Figure 1.
Platelet-specific loss of both PITPs impairs ex vivo platelet aggregation/secretion and spreading. (A) Western blot analysis of PITP expression in human platelets compared with their mouse counterparts. (B) Western blot–based densitometry quantification of individual PITP isoforms in human platelets. (C) Schematic representation of the conditional targeting strategy for Pitpβ. A 1.89 kb genomic DNA of PITPβ (which includes exons 4-6) was targeted by the insertion of loxP recombination sites. The Neo cassette was removed by crossing with the FRT mice before further crossing with PF4-Cre transgenic mice. (D) Western blot of platelet lysates demonstrating the specific deletion of the PITPβ isoform in Pitpβfl/flPf4-Cre+ mice. (E) Complete blood count analyses show mild thrombocytopenia in Pitpβfl/flPf4-Cre+ mice and more severe thrombocytopenia in Pitpαfl/fl/βfl/flPf4-Cre+ mice compared with their respective littermate controls (n = 6 for Pitpβfl/flPf4-Cre- mice; n = 8 for Pitpβfl/flPf4-Cre+ mice; n = 13 for Pitpαfl/fl/βfl/flPf4-Cre- mice; and n = 9 for Pitpαfl/fl/βfl/flPf4-Cre+ mice; error bars are standard deviation(s.d.); P values are shown obtained from unpaired t test). (F,G) Ex vivo analysis of platelet aggregation and dense granule secretion. PITPβ-null platelets aggregate normally, but dense granule secretion was impaired in response to low-dose thrombin (0.05 U/mL) and collagen (10 μg/mL), as measured by adenosine triphosphate release (F). Deleting both PITP isoforms increased the severity of aggregation and secretion defects (G). Adenosine triphosphate secretion traces start at 100% and trend downward, and aggregation traces start at 0% and trend upward. Traces are representative of 5 separate experiments per condition. (H) Spreading of PITPβ-null and PITPα/β-null platelets on fibrinogen after stimulation with thrombin (0.025 U/mL) revealed that PITPα/β-null platelets had a spreading defect, whereas PITPβ-null platelets spread normally. (I) PITPα/β-null platelet spreading was quantified as the total cumulative area of platelets per field (n = 3 per group). (J) The number of adherent PITPα/β-null platelets was quantified as the average number per field under a 100× microscope. (n = 5 per group; error bars represent s.d.; P values are shown, unpaired t test).

Platelet-specific loss of both PITPs impairs ex vivo platelet aggregation/secretion and spreading. (A) Western blot analysis of PITP expression in human platelets compared with their mouse counterparts. (B) Western blot–based densitometry quantification of individual PITP isoforms in human platelets. (C) Schematic representation of the conditional targeting strategy for Pitpβ. A 1.89 kb genomic DNA of PITPβ (which includes exons 4-6) was targeted by the insertion of loxP recombination sites. The Neo cassette was removed by crossing with the FRT mice before further crossing with PF4-Cre transgenic mice. (D) Western blot of platelet lysates demonstrating the specific deletion of the PITPβ isoform in Pitpβfl/flPf4-Cre+ mice. (E) Complete blood count analyses show mild thrombocytopenia in Pitpβfl/flPf4-Cre+ mice and more severe thrombocytopenia in Pitpαfl/fl/βfl/flPf4-Cre+ mice compared with their respective littermate controls (n = 6 for Pitpβfl/flPf4-Cre- mice; n = 8 for Pitpβfl/flPf4-Cre+ mice; n = 13 for Pitpαfl/fl/βfl/flPf4-Cre- mice; and n = 9 for Pitpαfl/fl/βfl/flPf4-Cre+ mice; error bars are standard deviation(s.d.); P values are shown obtained from unpaired t test). (F,G) Ex vivo analysis of platelet aggregation and dense granule secretion. PITPβ-null platelets aggregate normally, but dense granule secretion was impaired in response to low-dose thrombin (0.05 U/mL) and collagen (10 μg/mL), as measured by adenosine triphosphate release (F). Deleting both PITP isoforms increased the severity of aggregation and secretion defects (G). Adenosine triphosphate secretion traces start at 100% and trend downward, and aggregation traces start at 0% and trend upward. Traces are representative of 5 separate experiments per condition. (H) Spreading of PITPβ-null and PITPα/β-null platelets on fibrinogen after stimulation with thrombin (0.025 U/mL) revealed that PITPα/β-null platelets had a spreading defect, whereas PITPβ-null platelets spread normally. (I) PITPα/β-null platelet spreading was quantified as the total cumulative area of platelets per field (n = 3 per group). (J) The number of adherent PITPα/β-null platelets was quantified as the average number per field under a 100× microscope. (n = 5 per group; error bars represent s.d.; P values are shown, unpaired t test).

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