Phosphatidylserine-rich platelet blebs enhance hemostasis after mucous membrane injury but not after skin or vascular injury: Studies with ANO6-deficient mice

Abstract Platelets expose high levels of phosphatidylserine (PS) on large membrane blebs in response to strong agonists. These annexin A5-positive platelets are termed “procoagulant platelets.” Formation of PS-rich platelet blebs is impaired in people and mice deficient in functional TMEM16F/ANO6, a seven transmembrane domain protein that acts as a channel for PS and other phospholipids. Deficiency of ANO6 is the molecular cause of Scott Syndrome, a disorder characterized by mucous membrane bleeding and decreased platelet-supported thrombin generation but normal bleeding time. We wished to further probe the impact of PS-rich platelet blebs on hemostasis utilizing an ANO6 deficient mouse model. ANO6 heterozygous mice were bred on a pure C57Bl/6J genetic background with a gene trap between exons one and two (ANO6-/+). ANO6-/+ intercross produced no viable null pups at weaning. Timed matings demonstrated the expected mendelian ratio of ANO6-/- mice at E18.5 indicating perinatal lethality. This was partially rescued by outcrossing mice onto 129/SvJ mice. ANO6-/- mice on a 50/50 mixed genetic background (C57BL/6J and 129/SvJ) exhibited decreased PS exposure on platelets in response to TRAP4, thrombin (IIa), convulxin (CVX), TRAP4 + CVX, and A23187, and prolonged tail bleeding times compared to WT littermate controls. These data are consistent with published data for mice on mixed genetic background. However, after mice were backcrossed more than 4 generations into the C57BL/6 background the phenotype changed. To preserve the rescue genes, ANO6-/- mice were backcrossed with C57BL6 to produce the next ANO6+/- generation. The resulting ANO6+/- mice were then crossbred to produce the next generation of ANO6-/- mice that could be further backcrossed to C57BL6. This alternating mating pattern was repeated to produce ANO6-/- mice on the C57Bl6 genetic background with the rescue genes. Subsequent experiments on these mice, demonstrated no difference in PS exposure on resting platelets or in response to IIa. PS exposure was impaired on the “high PS” fraction of platelets following IIa + CVX or A23187 stimulation. The fraction of platelets in the high PS gate was not different but the quantity of PS exposed was 50-75% lower. In a novel 3-stage assay, where platelets support factor Xa generation followed by IIa generation, we found approximately 4x more IIa generated by +/+ platelets vs. -/- platelets with platelet stimulation by TRAP4 + CVX. ANO6-/- mice exhibited normal tail bleeding times using 3 different techniques. Likewise, bleeding times and blood loss from jugular vein puncture were comparable to wild-type littermate controls. Similarly, IIa generation and plasma clotting times were the same when supported by -/- mice vs. +/+ mice. These data demonstrate that ANO6 deficiency does not result in an overt bleeding phenotype and suggest strain-specific modifiers of these assays on a mixed background. We hypothesized that the hemostatic deficit of -/- mice would be uncovered when coagulation was triggered by higher concentrations of initiators that overwhelm intrinsic anticoagulation pathways. Therefore, we evaluated plasma clotting with increasing concentrations of factor XIa (intrinsic) or factor Xa (extrinsic pathway). Under these conditions, clotting was faster and +/+ platelets accelerated coagulation more than -/- platelets after TRAP4 + CVX and A23187 stimulation. To evaluate hemostasis when coagulation pathways are plausibly upregulated, we developed a mucous membrane bleeding assay in mice. Gastric ulcers were induced by indomethacin via gastric gavage. Gastric bleeding was evaluated over the next 64 hr using a chemiluminescence assay for heme in mouse feces. ANO6-/- mice lost twice as much blood as littermate controls (p < 0.05). Together these results indicate that the mouse model emulates Scott syndrome with excess mucous membrane bleeding and decreased platelet thrombin generation but normal bleeding time. However, there is no perinatal lethality reported in humans. Our data suggest that formation of PS-rich platelet blebs and the quantity of PS on the blebs is not rate-limiting for ordinary hemostasis. These data suggest the presence of an uncharacterized mechanism of coagulation suppression on PS-rich platelet membranes when the level of coagulation initiation is low or moderate.

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