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The von Willebrand factor (vWF) aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein 1b (GPIb) at high shear, may deliver a site-specific antithrombotic effect. We investigated the efficiency of ARC1779 on platelet function in patients with coronary artery disease (CAD) on double antiplatelet therapy. Blood from patients taking aspirin and clopidogrel and from normal volunteers was treated ex vivo with ARC1779 or abciximab, either prior to perfusion (pretherapy) or 10 minutes following the initiation of perfusion (posttherapy) on damaged arteries. Under pre- but not posttherapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nmol/L and by abciximab (100 nmol/L) versus placebo (4.8, 3.8, and 2.9 vs 7.3 platelets × 106/cm2, P < .05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation, P-selectin expression, and platelet−leukocyte binding. These proof-of-concept data may constitute the framework for randomized clinical investigations of this novel antiplatelet therapy among patients with CAD.

Summary. Background and objectives: Anti-heat shock protein (HSP)60 autoantibodies are associated with atherosclerosis and are known to affect endothelial cells in vitro. However, their role in thrombus formation remains unclear. We hypothesized that anti-HSP60 autoantibodies could potentiate thrombosis, and evaluated the effect of anti-murine HSP60 antibodies in a ferric chloride (FeCl3)-induced murine model of carotid artery injury. Methods: Anti-HSP60, or control, IgG was administered to BALB/c mice 48 h prior to inducing carotid artery injury, and blood flow was monitored using an ultrasound probe. Results: Thrombus formation was more rapid and stable in anti-HSP60 IGG-treated mice than in controls (blood flow = 1.7% ± 0.6% vs. 34% ± 12.6%, P = 0.0157). Occlusion was complete in all anti-HSP60 IgG-treated mice (13/13), with no reperfusion being observed. In contrast, 64% (9/14) of control mice had complete occlusion, with reperfusion occurring in 6/9 mice. Thrombi were significantly larger in anti-HSP60 IgG-treated mice (P = 0.0001), and contained four-fold more inflammatory cells (P = 0.0281) than in controls. Non-injured contralateral arteries of anti-HSP60 IgG-treated mice were also affected, exhibiting abnormal endothelial cell morphology and significantly greater von Willebrand factor (VWF) and P-selectin expression than control mice (P = 0.0024 and P = 0.001, respectively). Conclusions: In summary, the presence of circulating anti-HSP60 autoantibodies resulted in increased P-selectin and VWF expression and altered cell morphology in endothelial cells lining uninjured carotid arteries, and promoted thrombosis and inflammatory cell recruitment in FeCl3-injured carotid arteries. These findings suggest that anti-HSP60 autoantibodies may constitute an important prothrombotic risk factor in cardiovascular disease in human vascular disease.

1 P-selectin is involved, with P-selectin glycoprotein (GP)-ligand-1 (PSGL-1), in platelet/leukocyte interactions during thrombo-inflammatory reactions; it also stabilizes platelet aggregates. Its antagonism accelerates thrombolysis and enhances the anti-aggregatory effects of GPIIb–IIIa inhibitors. This study was designed to investigate the mechanisms of P-selectin-mediated platelet aggregation. 2 In freshly isolated human platelets, P-selectin translocation after thrombin stimulation increased rapidly to 48, 72, and 86% positive platelets after 60, 120, and 300 s, respectively. Platelet aggregation at 60 s post-stimulation averaged 46.7±1.9% and its extent followed closely the kinetics of P-selectin translocation. 3 Pre-treatment of platelets with P-selectin antagonists, a recombinant PSGL-1 (rPSGL-Ig) or a blocking monoclonal antibody, significantly delayed platelet aggregation in a dose-dependent manner. At 100 μg ml−1 of rPSGL-Ig, platelet aggregation was completely inhibited up to 60 s post-stimulation and increased thereafter to reach maximal aggregation at 5 min. The second phase of platelet aggregation, in the presence of rPSGL-Ig, was completely prevented by the addition of a GPIIb–IIIa antagonist (Reopro) at 60 s, whereas its addition in the absence of rPSGL-Ig was without any significant effect. 4 Combination of rPSGL-Ig with Reopro or with an inhibitor of Pi3K (LY294002), which reduces GPIIb–IIIa activation, showed to be more effective in inhibiting platelet aggregation, in comparison to the effects observed individually. 5 rPSGL-Ig blocks P-selectin, whereas Reopro and LY294002 block GPIIb–IIIa and its activation, respectively, without a major effect on the percentage of platelets expressing P-selectin. 6 In summary, platelet P-selectin participates with GPIIb–IIIa in the initiation of platelet aggregation. Its inhibition, with rPSGL-Ig, delays the aggregation process and increases the anti-aggregatory potency of Reopro. Thus, combination of P-selectin and GPIIb–IIIa antagonism may constitute a promising therapeutic option in the management of thrombotic disorders. British Journal of Pharmacology (2006) 148, 299–305. doi:10.1038/sj.bjp.0706734

SummaryPlatelets and neutrophils constitute a high source of metalloproteinases (MMPs), and their interactions via P-selectin and Pselectin- glycoprotein-ligand-1 (PSGL-1) are involved in thrombosis, vascular remodelling, and restenosis. We investigated the impact of these interactions on platelet MMP-2 secretion and function in platelet and neutrophil aggregation. The secretion of MMP-2 from human platelets was significantly increased threefold after thrombin activation, and enhanced two-fold in the presence of neutrophils. Neutrophil supernatant had no effect on platelet MMP-2 secretion. While no MMP-2 was detected in the supernatant of neutrophils, a high amount of MMP-9 was released by neutrophils, and remained unchanged upon thrombin activation or in the presence of platelets. Platelet P-selectin, which increased significantly after activation, triggered platelet binding to neutrophils that was completely inhibited by P-selectin or PSGL-1 antagonists, and was reduced by 50% with a GPIIb/ IIIa antagonist. P-selectin or PSGL-1 antagonism abolished the enhanced secretion of platelet MMP-2 in the presence of neutrophils and reduced platelet-neutrophil aggregation. Platelet activation and binding to neutrophils enhance the secretion of platelet MMP-2 via an adhesive interaction between P-selectin and PSGL-1, which contribute to increase platelet-neutrophil aggregation.

Platelet activation with GPIIb/IIIa binding to fibrinogen, aggregation and interaction with leukocytes constitute the principal mediator of thrombosis. Although the clinical benefits of GPIIb/IIIa antagonists have been documented, the relationship between their anti-platelet properties, platelet activation and binding to leukocytes is still debated. We investigated the effects of abciximab, tirofiban, roxifiban, and an anti-P-selectin blocking monoclonal antibody (Mab) on isolated human platelet aggregation using optical aggregometer, and on platelet P-selectin and GPIIb/IIIa expression, and platelet-neutrophil binding using flow cytometry. Thrombin at 0.025 U/ml induced maximal platelet aggregation (76.3 +/- 2.6%), P-selectin expression (88.5 +/- 4%), GPIIb/IIIa activation (PAC-1 binding, 86.2 +/- 8.9%) and platelet-neutrophil binding (58.0 +/- 6.4%). The GPIIb/IIIa antagonists inhibited in a concentration-dependent manner platelet aggregation (IC50 of 100 nM for abciximab and tirofiban and 50 nM for roxifiban) and PAC-1 binding, without any effect on P-selectin. None of these agents affected significantly platelet-neutrophil binding, whereas an anti-P-selectin Mab abolished this binding and amplified the effect of abciximab on platelet aggregation. These results indicate that the effects of these GPIIb/IIIa antagonists on platelet aggregation are not related to inhibition of platelet activation, as P-selectin levels and platelet-neutrophil binding remained unaffected, and highlight the participation of P-selectin with GPIIb/IIIa in platelet aggregation.

Background —P-selectin mediates leukocyte recruitment to activated platelets and endothelium through its high-affinity receptor P-selectin glycoprotein ligand-1 (PSGL-1). Platelet and leukocyte activation and binding have been reported after coronary angioplasty and were correlated with restenosis. We investigated the effect of a recombinant soluble PSGL-1 (rPSGL-Ig) on the adhesion of platelets and neutrophils and the development of restenosis after double arterial injury. Methods and Results —Four weeks after angioplasty of both carotid arteries in pigs, a second angioplasty was performed at the same sites, 15 minutes after a single administration of vehicle or rPSGL-1 (1 mg/kg IV). Animals were euthanized 1 hour, 4 hours, 1 week, or 4 weeks later. Adhesion of autologous 51 Cr-platelets and 111 In-neutrophils was quantified and histological/morphometric analyses were performed. Although rPSGL-Ig did not affect adherence of these cells 1 hour after injury, it significantly reduced the adhesion of platelets (50% at 4 hours and 85% at 1 week) and neutrophils (50% at 4 hours and 78% at 1 week) to deeply injured arteries. At 4 weeks, the residual lumen was 63% larger in rPSGL-Ig–treated arteries as compared with control arteries (6.1±0.6 versus 3.8±0.1 mm 2 ; P 2 in control). The ratio of the external elastic lamina of injured to uninjured reference segments was >1 in treated arteries and Conclusions —P-selectin antagonism with rPSGL-Ig inhibits early platelet/leukocyte adhesion on injured arteries and reduces restenosis through a positive impact on vascular remodeling. Hence, rPSGL-Ig may have potential in the prevention of restenosis.

Introduction Platelet P-selectin is a thrombo-inflammatory molecule involved in platelet activation and aggregation. This may occur via the adhesive function of P-selectin and its potential capacity to trigger intracellular signaling. However, its impact on platelet function remains elusive. This study was therefore designed to investigate the relationship between the signaling potential of platelet P-selectin and its function in platelet physiology. Methods and Results Human and mouse platelets were freshly isolated from whole blood. Platelet activation was assessed using flow cytometry and western blot analysis, while platelet physiological responses were evaluated through aggregation, microaggregate formation and in a thrombosis model in wild-type and P-selectin-deficient (CD62P−/−) mice. Interaction of P-selectin with its high-affinity ligand, a recombinant soluble form of P-Selectin Glycoprotein Ligand-1 (rPSGL-1), enhances platelet activation, adhesion and microaggregate formation. This augmented platelet microaggregates requires an intact cytoskeleton, but occurs independently of platelet αIIbβ3. Thrombus formation and microaggregate were both enhanced by rPSGL-1 in wild-type, but not in CD62P−/− mice. In addition, CD62P−/− mice exhibited thrombosis abnormalities without an αIIbβ3 activation defect. Conclusions This study demonstrates that the role of platelet P-selectin is not solely adhesive; its binding to PSGL-1 induces platelet activation that enhances platelet aggregation and thrombus formation. Therefore, targeting platelet P-selectin or its ligand PSGL-1 could provide a potential therapeutic approach in the management of thrombotic disorders.

Objective— CD40 ligand is a thromboinflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40 ligand (sCD40L), which has been shown to influence platelet activation, although its exact functional impact on platelets and the underlying mechanisms remain undefined. We aimed to determine the impact and the signaling mechanisms of sCD40L on platelets. Methods and Results— sCD40L strongly enhances platelet activation and aggregation. Human platelets treated with a mutated form of sCD40L that does not bind CD40, and CD40 −/− mouse platelets failed to elicit such responses. Furthermore, sCD40L stimulation induces the association of the tumor necrosis factor receptor–associated factor-2 with platelet CD40. Notably, sCD40L primes platelets through activation of the small GTPase Rac1 and its downstream target p38 mitogen-activated protein kinase, which leads to platelet shape change and actin polymerization. Moreover, sCD40L exacerbates thrombus formation and leukocyte infiltration in wild-type mice but not in CD40 −/− mice. Conclusion— sCD40L enhances agonist-induced platelet activation and aggregation through a CD40-dependent tumor necrosis factor receptor–associated factor-2/Rac1/p38 mitogen-activated protein kinase signaling pathway. Thus, sCD40L is an important platelet primer predisposing platelets to enhanced thrombus formation in response to vascular injury. This may explain the link between circulating levels of sCD40L and cardiovascular diseases.

Various antioxidants, including polyphenols, prevent the development of atherosclerosis in animal models, contrasting with the failure of antioxidants to provide benefits in patients with established atherosclerosis. We therefore tested in a mouse model the hypothesis that although catechin is atheroprotective in prevention, catechin brings no global vascular protection when initiated after established atherosclerosis, because aging associated with dyslipidemia has induced irreversible dysfunctions. To this end, LDLr−/−; hApoB+/+ atherosclerotic (ATX, 9 mo old) and pre-ATX (3 mo old) male mice were treated with catechin (30 mg·kg−1·day−1) up to 12 mo of age. Vascular function and endothelium/leukocyte interactions were studied at 12 mo old. The renal artery endothelium-dependent dilations were impaired with age whereas adhesion of leukocytes onto the native aortic endothelium was increased ( P < 0.05). Aortic oxidative stress [reactive oxygen species (ROS)] increased ( P < 0.05) at 3 mo in ATX and at 12 mo in wild-type mice. Aorta mRNA expression of NADPH oxidase increased, whereas that of manganese superoxide dismutase decreased in 12-mo-old ATX mice only. In mice with established ATX, catechin (from 9 to 12 mo) reduced ( P < 0.05) by ∼60% ROS without affecting plaque burden. Notably, catechin worsened endothelial dysfunction and further increased leukocyte adhesion ( P < 0.05) in ATX mice. In contrast, the same catechin treatment reversed all age-related dysfunctions in wild-type mice. On the other hand, in pre-ATX mice treated for 9 mo with catechin, plaque burden was reduced by 64% ( P < 0.05) and all vascular markers were normalized to the 3-mo-old values. These results demonstrate that an antioxidant treatment is deleterious in mice with established atherosclerosis.

Background— Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Methods and Results— Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride–induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Conclusions— Peripheral blood mononuclear cell–derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis.

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCdelta, but not PKCalpha or PKCbeta, is required for collagen-induced phospholipase C-dependent signaling, activation of alpha(IIb)beta(3), and platelet aggregation. Analysis of PKCdelta phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by alpha(IIb)beta(3) outside-in signaling. Moreover, PKCdelta triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A(2), which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCdelta signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A(2).

Angiopoietin-1 (Ang1) and -2 (Ang2) are endothelial growth factors that bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by the recruitment of pericytes. In contrast, Ang2 is classically considered as a Tie2 antagonist, counteracting the stabilizing action of Ang1. Inflammation exists in a mutually dependent association with angiogenesis and we have therefore studied the capacity of angiopoietins to modulate proinflammatory activities, namely P-selectin translocation and neutrophil adhesion onto endothelial cells. We observed that both Ang1 and Ang2 increased these biologic activities. Furthermore, combination of Ang1/Ang2 induced an additive effect on neutrophil adhesion but not on P-selectin translocation. In an attempt to clarify this phenomenon, we found that angiopoietins can directly activate neutrophils through Tie2 signaling as well as modulate platelet-activating factor (PAF) synthesis and β2 integrin functional up-regulation. Together, our data demonstrate that angiopoietins could promote acute recruitment of leukocytes, which might contribute to facilitate vascular remodeling and angiogenesis.

The selectin family of cell-adhesion molecules contributes to the interactions of leukocytes and platelets at the site of vascular injury. Such interactions enhance inflammatory reactions and thrombus formation during the arterial response to injury. In this study, we investigated the effects of a selectin inhibitor (Fucoidan) on platelet and neutrophil interactions after arterial injury produced by angioplasty in pigs. [ 51 Cr]-platelet deposition and [ 111 In]-neutrophil adhesion were quantified on intact, mildly, and deeply injured carotid arterial segments, produced by balloon dilation in control (saline, n = 7) and Fucoidan-treated (i.v.; 1 mg/kg, n = 6; 5 mg/kg, n = 5) pigs. In the control group, platelet deposition (×10 6 /cm 2 ) was influenced by the severity of injury and increased significantly (p < 0.05) from 0.06 ± 0.06 on intact endothelium to 3.8 ± 0.6 and 33.6 ± 4.9 on mildly and deeply injured segments, respectively. Fucoidan, 1 mg/kg, had no significant effect, although doses of 5 mg/kg reduced platelet deposition by 73% on deeply injured segments. The level of neutrophil adhesion (×10 3 /cm 2 ) was also influenced by the severity of injury: it increased in the control group from 8.8 ± 2.5 on intact endothelium to 226.6 ± 45.5 and 397.4 ± 61.3 on mildly and deeply injured arterial segments, respectively (p < 0.05). Again, 1 mg/kg Fucoidan had no effect, although doses of 5 mg/kg reduced neutrophil adhesion by 92% and by 84% on mildly and deeply injured segments, respectively. The effects of Fucoidan were associated with a 51% decrease in the vasoconstrictive response at the site of arterial injury. However, Fucoidan had no significant effect on either platelet aggregation or activated clotting time (ACT). In the in vitro perfusion experiments, Fucoidan inhibited both isolated platelet, and neutrophil, adhesion to damaged arterial surfaces. This inhibition was more pronounced in experiments using mixed cell preparations, indicating that Fucoidan interferes with platelet and neutrophil interactions. These results highlight the importance of selectins in the acute physiopathologic reactions related to platelet-neutrophil interactions after arterial injury.

Abstract —The adhesion of neutrophils to damaged arterial surfaces is increased in the presence of platelets by a mechanism implicating platelet P-selectin. Such interactions may enhance thrombus formation and the vascular response to injury. In this study, we investigated the effects of a selectin blocker (CY-1503), an analogue of sialyl Lewis x , on platelet and neutrophil interactions after arterial injury produced by angioplasty in pigs. 51 Cr-platelet deposition and 111 In-neutrophil adhesion were quantified on intact, mildly and deeply injured carotid arterial segments, produced by balloon dilation, in control (saline, n=8) and treated (CY-1503, 15 mg/kg IV, n=7) pigs. The hematological parameters, the aggregation of whole blood in response to adenosine diphosphate, and the activating clotting time, as well as the heart rate and mean arterial blood pressure, were similar among groups and were not influenced significantly by CY-1503. The level of platelet and neutrophil adhesion increased significantly with the severity of arterial injury but was not influenced by CY-1503 on intact and mildly injured arterial segments. However, at the site of deep arterial injury, CY-1503 treatment was associated with a 58% reduction ( P 3 neutrophils/cm 2 in the control group to 186.8±38.7×10 3 neutrophils/cm 2 in the CY-1503–treated group, whereas platelet deposition remained unchanged (43.4±15.6×10 6 platelets/cm 2 versus 50.1±12.2×10 6 platelets/cm 2 in the control group). In in vitro adhesion experiments, using isolated platelet and neutrophil suspensions, we found that CY-1503 interfered with the adhesion of neutrophils to damaged arterial surfaces only in the presence of platelets. In contact with thrombogenic arterial surfaces, adherent and activated platelets supports neutrophil adhesion at the site of deep injury by an adhesive interaction involving neutrophil sialyl Lewis x . The inhibitory effect of CY-1503 on neutrophil interaction with adherent platelets may be clinically relevant in patients undergoing percutaneous transluminal coronary angioplasty where platelet and neutrophil interactions may enhance the acute and chronic arterial response to injury.

A controlled redox environment is essential for vascular cell maturation and function. During aging, an imbalance occurs, leading to endothelial dysfunction. We hypothesized that, according to the concept of hormesis, exposure to physiologic oxidative stress during the maturation phase of the endothelium will activate protective pathways involved in stress resistance. C57Bl/6 mice were treated with the polyphenol catechin for the last 3 (post-maturation) or 9 months prior study at 12 months of age. Endothelial dysfunction, assessed by acetylcholine-induced dilations of isolated renal arteries, was present at 12 months (Pphox and reduced MnSOD (P