Todd M. Getz

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.

BACKGROUND: Platelet (PLT) storage has been limited to 5 days at room temperature due to metabolic decline and risk for bacterial contamination. Refrigeration preserves PLT metabolism and function as well as limits bacterial growth; however, cold storage of PLTs also leads to aggregate formation. We hypothesized that storage of PLT concentrates at 4°C leads to glycoprotein (GP)IIb-IIIa activation and thus aggregate formation through fibrinogen binding and that this could be prevented by storing PLTs in PLT additive solution (PAS) without compromising PLT function. STUDY DESIGN AND METHODS: Apheresis PLTs in plasma (AP) or apheresis PLTs in PAS were stored at 22 or 4°C for up to 15 days. Measurements include PLT counts, blood gases, aggregation response, flow cytometry analysis of integrin levels, activation markers, and microparticle formation. RESULTS: Storage of AP 4°C led to a gradual decline in PLT count and an increase in aggregate formation that was mediated by intracellular calcium leak and fibrinogen receptor activation. Storage of PAS at 4°C prevented aggregate formation due to dilution of plasma fibrinogen. PAS stored at 4°C maintained aggregation responses to multiple agonists better than 22°C controls. CONCLUSION: Storage of AP at 4°C leads to low level GPIIb-IIIa activation and results in aggregate formation over time. Separating the PLTs from the plasma component and storing them in PAS at 4°C resolves aggregate formation and preserves the metabolic and functional responses of these stored PLTs.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

Aromatic residues are relatively rare within the collagen triple helix, but they appear to play a specialized role in higher-order structure and function. The role of aromatic amino acids in the self-assembly of triple-helical peptides was investigated in terms of the kinetics of self-association, the nature of aggregated species formed, and the ability of these species to activate platelet aggregation. The presence of aromatic residues on both ends of a type IV collagen model peptide is observed to greatly accelerate the kinetics of self-association, decreasing the lag time and leading to insoluble, well-defined linear fibrils as well as small soluble aggregates. Both macroscopic visible aggregates and small multimolecular complexes in solution are capable of inducing platelet aggregation through the glycoprotein VI receptor on platelets. Proline-aromatic CH...pi interactions are often observed within globular proteins and in protein complexes, and examination of molecular packing in the crystal structure of the integrin binding collagen peptide shows Phe interacts with Pro/Hyp in a neighboring triple-helical molecule. An intermolecular interaction between aromatic amino acids and imino acids within the triple helix is also supported by the observed inhibitory effect of isolated Phe amino acids on the self-association of (Pro-Hyp-Gly)(10). Given the high fraction of Pro and Hyp residues on the surface of collagen molecules, it is likely that imino acid-aromatic CH...pi interactions are important in formation of higher-order structure. We suggest that the catalysis of type I collagen fibrillogenesis by nonhelical telopeptides is due to specific intermolecular CH...pi interactions between aromatic residues in the telopeptides and Pro/Hyp residues within the triple helix.