Platelet PECAM-1 inhibits thrombus formation in vivo.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

Antibody cross-linking of human platelet P-selectin induces calcium entry by a mechanism dependent upon Fcgamma receptor IIA.

It is established that antibody-induced cross-linking of platelet surface receptors is able to activate platelets in a manner dependent upon FcgammaRIIA. This has not, however, previously been shown for the adhesion receptor P-selectin, and since there is evidence that P-selectin may couple to activation events, it was important to address whether antibody cross-linking of this receptor induced signalling events, and whether this was dependent on FcgammaRIIA. Here we show that although addition of soluble P-selectin ligand rPSGL-Ig alone is not able to induce calcium signalling, further addition of a full-length rabbit anti human IgG leads to a sustained rise in [Ca 2+ ]i. This was due to an increase in the frequency and amplitude of transient calcium spiking in single platelets. The response was dependent upon engagement of both P-selectin and FcgammaRIIA since blocking anti-bodies to either receptor inhibited the response. The calcium rise is mediated primarily by induction of a calcium entry mechanism involving the Na(+)-Ca(2+) exchanger operating in reverse mode, since it was blocked by inhibitors of Na(+)-Ca(2+) exchange, bepridil and 5 mM NiCl(2).

GPIb potentiates GPVI-induced responses in human platelets.

Platelet adhesion to vascular subendothelial proteins at the site of blood vessel injury is critical for initiating haemostasis. Collagen is a major matrix protein that binds plasma von Willebrand factor (vWF) when the endothelium becomes damaged and therefore in vivo platelets are likely to encounter both of these agonists simultaneously, through glycoprotein VI (GPVI) and alpha2beta1 receptors for collagen and GPIb-V-IX and alphaIIbbeta3 receptors for vWF. We hypothesised a potentiatory role for vWF that would synergise with collagen leading to functional activation and show this to be the case for platelet aggregation, 5-HT secretion and calcium responses. Synergy between these two agonists is likely to involve receptors GPVI and GPIb-V-IX, for collagen and vWF, respectively, since 5-HT secretion in response to collagen is potentiated by vWF in the presence of either EGTA or EDTA, which prevent binding to integrins alphaIIbbeta3 (EGTA) or both alphaIIbbeta3 and alpha2beta1 (EDTA). In addition, vWF is also able to potentiate 5-HT secretion responses to collagen-related peptide, confirming that GPVI is able to support synergy with vWF. These findings are important in that they reveal a novel role for vWF in platelet activation as a potentiator of platelet activation by collagen.

Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin.

Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocyte-derived microparticles in human platelet-poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor- and PSGL-1-containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P-selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking.

Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.

We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. We observed platelet deposition, tissue factor accumulation and fibrin generation after laser-induced endothelial injury in a single developing thrombus. The initiation of blood coagulation in vivo entailed the initial accumulation of tissue factor on the upstream and thrombus-vessel wall interface of the developing thrombus. Subsequently tissue factor was associated with the interior of the thrombus. Tissue factor was biologically active, and was associated with fibrin generation within the thrombus.