Previous studies in our laboratory have shown that platelets are essential for the migration of eosinophils into the lungs of allergic mice, and that this is dependent on the functional expression of platelet P-selectin. We sought to investigate whether the same is true for nonallergic, acute inflammatory stimuli administered to distinct anatomic compartments. Neutrophil trafficking was induced in two models, namely zymosan-induced peritonitis and LPS-induced lung inflammation, and the platelet dependence of these responses investigated utilizing mice rendered thrombocytopenic. The relative contribution of selectins was also investigated. The results presented herein clearly show that platelet depletion (>90%) significantly inhibits neutrophil recruitment in both models. In addition, we show that P-selectin glycoprotein ligand-1, but not P-selectin, is essential for neutrophil recruitment in mice in vivo, thus suggesting the existence of different regulatory mechanisms for the recruitment of leukocyte subsets in response to allergic and nonallergic stimuli. Further studies in human blood demonstrate that low-dose prothrombotic and pro-inflammatory stimuli (CCL17 or CCL22) synergize to induce platelet and neutrophil activation, as well as the formation of platelet-neutrophil conjugates. We conclude that adhesion between platelets and neutrophils in vivo is an important event in acute inflammatory responses. Targeting this interaction may be a successful strategy for inflammatory conditions where current therapy fails to provide adequate treatment.

Glycoprotein Ib-IX-V (GPIb-IX-V) mediates platelet tethering to von Willebrand factor (VWF), recruiting platelets into the thrombus, and activates integrin ␣IIb␤3 through a pathway that is dependent on Src kinases. In addition, recent reports indicate that activation of ␣IIb␤3 by VWF is dependent on protein kinase G (PKG) and mitogen-activated protein (MAP) kinases. The present study compares the importance of these signaling pathways in the activation of ␣IIb␤3 by GPIb-IX-V. In contrast to a recent report, VWF did not promote an increase in cyclic guanosine monophosphate (cGMP), while agents that elevate cGMP, such as the nitrous oxide (NO) donor glyco-SNAP-1 (N-(␤-Dglucopyranosyl)-N 2 -acetyl-S-nitroso-D,Lpenicillaminamide) or the type 5 phosphosdiesterase inhibitor, sildenafil, inhibited rather than promoted activation of ␣IIb␤3 by GPIb-IX-V and blocked aggregate formation on collagen at an intermediate rate of shear (800 s ؊1 ). Additionally, sildenafil increased blood flow in a rabbit model of thrombus formation in vivo. A novel inhibitor of the MAP kinase pathway, which is active in plasma, PD184161, had no effect on aggregate formation on collagen under flow conditions, whereas a novel inhibitor of Src kinases, which is also active in plasma, PD173952, blocked this response. These results demonstrate a critical role for Src kinases but not MAP kinases in VWF-dependent platelet activation and demonstrate an inhibitory role for cGMP-elevating agents in regulating this process. IntroductionGlycoprotein Ib-IX-V (GPIb-IX-V), the receptor for von Willebrand factor (VWF), plays a critical role in thrombus formation in damaged blood vessels under high shear. [1][2][3][4] A fast on-rate of association between VWF and GPIb-IX-V allows the adhesion protein to tether (or capture) rapidly flowing platelets into the developing thrombus. A fast off-rate of dissociation, however, means that platelets rapidly detach from VWF, unless integrins such as ␣IIb␤3 or ␣2␤1 are activated by intracellular signals, thereby enabling them to bind their ligands and mediate stable adhesion and thrombus growth. Several surface receptors are able to mediate integrin activation, including the collagen receptor GPVI, the G protein-coupled receptors for adenosine diphosphate (ADP) and thromboxanes, P2Y 1 , P2Y 12 , and thromboxane prostanoid (TP) receptors. 5 Significantly, these receptors act in synergy to mediate integrin activation and thrombus growth. [6][7][8][9] It is recognized that GPIb-IX-V is also able to stimulate activation of ␣IIb␤3. However, the GPIb-IX-V complex generates a much weaker signal than many of the other agonists involved in thrombus formation, including collagen and ADP, thereby questioning the significance of this event. Indeed, the extent of activation is heavily dependent on experimental conditions, with activation being more readily seen in plasma than in washed platelets for reasons that remain unclear. [10][11][12][13][14][15] It is also difficult to ascertain the significance of activation of ␣IIb␤3 by GPIb...

It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) gamma-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR gamma-chain, Syk and PLCgamma2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCgamma2 nor functional responses, such as Ca(2+) elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbalpha in washed platelets does not promote functional responses.