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Platelet-type von Willebrand disease is an inherited platelet disorder characterized by thrombocytopenia with large platelets caused by gain-of-function variants in GP1BA leading to enhanced GPIbα-von Willebrand factor (vWF) interaction. GPIbα and vWF play a role in megakaryocytopoiesis, thus we aimed to investigate megakaryocyte differentiation and proplatelet-formation in platelet-type von Willebrand disease using megakaryocytes from a patient carrying the Met239Val variant and from mice carrying the Gly233Val variant. Platelet-type von Willebrand disease megakaryocytes bound vWF at an early differentiation stage and generated proplatelets with a decreased number of enlarged tips compared to control megakaryocytes. Moreover, they formed proplatelets upon contact with collagen, differently from normal megakaryocytes. Similarly, collagen triggered megakaryocytes showed defective activation of the RhoA-MLC2 axis, which prevents proplatelet formation, and increased phosphorylation of Lyn, which acts as a negative regulator of GPVI signaling, thus preventing ectopic proplatelet-formation on collagen. Consistently, human and murine bone marrow contained an increased number of extravascular platelets compared to controls. In addition, platelet survival of mutant mice was shortened compared to control mice, and the administration of desmopressin, raising circulating vWF, caused a marked drop in platelet count. Taken together, these results show for the first time that thrombocytopenia in platelet-type von Willebrand disease is due to the combination of different pathogenic mechanisms, i.e. the formation of a reduced number of platelets by megakaryocytes, the ectopic release of platelets in the bone marrow, and the increased clearance of platelet/vWF complexes.

Introduction Platelets participate in inflammatory disorders through a variety of different functional responses, including chemotaxis, platelet-leukocyte complex formation and facilitation of leukocyte recruitment that are thought to be distinct from platelet aggregation. This may account for why classical anti-platelet drugs have failed to ameliorate inflammatory disorders where platelets are known to participate, suggesting that distinct pathways may control inflammatory and haemostatic functions of platelets. In the present study, we have therefore investigated the effect of different stimuli on several different functions of platelets preferentially involved either in haemostasis or in inflammation. Materials and methods Human platelets were stimulated with either inflammatory (fMLP, histamine, IL-1β, LPS, MDC/CCL22, SDF-1α/CXCL12 and 5-HT) or haemostatic (ADP, collagen, convulxin, epinephrine, TRAP-6 and U46619) stimuli. Aggregation, platelet-leukocyte complex formation, platelet migration and platelet protein phosphorylation were assessed. Results Haemostatic stimuli induced platelet aggregation, whilst inflammatory agonists induced platelet migration. The haemostatic stimuli, with the exception of epinephrine, and some of the inflammatory stimuli induced platelet-leukocyte complex formation, even if to a different extent. Furthermore, inflammatory stimuli induced a shorter lasting profile of platelet protein phosphorylation compared with haemostatic stimuli. Conclusions Stimulation of platelets with inflammatory stimuli triggers the activation of non haemostatic functions different from those induced by haemostatic stimuli, supporting the existence of alternative platelet responses depending on the stimulus (haemostatic or inflammatory). A deeper understanding of the biochemical pathways behind these functional differences may lead to the development of novel therapeutic options targeting the inflammatory actions of platelets, without affecting their critical role in haemostasis.

Over the past few decades the central role that platelets play in cancer development and progression, and especially in metastasis, has been elucidated. The molecular mechanisms responsible for initiating and mediating tumor cell-induced platelet aggregation and secretion have been largely unravelled. Considerable mechanistic insight into how platelets contribute to tumor angiogenesis, immunoevasion and cancer cell invasion have been clarified and, consequently, platelets have been identified as potential new drug targets for cancer therapy. 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 antiplatelet drugs currently used for cardiovascular disease and of new and novel strategies.

The role of platelets as inflammatory cells is now well established. Given the peculiar characteristics of the lung circulation, with a broad capillary bed, platelets are especially involved with the physiology of the lungs and play a key role in a number of inflammatory lung disorders. The platelet precursors, megakaryocytes, are detected in the lung microcirculation; moreover platelets with their endothelium-protective and vascular reparative activities contribute to the lung capillary blood barrier integrity. Given the function of the lungs as first wall against pathogen invasion, platelets participate in immune defence of the normal lung. On the other hand, platelets may turn into effectors of the inflammatory reaction of the lungs to allergens, to infectious agents, to chemical agents and may contribute strongly to the perpetuation of chronic inflammatory reactions, largely by their ability to interact with other inflammatory cells and the endothelium. In this chapter we provide an overview of the role of platelets in several inflammatory lung disorders discussing the pathophysiologic bases of platelet involvement in these conditions and the experimental and clinical evidence for a role of platelets in lung diseases.

Platelets, beyond their well-described role in haemostasis and thrombosis, act as inflammatory cells playing an active role in several inflammatory conditions. As observed with other inflammatory cells platelets can migrate in vitro, either randomly or in the direction of a chemotactic agent, and in vivo, into inflammed tissues in response to different stimuli. In this chapter we will summarize the current knowledge about the mechanisms that regulate platelet chemotaxis, the evidence for the ability of platelets to migrate in vitro and in vivo, and the mechanisms by which platelets influence chemotaxis of other cells.

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.

The amyloid precursor protein (APP), primarily known as the precursor of amyloid peptides that accumulate in the brain of patients with Alzheimer disease, is abundant in platelets, but its physiological function remains unknown. In this study, we investigated the role of APP in hemostasis and thrombosis, using APP knockout (KO) mice. Ex vivo aggregation, secretion, and integrin αIIbβ3 inside-out activation induced by several agonists were normal in APP-deficient platelets, but the number of circulating platelets was reduced by about 20%, and their size was slightly increased. Tail bleeding time was normal, and in vivo, the absence of APP did not alter thrombus formation in the femoral artery. In contrast, in a model of vein thrombosis induced by flow restriction in the inferior vena cava, APP-KO mice, as well as chimeric mice with selective deficiency of APP in blood cells, developed much larger thrombi than control animals, and were more sensitive to embolization. Consistent with this, in a pulmonary thromboembolism model, larger vessels were occluded. APP-KO mice displayed a shorter APTT, but not PT, when measured in the presence of platelets. Moreover, the activity of factor XIa (FXIa), but not FXIIa, was higher in APP-KO mice compared with controls. APP-KO mice presented a higher number of circulating platelet-leukocyte aggregates, and neutrophils displayed a greater tendency to protrude extracellular traps, which were more strongly incorporated into venous thrombi. These results indicate that platelet APP limits venous thromboembolism through a negative regulation of both fibrin formation and neutrophil function.

Background Subconjunctival hemorrage (SCH) is a frequent, mild bleeding manifestation and a common cause of consultation. Hemostatic alterations are possible causes of SCH but their role and prevalence is unknown. We assessed the prevalence of hemostatic abnormalities in patients with spontaneous, recurrent SCH to clarify the role of the hemostasis laboratory in this clinical setting. Methods A total of 105 SCH patients (21-78 years, 65 females) with no identifiable cause (hypertension-trauma-conjunctivitis) or concomitant treatments (NSAIDs- aspirin-oral anticoagulants-antiplatelet agents) and 53 age and sex-matched healthy controls (HCs) (22-72 years, 29 females) were evaluated for skin bleeding time, PFA-100®, blood clotting screening, platelet count, light transmission aggregomery, VWF:Ag, VWF:RCo, RIPA, FVIII activity, FXIII antigen and activity and ISTH Bleeding Severity Score (BSS). Results Prevalence of hemostatic abnormalities was not higher in the SCH population than in HCs BSS was 0.83 (95% CI 0.62-1.06) in SCH and 0.66 (0.37-0.95) in HC (p=NS). Type I Von Willebrand disease was diagnosed in one SCH and none HC patients, a prevalence not significantly different (p=NS by χ2). Conclusions The prevalence of hemostatic alterations in patients with recurrent, spontaneous SCH is not different from the general population; hemostatic screening or second level tests are of no use in patients with recurrent SCH and no other bleedings.

Background Clinical studies reveal platelet activation in patients with asthma, allergic rhinitis, and eczema. This is distinct from platelet aggregation, which is critical for the maintenance of hemostasis and in which a role for platelet purinergic receptors is well documented. However, purines are also essential for inflammatory cell trafficking in animal models of allergic lung inflammation, which are known to be platelet dependent, yet the role of purines in the platelet activation accompanying inflammation is unknown. Objectives We investigated whether the involvement of purine activation of platelets during allergic inflammation is distinct from purine involvement in platelet aggregation. Methods BALB/c mice were sensitized to ovalbumin and subsequent airway ovalbumin challenge. Bronchoalveolar lavage fluid was analyzed for inflammatory cells, and blood samples were assessed for platelet activation. The role of platelet purinergic receptors and associated signaling mechanisms (RhoA) were assessed. Results P2Y 1 , but not P2Y 12 or P2X 1 , antagonism inhibited pulmonary leukocyte recruitment. The formation of platelet-leukocyte complexes in vivo and platelet/P-selectin–dependent polymorphonuclear cell migration in vitro were exclusively platelet P2Y 1 receptor dependent. Furthermore, platelet P2Y 1 activation resulted in RhoA activity in vivo after allergen challenge, and RhoA signaling in platelets through P2Y 1 stimulation was required for platelet-dependent leukocyte chemotaxis in vitro . Leukocyte recruitment in thrombocytopenic mice remained suppressed after reinfusion of platelets pretreated with a P2Y 1 antagonist or a Rho-associated kinase 1 inhibitor, confirming the crucial role of platelet P2Y 1 receptor and subsequent activation of RhoA. Conclusion RhoA signaling downstream of platelet P2Y 1 , but not P2Y 12 , represents a clear dichotomy in platelet activation during allergic inflammation versus hemostasis.

Highly active antiretroviral therapy (HAART) has transformed human immunodeficiency virus (HIV) infection into a chronic condition, which has allowed the infected population to age and become prone to chronic degenerative diseases common to the general population, including atherosclerotic cardiovascular disease, and coronary artery disease (CAD). Possible causative mechanisms of HIV-associated CAD are related to classic cardiovascular risk factors, such as dyslipidemia, insulin resistance, and fat redistribution, which may be due to either HIV infection or to HAART-associated toxicity. However, other mechanisms are emerging as crucial for the cardiovascular complication of HIV and HAART. This article analyzes the effects of HIV and HAART on endothelial function, endothelium-leukocyte interactions, and platelets as possible mechanisms of enhanced cardiovascular risk.

SummaryPurified active matrix metalloproteinase-2 (MMP-2) is able to promote platelet aggregation. We aimed to assess the role of MMP-2 expressed in atherosclerotic plaques in the platelet-activating potential of human carotid plaques and its correlation with ischaemic events. Carotid plaques from 81 patients undergoing endarterectomy were tested for pro-MMP-2 and TIMP-2 content by zymography and ELISA. Plaque extracts were incubated with gel-filtered platelets from healthy volunteers for 2 minutes before the addition of a subthreshold concentration of thrombin receptor activating peptide-6 (TRAP-6) and aggregation was assessed. Moreover, platelet deposition on plaque extracts immobilised on plastic coverslips under high shear-rate flow conditions was measured. Forty-three plaque extracts (53%) potentiated platelet aggregation (+233 ± 26.8%), an effect prevented by three different specific MMP-2 inhibitors (inhibitor II, TIMP-2, moAb anti-MMP-2). The pro-MMP-2/TIMP-2 ratio of plaques potentiating platelet aggregation was significantly higher than that of plaques not potentiating it (3.67 ± 1.21 vs 1.01 ± 0.43, p

Objective— dl -Nebivolol, a selective β1-adrenergic receptor antagonist, besides its hypotensive activity exerts vasodilatory and platelet inhibitory effects in vitro by a mechanism involving nitric oxide (NO). Our aim was to evaluate whether nebivolol exerts in vivo antithrombotic effects, to unravel the mechanism of this action and to clarify the relative roles of its 2 enantiomers: d - and l -nebivolol. Methods and Results— In wild-type mice, dl -nebivolol, l -nebivolol, and d -nebivolol, but not bisoprolol, reduced mortality consequent to platelet pulmonary thromboembolism induced by the intravenous injection of collagen plus epinephrine (−44%, −45%, −29%, respectively; P −/− mice only dl -nebivolol and d -nebivolol were effective. dl -Nebivolol, l - and d -nebivolol reduced photochemical damage-induced femoral artery thrombosis in wild-type mice, whereas in eNOS −/− mice only dl -nebivolol and d -nebivolol were active. Moreover, dl -nebivolol and l -nebivolol increased plasma, urinary-, and platelet-derived nitrites and nitrates (NOx), NO degradation products, in wild-type but not in eNOS −/− mice. In vivo platelet activation, assessed by platelet P-selectin expression, was reduced by dl -nebivolol and l - and d -nebivolol in wild-type mice but only by dl -nebivolol and d -nebivolol in eNOS −/− mice. In bone marrow–transplanted, chimeric mice with only blood cells, and not the endothelium, producing NO dl -nebivolol and l -nebivolol maintained their antithrombotic activity, whereas they lose it in chimeras with only endothelium, and not blood cells, producing NO. In vitro, with isolated platelets, dl -nebivolol and l -nebivolol, but not d -nebivolol and bisoprolol, increased platelet cGMP and NOx formation. Treatment with dl -nebivolol and l -nebivolol increased phophorylated eNOS in platelets. Conclusions— Our data show that dl -nebivolol exerts an antithrombotic activity by stimulating the formation of NO by platelets, and that this effect is generated by its l -enantiomer, whereas the d -enantiomer exerts a weak antiplatelet effect because of β−adrenergic receptor–independent stimulation of adenyly cyclase. These results confirm that platelet-derived NO plays a role in thrombosis prevention and it may represent a target of pharmacological intervention.

SummaryAntiphospholipid 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-β2 glycoprotein I (anti-β2GPI) antibodies isolated from APS patient in either quiescent or catastrophic phase and to investigate ex vivo platelet and endothelial activation in patients with quiescent or catastrophic APS. Anti-β2GPI 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-β2GPI antibodies co-incubated with platelets enhanced TRAP-6-induced platelet P-sel expression. Notably, anti-β2GPI 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.

In the present study, we used a knockout murine model to analyze the contribution of the Ca2+-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin αIIbβ3 and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A2 production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA2 phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein–coupled receptors in supporting platelet aggregation and thrombus formation.

Thrombolytic therapy is the cornerstone of treatment of acute atherothrombotic ischemic stroke but is associated with brain hemorrhage; antiplatelet therapy has limited efficacy and is still associated with intracranial bleeding. Therefore, new antithrombotic approaches with a better efficacy/safety ratio are required. We have assessed the effect of ALX-0081, a Nanobody against the A1 domain of von Willebrand factor (VWF) that blocks VWF binding to GPIb, of the thrombolytic agent recombinant tissue plasminogen activator (rtPA), and of the GPIIb/IIIa antagonist tirofiban, in a middle cerebral artery (MCA) thrombosis model in guinea pigs. Drugs were administered before, immediately after, or 15 or 60 minutes after the total occlusion of the MCA. ALX-0081 prevented MCA thrombosis and induced reperfusion when given immediately after and 15 minutes after complete occlusion and reduced brain damage without inducing hemorrhage, whereas tirofiban prevented thrombosis but did not induce reperfusion and induced striking brain hemorrhage. rtPA also induced reperfusion when given 60 minutes after occlusion but provoked brain hemorrhage. Skin bleeding time was not modified or was moderately prolonged by ALX-0081, whereas tirofiban and rtPA prolonged it. The inhibition of the GPIb-VWF axis in guinea pigs prevents cerebral artery thrombosis and induces early reperfusion without provoking intracerebral bleeding thus reducing brain infarct area.

Stroke is the third most common cause of death in the industrialized countries and adequate primary and secondary prevention strategies are mandatory. In addition to lifestyle-changes and correction of cardiovascular risk factors, the mainstay of the atherothrombotic stroke prevention is represented by antiplatelet treatment. Although aspirin and thienopyridines have proved their efficacy in the prevention of arterial thrombotic events, limited efficacy, increased risk of bleeding, significant inter-individual variability in the response and extended duration of action that cannot be reversed if the need for haemostasis or emergency surgery arises represent major limitations of these drugs. Moreover, despite recommendations and guidelines about stroke prevention, registries data clearly suggest an underuse of antiplatelet drugs, mainly because of bleeding episodes fear. At variance with newer anticoagulant drugs, which showed a series of advantages as compared with the traditional warfarin treatment, newer antiplatelet drugs have only partially overcome these limitations. Although ad hoc studies on their efficacy in the stroke prevention are currently lacking, newer antiplatelet agents (mainly ticagrelor and prasugrel) do not provide a significant better protection over and above aspirin and/or clopidogrel in the prevention of atherothrombotic stroke. In addition, a significantly increased bleeding risk has been reported in subjects receiving these new thienopyridines. According to these data, the identification of further molecular targets is needed, in order to design future antiplatelet drugs. In this review, after summarizing major literature data about traditional and newer antiplatelet drugs, we will specifically focus on novel potential target candidates for antiplatelet therapy.

Objective To assess the role of matrix metalloproteinase 2 (MMP-2) in the evolution of septic arthritis induced by group B streptococci (GBS) in mice. Methods Mice deficient in MMP-2 (MMP-2−/−) and wild-type controls were injected intravenously with 1 × 107 colony-forming units of type IV GBS (strain 1/82). Levels of MMP-2, mortality rates, evolution of arthritis, bacterial clearance, joint histopathologic features, and production of cytokines and chemokines were examined in both experimental groups of mice on days 3, 6, and 9 after infection. Results MMP-2 was produced during GBS infection. Disruption of the gene for MMP-2 resulted in a decrease in the incidence and severity of arthritis, as demonstrated by both clinical and histologic findings, without affecting mortality rates. Amelioration of arthritis was accompanied by a dramatic reduction in the local production of interleukin-1β (IL-1β), IL-6, macrophage inflammatory protein 1α (MIP-1α), and MIP-2 and a reduced bacterial burden. Conclusion MMP-2, produced early during GBS infection in mice, is involved in the degradation of extracellular matrix components at the level of the joint. This degradation is the first step in a cascade of events (joint invasion by GBS, extravasation and accumulation of inflammatory cells, proinflammatory cytokine production), all of which contribute to the damage of articular tissue. Thus, MMP-2 should be regarded as a potential therapeutic target in GBS-induced arthritis.

Aims The aim of the present study was to assess whether the addition of a nitric oxide (NO)-donating moiety to atorvastatin enhances anti-inflammatory and anti-atherogenic effects in an animal model of endothelial dysfunction, systemic peroxidation and inflammation, and accelerated atherosclerosis. Methods and results Low-density lipoprotein receptor (LDLR)−/− mice kept on a high-fat diet (HFD) for 16 weeks underwent photochemical injury to the femoral artery with the local production of oxygen radicals. HFD markedly enhanced cholesterol, inflammatory biomarkers in plasma and in the femoral arterial wall, and atherosclerotic lesions in the aortic arch; inflammation and atherosclerosis were further increased by photochemically generated oxygen radicals. Treatment with the NO-donating atorvastatin NCX 6560 (11.7 mg/kg) was significantly more effective than atorvastatin (10 mg/kg) in reducing the following parameters: lipid-rich lesions in the aortic arch (surface covered: atorvastatin = 24 ± 5%; NCX 6560 = 14.7 ± 3.9%; P < 0.05); the production of radical oxygen species in the aorta (dichlorofluorescein fluorescence intensity per milligram of protein: atorvastatin = 2419 ± 136.7; NCX 6560 = 1766 ± 161.2; P < 0.05); femoral artery intima/media thickness (atorvastatin = 1.2 ± 0.11; NCX 6560 = 0.3 ± 0.14; P < 0.05); circulating interleukin-6 (atorvastatin = 34.3 ± 6.8 pg/mL; NCX 6560 = 17.7 ± 14.4 pg/mL; P < 0.05); and matrix metalloproteinase 2 in the arterial wall (atorvastatin = 55.2 ± 1.9 ng/µg of proteins; NCX 6560 = 45.8 ± 2.6 ng/µg of proteins; P < 0.05). Conclusion In conditions of severe endothelial dysfunction, systemic peroxidation and inflammation, and accelerated atherosclerosis, atorvastatin, even at high doses, displays suboptimal anti-atherogenic and anti-inflammatory effects, while the addition of a NO-donating property confers enhanced anti-atherogenic and anti-inflammatory effects.

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 b-thromboglobulin (b-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 b-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.

SummaryThe 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.

Abnormalities of platelet function or structure are a hallmark of chronic myeloproliferative disorders (MPD). In vivo platelet activation with the release of alpha- and delta-granules in the circulation is one of the most frequently described alterations in MPD. Platelets contain and release upon activation also lysosomes, and in particular beta-N-acetylhexosaminidase (Hex). We have assessed whether the content and in vivo release of Hex of platelets from MPD patients is altered.Twenty-three MPD patients were compared with 19 age- and sex-matched healthy controls. The activity of platelet beta-N-acetylhexosaminidase was measured in plasma, serum and in the capillary blood emerging from the skin wound inflicted for the measurement of the bleeding time. Lysosome integral membrane protein (LIMP or CD63), lysosome-associated membrane protein (LAMP-2 or CD107b) and P-selectin were evaluated by flow cytometry. Platelet aggregation in vitro and the release of beta-N-acetylhexosaminidase, ATP and beta-thromboglobulin were performed to study platelet reactivity.Hex levels in plasma were significantly higher in MPD than in controls while the release of Hex in the bleeding time blood, i.e. at a localized site of in vivo platelet plug formation, was lower in MPD and the platelet content of Hex was reduced. These changes were accompanied by in vivo platelet activation. Finally, the isoenzymatic pattern of Hex was altered in platelets of MPD patients, with a reduced amount of the Hex A isoform as compared with controls.bMPD patients present an altered platelet Hex content and release; prospective studies to assess whether altered platelet Hex is related to thrombotic/hemorrhagic complications and/or tissue fibrosis in MPD are warranted.

SummaryClopidogrel plus aspirin is the treatment of choice for patients undergoing percutaneous, coronary interventions with stenting, but it does not prevent restenosis. NCX-4016, a nitric oxide-releasing aspirin (nitroaspirin), exerts a wider range of antiplatelet actions compared to aspirin, superior antithrombotic activity and reduces restenosis after arterial injury in animals. The aim of the present study was to compare the combination of nitroaspirin plus clopidogrel with aspirin plus clopidogrel in a model of platelet pulmonary thromboembolism, bleeding and intimal thickening in mice. Drugs were administered orally for 5 days; the antithrombotic effects were evaluated against collagen plus epinephrine-induced pulmonary thromboembolism, the haemorrhagic effects by tail transection bleeding time and the effects on neointima proliferation by histomorphology of photochemically injured femoral arteries. Lung platelet emboli were reduced significantly and more effectively by nitroaspirin plus clopidogrel (-56%, p< 0.05 vs control) than by aspirin plus clopidogrel (-26%, p< 0.05 vs control). Ex vivo platelet aggregation was inhibited maximally by nitroaspirin plus clopidogrel. Aspirin plus clopidogrel strikingly prolonged the bleeding time while nitroaspirin plus clopidogrel induced a lesser prolongation. Nitroaspirin plus clopidogrel significantly reduced intimal thickening of the femoral artery while aspirin plus clopidogrel was ineffective. Nitroaspirin plus clopidogrel is more effective and less prohaemorrhagic than aspirin plus clopidogrel in mice; provided these data are confirmed in other animal models, nitroaspirin plus clopidogrel may represent a new regimen to be tested in patients undergoing coronary revascularization procedures.

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.

Momi, S., Perito, S., Mezzasoma, A. M., Bistoni, F., and Gresele, P. 2000. Involvement of platelets in experimental mouse trypanosomiasis: Evidence of mouse platelet cytotoxicity against Trypanosoma equiperdum. Experimental Parasitology95, 136–143. 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.

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.