Stefania Momi

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.

Matrix metalloproteinases and peripheral arterial disease

Matrix metalloproteinases (MMPs), a family of enzymes that degrade extracellular matrix, are emerging as important modulators of atherothrombosis. MMPs are produced by inflammatory cells; some of them are also released by activated platelets and play a crucial role in the remodeling processes, leading to atherosclerotic plaque formation, plaque rupture, arterial aneurysm development, and critical limb ischemia. Independent from their matrix degrading activity, MMPs also regulate some cell functions relevant to atherothrombosis, such as platelet activation, neutrophil activation, and vascular reactivity. Plasma levels of some MMPs are increasingly being recognized as a biomarker of atherosclerosis and cardiovascular risk. In peripheral arterial disease, MMPs have been shown to be involved in angiogenesis, arteriogenesis, and the development of arterial calcifications. Increased plasma levels of some MMPs (MMP-2, MMP-9) have been correlated with PAD development and severity. Single nucleotide polymorphisms of the genes encoding for some MMPs have also been associated with the risk of developing peripheral arterial disease and critical limb ischemia. Large prospective observational studies are needed to further demonstrate the role of MMPs in PAD. In perspective, pharmacologic targeting of the expression or activity of MMPs may represent a novel, attractive approach for the treatment of peripheral arterial disease.

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.

Interactions of gallic acid, resveratrol, quercetin and aspirin at the platelet cyclooxygenase-1 level. Functional and modelling studies.

While resveratrol and quercetin possess antiplatelet activity, little is known on the effect of gallic acid on platelets. We studied the interactions of these three different polyphenols among themselves and with aspirin, at the level of platelet cyclooxygenase-1 (COX-1). Both functional (in vitro and in vivo) and molecular modelling approaches were used. All three polyphenols showed comparable antioxidant activity (arachidonic acid [AA]-induced intraplatelet ROS production); however, resveratrol and quercetin, but not gallic acid, inhibited AA-induced platelet aggregation. Gallic acid, similarly to salicylic acid, the major aspirin metabolite, prevented inhibition of AA-induced platelet function by aspirin but, at variance with salicylic acid, also prevented inhibition by the other two polyphenols. Molecular modelling studies, performed by in silico docking the polyphenols into the crystal structure of COX-1, suggested that all compounds form stable complexes into the COX-1 channel, with slightly different but functionally relevant interaction geometries. Experiments in mice showed that gallic acid administered before aspirin, resveratrol or quercetin fully prevented their inhibitory effect on serum TxB(2). Finally, a mixture of resveratrol, quercetin and gallic acid, at relative concentrations similar to those contained in most red wines, did not inhibit platelet aggregation, but potentiated sub-inhibitory concentrations of aspirin. Gallic acid interactions with other polyphenols or aspirin at the level of platelet COX-1 might partly explain the complex, and possibly contrasting, effects of wine and other components of the Mediterranean diet on platelets and on the pharmacologic effect of low-dose aspirin.

Endothelium, venous thromboembolism and ischaemic cardiovascular events.

The association between venous thromboembolism and arterial thrombosis has emerged as consistent clinical observation in the last few years. While several experimental, epidemiological and pharmacologic studies support this association, the initial pathophysiological mechanism linking these two clinical conditions remains to be established. This review discusses the pathophysiological bases and a number of experimental and clinical observations suggesting that the common link between venous thromboembolism and arterial thrombosis is represented by a dysfunctional endothelium.

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression1

Summary.  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 μm, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 μm). 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 μm), 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.

Platelets release matrix metalloproteinase-2 in the coronary circulation of patients with acute coronary syndromes: possible role in sustained platelet activation

AIMS To investigate whether selected matrix metalloproteinases (MMPs) are released in the coronary circulation of patients with acute coronary syndrome (ACS), whether this release is related to platelet activation, and whether it contributes to sustained platelet activation. METHODS AND RESULTS Blood from the aorta (Ao) and the coronary sinus (Cs) was obtained from 21 controls (non-cardiac chest pain), 24 stable angina (SA), and 30 ACS patients, before performing percutaneous transluminal coronary angioplasty. Selected MMPs, some platelet activation- and atheroma-related markers, and the platelet activation-potentiating activity of plasma were measured. Total MMP-2, active MMP-2, and MMP-9 were released in the coronary circulation of patients with ACS, but not of those with SA or controls. Similarly, transcoronary gradients of β-thromboglobulin (β-TG) and platelet factor 4, two platelet-specific proteins, and of soluble CD40L and secretory phospholipase A₂ (sPLA₂), markers of inflammation and platelet activation, were higher in ACS patients than in the other groups. In contrast, plasma monocyte chemoattractant protein-1, a platelet-unrelated marker of atherogenesis, was not increased in the Cs compared with Ao in any of the groups. Transcoronary gradients of both β-TG and sPLA₂ correlated with those of total and active MMP-2 in ACS, but not in controls or SA. Plasma from the Cs of ACS patients potentiated platelet activation, an effect suppressed by the specific MMP-2-inhibitor, tissue inhibitor of MMP-2 (TIMP-2). CONCLUSION Matrix metalloproteinase-2 is released in the coronary circulation of ACS patients, derives in part from activated platelets, and may contribute to sustained intracoronary platelet activation.

Potentiation and priming of platelet activation: a potential target for antiplatelet therapy

Ischemic cardiovascular events represent the leading cause of mortality and morbidity worldwide, and platelet aggregation and thrombus formation are the main effectors of acute arterial ischemic events. Although antiplatelet therapy is the cornerstone of antithrombotic treatment of ischemic cardiovascular disorders, available antiplatelet agents have less than satisfactory efficacy; thus, the identification of novel potential target candidates for antiplatelet therapy is highly warranted. Recent evidence suggests that several molecules that amplify the aggregation response of platelets to activating stimuli, which are either released by platelets (potentiating molecules) or present in the milieu before platelets get activated (primers), play a major role in pathologic thrombus formation without being significantly involved in primary haemostasis. These molecules appear to be a particularly appealing novel potential pharmacologic target for antiplatelet therapy. Here, we review the present knowledge on some molecules acting as potentiators or primers of platelet activation and discuss their possible pharmacologic modulation for antithrombotic purposes.

Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo

1. 1. Momi, 2. et al . 2009. J. Exp. Med. doi: 10.1084/jem.20090687 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20090687%26rft_id%253Dinfo%253Apmid%252F19808257%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%

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.