Shear-Induced Platelet Activation is Sensitive to Age and Calcium Availability: A Comparison of Adult and Cord Blood.

INTRODUCTION: Antiplatelet therapy for neonates and infants is often extrapolated from the adult experience, based on limited observation of agonist-induced neonatal platelet hypoactivity and poor understanding of flow shear-mediated platelet activation. Therefore, thrombotic events due to device-associated disturbed flow are inadequately mitigated in critically ill neonates with indwelling umbilical catheters and infants receiving cardiovascular implants. METHODS: Whole blood (WB), platelet-rich plasma (PRP), and gel-filtered platelets (GFP) were prepared from umbilical cord and adult blood, and exposed to biochemical agonists or pathological shear stress of 70 dyne/cm2. We evaluated α-granule release, phosphatidylserine (PS) scrambling, and procoagulant response using P-selectin expression, Annexin V binding, and thrombin generation (PAS), respectively. Activation modulation due to depletion of intracellular and extracellular calcium, requisite second messengers, was also examined. RESULTS: Similar P-selectin expression was observed for sheared adult and cord platelets, with concordant inhibition due to intracellular and extracellular calcium depletion. Sheared cord platelet Annexin V binding and PAS activity was similar to adult values in GFP, but lower in PRP and WB. Annexin V on sheared cord platelets was calcium-independent, with PAS slightly reduced by intracellular calcium depletion. CONCLUSIONS: Increased PS activity on purified sheared cord platelets suggest that their intrinsic function under pathological flow conditions is suppressed by cell-cell or plasmatic components. Although secretory functions of adult and cord platelets retain comparable calcium-dependence, PS exposure in sheared cord platelets is uniquely calcium-independent and distinct from adults. Identification of calcium-regulated developmental disparities in shear-mediated platelet function may provide novel targets for age-specific antiplatelet therapy.

Assessment of neonatal, cord, and adult platelet granule trafficking and secretion.

Despite the transient hyporeactivity of neonatal platelets, full-term neonates do not display a bleeding tendency, suggesting potential compensatory mechanisms which allow for balanced and efficient neonatal hemostasis. This study aimed to utilize small-volume, whole blood platelet functional assays to assess the neonatal platelet response downstream of the hemostatic platelet agonists thrombin and adenosine diphosphate (ADP). Thrombin activates platelets via the protease-activated receptors (PARs) 1 and 4, whereas ADP signals via the receptors P2Y1 and P2Y12 as a positive feedback mediator of platelet activation. We observed that neonatal and cord blood-derived platelets exhibited diminished PAR1-mediated granule secretion and integrin activation relative to adult platelets, correlating to reduced PAR1 expression by neonatal platelets. PAR4-mediated granule secretion was blunted in neonatal platelets, correlating to lower PAR4 expression as compared to adult platelets, while PAR4 mediated GPIIb/IIIa activation was similar between neonatal and adult platelets. Under high shear stress, cord blood-derived platelets yielded similar thrombin generation rates but reduced phosphatidylserine expression as compared to adult platelets. Interestingly, we observed enhanced P2Y1/P2Y12-mediated dense granule trafficking in neonatal platelets relative to adults, although P2Y1/P2Y12 expression in neonatal, cord, and adult platelets were similar, suggesting that neonatal platelets may employ an ADP-mediated positive feedback loop as a potential compensatory mechanism for neonatal platelet hyporeactivity.