Microparticles facilitate neutrophil/platelet crosstalk

In this issue of Blood, Pluskota and colleagues reveal a new prothrombotic pathway initiated by neutrophil-derived microparticles through the interaction of the leukocyte integrin Mac-1 (α_Mβ₂) on microparticles with its counterreceptor on platelets, GPIbα, the major ligand-binding subunit of the GPIb-IX-V complex (see figure). These findings have broad implications for the pathogenesis, monitoring, and/or future therapy of inflammatory/thrombotic disease.

All inflammatory/thrombotic vascular cells (leukocytes, platelets, endothelial cells) generate microparticles, small membrane-bound vesicles budded off from the parent cell that generally reflect that cell's surface-receptor profile. Microparticles are not just by-products of cellular activation or apoptosis, but are functional and modulate the activity of other cells. They also represent potentially selective biomarkers of diseases involving cellular perturbation.¹  One important role for leukocyte-derived microparticles is to deliver clot-promoting tissue factor rapidly to a developing thrombus.²  In this case, P-selectin glycoprotein ligand-1 (PSGL-1) on the microparticle targets activated platelets by binding platelet P-selectin. Activated platelets are also strongly procoagulant due to increased exposure of phosphatidyl serine, the expression of coagulation factor-binding receptors, the secretion of polyphos-phates, and through glycoprotein (GP) Ibα, which acts as a molecular scaffold local-izing coagulation factors such as high-molecular-weight kininogen, factors XI and XII, and thrombin.³  Interestingly, the present findings indicate that leukocyte-derived microparticles play another poten-tial role in thrombus development by acting as a novel platelet agonist targeting GPIbα. This provides a potential explanation of how chronic inflammation can perpetuate a prothrombotic phenotype.

Pluskota and colleagues show that microparticles derived from activated neutrophils express functional Mac-1 and act as platelet agonists by virtue of Mac-1 binding platelet GPIbα. GPIbα is a constitutively expressed platelet-adhesion receptor that initiates thrombus formation at arterial shear rates by binding von Willebrand factor (VWF) or other ligands.^3,4  Engagement of GPIbα by Mac-1–bearing microparticles leads to phosphorylation of the signaling protein Akt, which is an intermediary in GPIbα-dependent signaling downstream of phosphatidyl inositol (PI) 3-kinase activation, and upstream of surface expression of P-selectin and activation of α_IIbβ₃ (which binds fibrinogen and VWF and mediates platelet aggregation). P-selectin expressed on the activated platelets can bind PSGL-1, strengthening the association between the activated neutrophil microparticle and the platelets. It has been previously established that GPIbα directly binds activated leukocyte Mac-1,⁵  as well as P-selectin expressed on activated platelets/endothelial cells,^3,6  enabling GPIbα-mediated platelet-leukocyte-endothelial cell crosstalk; for example, platelets strongly promote vascular adhesion of leukocytes in atherogenesis.⁷  Binding of P-selectin on activated platelets or platelet microparticles to leukocyte PSGL-1 initiates signals activating Mac-1, thereby enabling integrin-dependent adhesion to endothelial cells via intercellular adhesion molecule-1 (ICAM-1), or to mural platelets via GPIbα (see figure).

One of the key findings from this study is that, whereas microparticles derived from unstimulated neutrophils contain relatively low levels of Mac-1 in an inactive state, microparticles derived from PMA- or PAF-activated neutrophils contain approximately10-fold enrichment of Mac-1 present in an activated state, where the insert or “I” domain in the α_M subunit is in a high-affinity ligand-binding form. This was established by binding of conformation-dependent antibodies or the Mac-1 ligand, fibrinogen. These relative levels of inactive/active Mac-1 in microparticles are thought to depend on lipid raft constituency before or after stimulation. This is because microparticles are likely derived from these membrane domains, and activated Mac-1 is enriched in rafts following neutrophil stimulation. There is thus a built-in differential adhesive capacity of microparticles from unstimulated versus activated neutrophils to facilitate interaction with either activated platelets (via PSGL-1/P-selectin) or resting platelets (via active Mac-1/GPIbα), respectively. This implies that microparticle-dependent platelet activation occurs under conditions where neutrophils are also activated; conversely, microparticles generated from quiescent neutrophils (expressing inactive Mac-1) would be limited to interacting only with activated platelets (expressing surface P-selectin).

What, then, is the relevance of these new findings to inflammation and thrombosis, and is there potential diagnostic value in measuring the proportion of total neutrophil-derived microparticles expressing activated Mac-1 as a biomarker for pathological inflammation/thrombosis? This remains to be determined, but the new discoveries by Pluskota and colleagues demonstrate a clear prothrombotic functional pathway for this microparticle subtype.