Platelet ITAM signaling is critical for vascular integrity in inflammation

Abstract

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

Thrombospondin 1 requires von Willebrand factor to modulate arterial thrombosis in mice

“…27 Anti-hIL-4R (clone 25463; R&D Systems) antibody at a concentration of 2.5 mg/g body weight was infused through retro-orbital plexus to deplete platelets from GPIba/human IL4R transgenic mice. After 2 hours, 5 3 10 8 platelets from WT or Tsp1 2/2 mice were injected through retro-orbital plexus, and mice were subjected to FeCl 3 injury model as described above.…”

“…To specifically determine whether TSP1 released from platelets contributes to arterial thrombosis in vivo, we depleted platelets from GPIba/human IL4R transgenic mice using anti-hIL4R antibody as described, 27,29 followed by transfusion of platelets from either WT or Tsp1 2/2 mice ( Figure 6A). GPIba/human IL4R transgenic mice lack murine GPIba, but they express the extracellular domain of human IL-4 receptor (fused to the transmembrane and cytoplasmic domains of human GPIba).…”

Platelet GPVI repairs its own damage

“…Significantly, this effect is independent of hemostasis. 3 They firstly confirmed the increase in bleeding in the rpA model in thrombocytopenic mice and further demonstrated that bleeding is also seen in GPVI-deficient mice, which have a normal platelet count. Moreover, they showed that bleeding was prevented by depletion of neutrophils by over 70%.…”

Platelets at the vascular interface

“…The authors suggest that the CLEC2-podoplanin interaction is required as a fail-safe in case GPVI is malfunctional, a hypothesis that is supported by the observation that megakaryocyte/platelet-specific deletion of CLEC2 in mice does not cause increased inflammatory hemorrhage, unless the mice are crossed with GPVI-deficient mice[67]. Interestingly, the preliminary studies by Rayes et al also suggest that platelets contribute to vascular integrity in LPS challenged lungs by a GPVI/CLEC2-independent but GPIbα-dependent mechanism, findings that are in conflict with previous findings [40]. Additional studies are required to clarify whether there is indeed an organ and/or inflammatory trigger-specific platelet response in this form of hemostasis.…”

“…Without platelets (severe thrombocytopenia), these vessels become leaky for RBCs, causing hemorrhage in the affected areas[62]. We have recently demonstrated that GPCRs are not critical for platelet function in this setting[40,65]. Instead, vascular integrity at sites of inflammation depends strongly on platelet ITAM signaling, mediated by GPVI and CLEC-2.…”