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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.

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.