Anja J. Gerrits

Platelet function tests, independent of platelet count, are associated with bleeding severity in ITP.

Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. To determine if differences in platelet function in ITP patients account for this variation in bleeding tendency, we conducted a single-center, cross-sectional study of pediatric patients with ITP. Bleeding severity (assessed by standardized bleeding score) and platelet function (assessed by whole blood flow cytometry) with and without agonist stimulation was evaluated in 57 ITP patients (median age, 9.9 years). After adjustment for platelet count, higher levels of thrombin receptor activating peptide (TRAP)-stimulated percent P-selectin- and activated glycoprotein (GP)IIb-IIIa-positive platelets were significantly associated with a lower bleeding score, whereas higher levels of immature platelet fraction (IPF), TRAP-stimulated platelet surface CD42b, unstimulated platelet surface P-selectin, and platelet forward light scatter (FSC) were associated with a higher bleeding score. Thus, platelet function tests related to platelet age (IPF, FSC) and activation through the protease activated receptor 1 (PAR1) thrombin receptor (TRAP-stimulated P-selectin, activated GPIIb-IIIa, and CD42b), independent of platelet count, are associated with concurrent bleeding severity in ITP. These tests may be useful markers of future bleeding risk in ITP.

Effects of eltrombopag on platelet count and platelet activation in Wiskott-Aldrich syndrome/X-linked thrombocytopenia

UNLABELLED Because Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) patients have microthrombocytopenia, hemorrhage is a major problem. We asked whether eltrombopag, a thrombopoietic agent, would increase platelet counts, improve platelet activation, and/or reduce bleeding in WAS/XLT patients. In 9 WAS/XLT patients and 8 age-matched healthy controls, platelet activation was assessed by whole blood flow cytometry. Agonist-induced platelet surface activated glycoprotein (GP) IIb-IIIa and P-selectin in WAS/XLT patients were proportional to platelet size and therefore decreased compared with controls. In contrast, annexin V binding showed no differences between WAS/XLT and controls. Eltrombopag treatment resulted in an increased platelet count in 5 out of 8 patients. Among responders to eltrombopag, immature platelet fraction in 3 WAS/XLT patients was significantly less increased compared with 7 pediatric chronic immune thrombocytopenia (ITP) patients. Platelet activation did not improve in 3 WAS/XLT patients whose platelet count improved on eltrombopag. IN CONCLUSION (1) the reduced platelet activation observed in WAS/XLT is primarily due to the microthrombocytopenia; and (2) although the eltrombopag-induced increase in platelet production in WAS/XLT is less than in ITP, eltrombopag has beneficial effects on platelet count but not platelet activation in the majority of WAS/XLT patients. This trial was registered at www.clinicaltrials.gov as #NCT00909363.

Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation

Abstract Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEFs inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma

Background Activated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma. Aims To determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity. Methods PRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin. Results PRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone. Conclusions PEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

High serum serotonin in sudden infant death syndrome

Significance Sudden infant death syndrome (SIDS), the leading cause of postneonatal infant mortality, is defined as the sudden death of an infant less than 1 y of age that remains unexplained after a complete autopsy and death scene investigation. Although SIDS has been associated with deficiencies in central (brainstem) serotonin (5-hydroxytryptamine, 5-HT), there are no known peripheral biomarkers for SIDS. Here we demonstrate increased serum serotonin levels in a subset (31%) of SIDS infants compared with control infants. These findings suggest the potential of a high serum serotonin level as a forensic biomarker at autopsy to differentiate SIDS deaths with serotonergic defects from other causes of sudden death and, importantly, as evidence of a peripheral 5-HT abnormality in SIDS. Sudden infant death syndrome (SIDS), the leading cause of postneonatal infant mortality, likely comprises heterogeneous disorders with the common phenotype of sudden death without explanation upon postmortem investigation. Previously, we reported that ∼40% of SIDS deaths are associated with abnormalities in serotonin (5-hydroxytryptamine, 5-HT) in regions of the brainstem critical in homeostatic regulation. Here we tested the hypothesis that SIDS is associated with an alteration in serum 5-HT levels. Serum 5-HT, adjusted for postconceptional age, was significantly elevated (95%) in SIDS infants (n = 61) compared with autopsied controls (n = 15) [SIDS, 177.2 ± 15.1 (mean ± SE) ng/mL versus controls, 91.1 ± 30.6 ng/mL] (P = 0.014), as determined by ELISA. This increase was validated using high-performance liquid chromatography. Thirty-one percent (19/61) of SIDS cases had 5-HT levels greater than 2 SDs above the mean of the controls, thus defining a subset of SIDS cases with elevated 5-HT. There was no association between genotypes of the serotonin transporter promoter region polymorphism and serum 5-HT level. This study demonstrates that SIDS is associated with peripheral abnormalities in the 5-HT pathway. High serum 5-HT may serve as a potential forensic biomarker in autopsied infants with SIDS with serotonergic defects.

Incomplete Reversibility of Platelet Inhibition Following Prolonged Exposure to Ticagrelor

Essentials Irreversible platelet inhibition persists after reversibly‐binding ticagrelor is discontinued. Reversibility of platelet inhibition by ticagrelor and its active metabolite was assessed. Incomplete recovery was observed after prolonged exposure to ticagrelor. Activated GPIIb‐IIIa and P‐selectin, not platelet reactivity index, showed irreversibility.

Platelet Function in ITP, Independent of Platelet Count, Is Consistent Over Time and Is Associated with Both Current and Subsequent Bleeding Severity

BACKGROUND Treatment decisions for patients with immune thrombocytopenia (ITP) are difficult because patients with similarly low platelet counts differ in their bleeding tendency. We recently reported that platelet function tests, independent of platelet count, are associated with concurrent bleeding severity, suggesting that these tests may be useful indicators of future bleeding in ITP. OBJECTIVES To test this hypothesis, we evaluated the consistency of these platelet function tests over time and their association with subsequent bleeding severity. METHODS Bleeding score and platelet biomarkers were evaluated in a cross-sectional study of children with ITP at two visits separated by a median of 10 months. RESULTS AND CONCLUSIONS Correlations between Visit 1 and Visit 2 results for immature platelet fraction, circulating and agonist-stimulated platelet surface P-selectin, and activated GPIIb-IIIa and GPIbα indicated consistency of the platelet phenotype over time. Consistent with our previous findings, platelet biomarkers at each visit were significantly associated with the concurrent bleeding score. Furthermore, increased P-selectin on circulating platelets and reduced agonist-stimulated P-selectin and activated GPIIb-IIIa-positive platelets at Visit 1 were significantly associated with bleeding scores at Visit 2 and remained significantly associated with bleeding severity after adjustment for platelet count. These results suggest a mechanistic link between desensitization of agonist receptors and increased bleeding severity. In summary, platelet function in ITP, independent of platelet count, is consistent over time and is associated with both concurrent and subsequent bleeding severity. These findings support further evaluation of platelet function testing to help guide patient management in ITP.

Tunable activation of therapeutic platelet-rich plasma by pulse electric field: Differential effects on clot formation, growth factor release, and platelet morphology

Background Activation of platelet-rich plasma (PRP) by pulse electric field (PEF) releases growth factors which promote wound healing (e.g., PDGF, VEGF for granulation, EGF for epithelialization). Aims To determine after PEF activation of therapeutic PRP: 1) platelet gel strength; 2) profile of released growth factors; 3) alpha- and T-granule release; 4) platelet morphology. Methods Concentrated normal donor PRP was activated by 5 μsec (long) monopolar pulse, ~4000 V/cm (PEF A) or 150 nsec (short) bipolar pulse, ~3000 V/cm (PEF B) in the presence of 2.5 mM (low) or 20 mM (high) added CaCl2. Clot formation was evaluated by thromboelastography (TEG). Surface exposure of alpha granule (P-selectin) and T-granule (TLR9 and protein disulfide isomerase [PDI]) markers were assessed by flow cytometry. Factors in supernatants of activated PRP were measured by ELISA. Platelet morphology was evaluated by transmission electron microscopy (TEM). Results Time to initial clot formation was shorter with thrombin (<1 min) than with PEF A and B (4.4–8.7 min) but clot strength (elastic modulus, derived from TEG maximum amplitude) was greater with PEF B than with either thrombin or PEF A (p<0.05). Supernatants of PRP activated with PEF A had higher EGF levels than supernatants from all other conditions. In contrast, levels of PF4, PDGF, and VEGF in supernatants were not significantly different after PEF A, PEF B, and thrombin activation. T-granule markers (TLR9 and PDI) were higher after thrombin than after PEF A or B with low or high CaCl2. By TEM, platelets in PEF-treated samples retained a subset of granules suggesting regulated granule release. Conclusion Pulse length and polarity can be modulated to produce therapeutic platelet gels as strong or stronger than those produced by thrombin, and this is tunable to produce growth factor profiles enhanced in specific factors important for different stages of wound healing.