Autoantibodies to heat shock protein 60 promote thrombus formation in a murine model of arterial thrombosis.

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.

P-selectin antagonism with recombinant p-selectin glycoprotein ligand-1 (rPSGL-Ig) inhibits circulating activated platelet binding to neutrophils induced by damaged arterial surfaces.

Neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial rolling and adhesion of neutrophils to P-selectin on activated endothelium and platelets. Platelet-neutrophil activation and binding occur in the blood of patients with arterial diseases, suggesting that arterial damage leads to these phenomena. We investigated the influence of endothelial surface integrity on circulating platelet activation and binding to neutrophils and the mechanism involved in these interactions. Expression of P-selectin on human platelets and their binding to neutrophils was determined by flow cytometry at baseline after thrombin activation and after exposure for 15 min to intact and damaged arterial surfaces in flow chambers. Expression of platelet P-selectin at baseline and after perfusion over intact endothelium averaged 13.8 +/- 1.2 and 12.7 +/- 1.8%, respectively, and increased significantly to 19.7 +/- 1.8% (P < 0.05) after perfusion over damaged arteries. In mixed neutrophil/platelet suspensions, the percentage of neutrophils that bind platelets increased significantly also, from 10.8 +/- 1.6% at baseline to 39.7 +/- 2.9% (P < 0.05) after perfusion over damaged arteries compared with 69.7 +/- 2.5% with thrombin. This binding was completely inhibited by a recombinant soluble PSGL-1 (rPSGL-Ig) and anti-P-selectin and PSGL-1-blocking monoclonal antibodies. The inhibitory effect of rPSGL-Ig correlated well with its binding to platelets (r = 0.98, P < 0.001). Circulating platelets are activated upon contact with damaged arteries, thereby enhancing their adhesive interactions with neutrophils via P-selectin and PSGL-1. Inhibition of this binding with rPSGL-Ig may constitute a target in the treatment of inflammatory and thrombotic reactions.

Recombinant soluble p-selectin glycoprotein ligand-1-Ig reduces restenosis through inhibition of platelet-neutrophil adhesion after double angioplasty in swine.

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:<0.002). The neointimal area was slightly reduced (0.5 in rPSGL-Ig versus 0.7 mm(2) in control). The ratio of the external elastic lamina of injured to uninjured reference segments was >1 in treated arteries and <1 in control arteries. 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.

Inhibition of platelet-neutrophil interactions by Fucoidan reduces adhesion and vasoconstriction after acute arterial injury by angioplasty in pigs.

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. [51Cr]-platelet deposition and [111In]-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 (x10(6)/cm2) 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 (x10(3)/cm2) 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.

Selectin blockade reduces neutrophil interaction with platelets at the site of deep arterial injury by angioplasty in pigs.

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 Lewisx, on platelet and neutrophil interactions after arterial injury produced by angioplasty in pigs.51Cr-platelet deposition and 111In-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<0.01) in neutrophil adhesion, from 446.7+/-72.6x10(3) neutrophils/cm2 in the control group to 186.8+/-38.7x10(3) neutrophils/cm2 in the CY-1503-treated group, whereas platelet deposition remained unchanged (43.4+/-15.6x10(6) platelets/cm2 versus 50.1+/-12.2x10(6) platelets/cm2 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 Lewisx. 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.