Assessment of Neonatal Platelet Dense Granule Trafficking Downstream of Protease-Activated Receptors and P2Y1/ P2Y12

Assessment
of neonatal platelet dense granule trafficking downstream of protease-activated
receptors and P2Y₁/ P2Y₁₂

Kendra
R. Jones,¹ Anh T. P. Ngo,² Annachiara Mitrugno,² Anne D. Rocheleau,² Sandra Baker-Groberg,² Joseph E. Aslan,² Stewart Worthington,³ Alysia Cox,³ Susan Lattimore,³ Michael Recht,³ Kristina M. Haley,³ Owen J. T. McCarty²

¹School of Chemical, Biological, and Environmental Engineering,
Oregon State University, Corvallis, OR

²Department of Biomedical Engineering, Oregon Health & Science
University, Portland, OR

³The Hemophilia Center, Oregon Health & Science University,
Portland, OR

Introduction:
Platelet
dense granule secretion follows activation of protease-activated receptors
(PARs) 1 and 4 in human platelets to result in ADP release with subsequent
signaling of the ADP receptors P2Y₁/P2Y₁₂ and amplification of
adult platelet activation and aggregation^2,3. Neonatal platelets
are hyporesponsive to multiple platelet agonists as compared to adult
platelets. These mechanisms are unknown, yet neonates do not manifest a
clinical bleeding tendency.  Our group has shown that neonatal platelets
(neonatelets) are hyporesponsive to the PAR1 agonist, thrombin receptor
activator peptide 6 (TRAP-6), in comparison to adult platelets. Therefore, we
hypothesize that the hyporesponsivity in neonates is due to either a deficiency
in platelet dense granule function and thus ADP release, and/or a lack of
synergy between P2Y₁/P2Y₁₂ and PARs. This study aimed to investigate: 1) dense granule
trafficking and release, and 2) PAR cleavage using small volume, whole blood
samples. Blood was tested using two novel assays developed by our group; a
super-resolution structured illumination microscopy (SR-SIM) and
chemiluminescent secretion assay. We also utilized fluorescent-activated cell
sorting (FACs) to assess synergy between P2Y₁₂ and PARs by detecting
markers of platelet activation.

Materials
and Methods: In accordance with an Institutional Review Board-approved
protocol, fresh adult blood was obtained from healthy donors by venipuncture
into 3.8% sodium citrate, and fresh full-term neonatal blood was collected at
24 hours of life via heel stick into trisodium citrate (0.38% w/v). Glass
bottom dishes were coated with Poly-L-lysine and seeded with adult or neonatal
whole blood previously fixed and diluted with 4% paraformaldehyde. Samples were
stained with platelet dense granule marker (CD63), PAR1 (ATAP2), PAR4 (14H6)
and imaged using 100x SR-SIM. Platelet dense granule release was measured as
light output generated following an ATP-luciferase reaction. Additionally, FACS
was performed using whole blood treated with TRAP-6, ADP, AYPGKF (PAR4
agonist), or a combination of ADP and TRAP-6/AYPGKF, and labeled for CD62P and
PAC-1. Total platelet glycoprotein (GP) expression was measured using a
BioCytex kit.

Results
and Discussion: Both dense granule trafficking via SR-SIM and the luminescent
assay suggest that neonatal dense granule secretion was unresponsive to
PAR1/PAR4 activation. Exogenous ADP appears to rescue the PARs response, but
not significantly when compared to the signal for ADP response alone.
Additionally, flow cytometry results demonstrate GP expression does not differ
in adults and neonates, indicating that the hyposensitivity to PAR1 agonists is
likely physiological.

Conclusion:
Our
hyposensitivity results suggest that neonatal dense granule trafficking and
secretion following PARs activation is impaired, when compared to adult
platelets. These results provide a potential explanation for hyposensitivity to
PAR agonists, and provides insight into platelet granules and synergy between
PARs and P2Y₁₂.

References:
(1)
Andrew M, et al. Blood 1992; 80:1998-2005. (2) Falker K, et al. Biochemical
Journal  2011; 436: 469-480. (3) Nylander S, et al. British
Journal of Pharmacology 2004; 142: 1325-1331. (4) Baker-Groberg SM,
et al. Thrombosis and Haemostasis; 14: 815-27.