Assessment of neonatal platelet adhesion, activation, and aggregation

Abstract

Background: Acquired and inherited bleeding disorders may present in the neonatal period with devastating lifelong effects. Diagnosing bleeding disorders in the neonatal population could aid in preventing and treating the associated complications. However, currently available platelet function testing is limited in neonates, owing to difficulties in obtaining an adequate blood volume, a lack of normal reference ranges, and an incomplete understanding of the neonatal platelet functional phenotype.

Objective: To develop small-volume, whole blood platelet function assays in order to quantify and compare neonatal and adult platelet function.

Methods and results: Peripheral blood was obtained from healthy, full-term neonates at 24 h of life. Platelet activation, secretion and aggregation were measured via flow cytometry. Platelet adhesion and aggregation were assessed under static and flow conditions. As compared with adult platelets, peripheral neonatal platelet P-selectin expression and integrin glycoprotein IIbIIIa activation were significantly reduced in response to the G-protein-coupled receptor (GPCR) agonists thrombin receptor activator peptide-6 (TRAP-6), ADP, and U46619, and the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway agonists collagen-related peptide (CRP) and rhodocytin. Neonatal platelet aggregation was markedly reduced in response to TRAP-6, ADP, U46619, CRP and rhodocytin as compared with adult platelets. The extents of neonatal and adult platelet adhesion and aggregate formation under static and shear conditions on collagen and von Willebrand factor were similar.

Conclusions: As compared with adult platelets, we found that neonatal platelet activation and secretion were blunted in response to GPCR or ITAM agonists, whereas the extent of neonatal platelet adhesion and aggregate formation was similar to that of adult platelets.

Keywords: neonate; platelet activation; platelet adhesiveness; platelet aggregation; platelet function tests.

Figure 1. Adult and neonatal whole blood platelet activation

Representative flow cytometry forward and side scatter dot plots of a whole blood sample from an adult (A) and a neonate (B). Representative histograms of PAC-1-FITC (C and D), CD62P-APC (E and F), and platelet factor 4 (PF4)-Alexa Fluor 350 (G and H) fluorescence intensity of adult and neonatal whole blood treated with adenosine 5′-diphosphate (ADP) + U46619 (10 μM; black line) or vehicle (gray line). Percent of platelets positive for PAC-1-FITC (I), CD62P-APC (J), and PF4-Alexa Fluor 350 (K) in response to thrombin receptor activator peptide-6 (TRAP-6; 10 μM), collagen related peptide (CRP; 10 μg/mL), ADP (10 μM), U46619 (10 μM), ADP+U46619 (10 μM), epinephrine (10 μM), calcium ionophore A23187 (10 μM), rhodocytin (300 nM), or vehicle treatment. Data are represented as mean ± SEM; N_adult = 6 and N_neonate = 8; *P < 0.05 with respect to vehicle treated samples; #P < 0.05 with respect to adult samples.

Figure 2. Adult and neonatal whole blood platelet aggregation

Representative CD31-FITC and CD31-eFluor 450 dot plots of adult (A) and neonatal (B) blood at t = 0 min (left) and t = 1 min (right) after treatment with thrombin receptor activator peptide-6 (TRAP-6; 10 μM). Double-labeled events (platelet aggregates) are shown in the upper right quadrant. Representative graph of percent double-labeled events of adult (C) and neonatal (D) platelets stimulated with TRAP-6 (10 μM) or vehicle at 0, 0.5, 1, 2, and 5 min. (E) Fold change of percent double-labeled events normalized to vehicle treatment of adult and neonatal blood stimulated with TRAP-6 (10 μM), collagen related peptide (CRP; 10 μg/mL), epinephrine (10 μM), adenosine 5′-diphosphate (ADP; 10 μM), U46619 (10 μM), ADP+U46619 (10 μM), rhodocytin (300 nM), or calcium ionophore A23187 (10 μM). Data are represented as mean ± SEM; N_adult = 3 and N_neonate = 4; *P < 0.05 with respect to vehicle treated samples; #P < 0.05 with respect to adult samples.

Figure 3. Adult and neonatal platelet adhesion and aggregation under static conditions

Representative differential interference contrast images and corresponding fluorescent CD41a-PE and CD62P-FITC images of adult and neonatal platelet aggregates formed on coverslips coated with 100 μg/mL von Willebrand factor (VWF) (A) or 100 μg/mL fibrillar collagen (B). Adult and neonatal citrated whole blood were pretreated with vehicle, thrombin receptor activator peptide-6 (TRAP-6; 10 μM), collagen related peptide (CRP; 10 μg/mL), adenosine 5′-diphosphate (ADP; 10 μM), U46619 (10 μM), ADP+U46619 (10 μM), epinephrine (10 μM), ristocetin (1 mg/mL), calcium ionophore A23187 (10 μM), or rhodocytin (300 nM) for 10 min prior to coverslip incubation. Mean volume of adult and neonatal platelet aggregates positive for CD62P-FITC formed on coverslips coated with VWF (C) or fibrillar collagen (D). Data are represented as mean ± SEM; N_adult = 3 and N_neonate = 3; *P < 0.05 with respect to vehicle treated samples.

Figure 4. Effect of glycoprotein (GP) IIbIIIa and GPIb inhibition on adult and neonatal platelet adhesion and aggregation under static conditions

Representative differential interference contrast images of adult and neonatal platelet aggregates formed on coverslips coated with 100 μg/mL von Willebrand factor (VWF) (A) or 100 μg/mL fibrillar collagen (B). Adult and neonatal citrated whole blood were incubated with a glycoprotein (GP) IIbIIIa inhibitor (eptifibatide; 20 μg/mL), a GPIb function blocking antibody (6D1; 20 μg/mL), or vehicle for 10 min at 25°C and then treated with thrombin receptor activator peptide-6 (TRAP-6; 10 μM), collagen related peptide (CRP; 10 μg/mL), adenosine 5′-diphosphate (ADP) + U46619 (10 μM), ristocetin (1 mg/mL), or vehicle for 10 min. Mean volume of adult and neonatal platelet aggregates positive for CD62P-FITC formed on coverslips coated with VWF (C) or fibrillar collagen (D). Data are represented as mean ± SEM; N_adult = 3 and N_neonate = 3; *P < 0.05 with respect to vehicle treated samples.

Figure 5. Adult and neonatal platelet adhesion and aggregation under fluid shear conditions

(A) Representative differential interference contrast images and corresponding fluorescent CD41a-PE and CD62P-FITC images of adult and neonatal platelet aggregates formed in a small-volume flow chamber coated with 100 μg/mL von Willebrand factor (VWF) or 100 μg/mL fibrillar collagen at 200 or 1500 s⁻¹ shear rate for 30 s. The black arrow denotes direction of blood flow. Mean percent surface area coverage of a 140 × 105 μm field of view of adult and neonatal platelet aggregates positive for CD41a-PE formed in flow chambers coated with VWF (B) or collagen (C). Data are represented as mean ± SEM; N_adult = 3 and N_neonate = 3; *P < 0.05 with respect to 200 s⁻¹ shear rate.

Figure 6. Effect of glycoprotein (GP) IIbIIIa and GPIb inhibition on adult and neonatal platelet adhesion and aggregation under fluid shear conditions

(A) Representative differential interference contrast images of adult and neonatal platelet aggregates formed in a small-volume flow chamber coated with 100 μg/mL von Willebrand factor (VWF) or 100 μg/mL fibrillar collagen at 200 or 1500 s⁻¹ shear rate for 30 s. Adult and neonatal citrated whole blood were incubated with a glycoprotein (GP) IIbIIIa inhibitor (eptifibatide; 20 μg/mL), a GPIb function blocking antibody (6D1; 20 μg/mL), or vehicle for 10 min at 25°C prior to perfusion through flow chambers. Mean percent surface area coverage of a 140 × 105 μm field of view of adult and neonatal platelet aggregates positive for CD41a-PE formed in flow chambers coated with VWF (B) or collagen (C). Data are represented as mean ± SEM; N_adult = 3 and N_neonate = 3; *P < 0.05 with respect to vehicle treated samples.