1 mg kg-1, i.v.) or unfractionated heparin (10 u kg-1, i.v.) administration.8. The results suggest that defibrotide may possess antithrombotic activity on thrombin-induced thromboembolism which, at least in the mouse, may be partially mediated via induction of the fibrinolytic pathway.