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 activation and inhibition in sickle cell disease (pains) study.

Platelet activation/aggregation in sickle cell disease (SCD) may promote tissue ischemia, suggesting that antiplatelet therapy may be useful. However, the assessment of platelet function and the effect of antiplatelet therapy in blood from SCD patients may be confounded by hemolysis with the release of adenosine 5'-diphosphate (ADP). Here we evaluate the levels of platelet activation markers in SCD adolescents vs. normal controls and compare, by multiple methods, the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Platelet activation markers in blood from SCD adolescents (n = 15) and healthy adults (n = 10), and the effect of R-138727 (0.1-10 µM) added in vitro, were evaluated with and without ADP stimulation. The circulating levels of platelet-monocyte and platelet-neutrophil aggregates were significantly higher (p < 0.01) in SCD patients than in healthy controls. R-138727, in a concentration-dependent manner, inhibited platelet function in both SCD patients and healthy subjects as judged by ADP-stimulated light transmission aggregation, VerifyNow P2Y12 assay, multiple electrode aggregometry, and flow cytometric analysis of platelet vasodilator-stimulated phosphoprotein, activated GPIIb-IIIa and P-selectin. The R-138727 IC50s for each assay were not significantly different in SCD vs. healthy subjects. In summary: (1) The high circulating levels of platelet-monocyte and platelet-neutrophil aggregates demonstrate in vivo platelet activation in SCD and may be useful as markers of the in vivo pharmacodynamic efficacy of antiplatelet therapy in SCD. (2) The similar in vitro R-138727 IC50s in SCD and healthy subjects suggest that the prasugrel dose-dependence for platelet inhibition in SCD patients will be similar to that previously observed in healthy subjects.

Activation of platelet-rich plasma by pulse electric fields: Voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin.

Activated platelet-rich plasma (PRP) has been used in the clinical settings of wound healing and regenerative medicine, with activation typically induced by the addition of bovine thrombin. To eliminate issues with availability, cost and potential side effects associated with bovine thrombin, ex vivo PRP activation using pulse electric fields (PEF) has been proposed and demonstrated. The present study characterizes the effect of PEF voltage and pulse width, in combination with a range of calcium concentrations, on clot formation, growth factor release, and serotonin (5-HT) release from dense granules. The main findings are: 1) increasing calcium concentrations with most PEF conditions leads to increased levels of PDGF and 5-HT release; 2) whether EGF levels increase or decrease with increasing calcium concentration depends on the specific PEF parameters; 3) the pattern of PDGF and EGF levels in supernatants suggest that these molecules are localized differently within platelets; 4) significant levels of PDGF, EGF, and 5-HT can be released without inducing clot formation or hemoglobin release. In conclusion, voltage, pulse width and calcium concentration can be used to control and tune the release of growth factors, serotonin and hemoglobin from PEF-activated PRP. Because growth factor requirements vary for different types of wounds and for wounds at different stages of healing, the unique balance of factors in supernatants of PEF-activated PRP may provide more clinically advantageous than the current standard of bovine thrombin-activated PRP.

Using extracellular calcium concentration and electric pulse conditions to tune platelet-rich plasma growth factor release and clotting.

Platelet-rich plasma (PRP) is an emerging autologous biologic method for wound healing. Clinicians apply PRP either topically (where it is activated ex-vivo before treatment by adding an external agent to trigger clotting and the release of growth factors that facilitate wound healing) or through injection (where it is activated in vivo at the injury site with no prior activation before injection). Because topical PRP activation typically utilizes bovine thrombin, which has significant potential side effects and high costs, recent studies have assessed the efficacy of combining extracellular calcium (EC) and electric pulses (EPs) to activate PRP. The potential to apply this novel technique to PRP both topically and internally via injection raises the question about the ability to tune the clotting time and growth factor release for a given application. While previous studies have assessed the impact of applying EPs of various durations either directly (conductive coupling) or indirectly (capacitive coupling) to PRP containing EC, no studies have assessed the tunability of this activation based on modifying EP parameters, EP delivery method (conductive or capacitive coupling), and the EC concentration. We hypothesize that tuning these parameters will modify intracellular calcium uptake to permit the control of growth factor release and clotting time, which are critical for optimizing PRP for either topical or internal clinical applications. A pilot study for a single donor demonstrates the potential for tunability as a function of the intensity of membrane manipulation and calcium concentration, which facilitate the increase of cytosolic calcium. This motivates future studies assessing EC and EP optimization and in vivo studies to determine the overall efficacy of this tunability for wound healing.

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.

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.