Dysregulation of platelet serotonin, 14-3-3, and GPIX in sudden infant death syndrome.

Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal infant mortality, but the underlying cause(s) are unclear. A subset of SIDS infants has abnormalities in the neurotransmitter, serotonin (5-hydroxytryptamine [5-HT]) and the adaptor molecule, 14-3-3 pathways in regions of the brain involved in gasping, response to hypoxia, and arousal. To evaluate our hypothesis that SIDS is, at least in part, a multi-organ dysregulation of 5-HT, we examined whether blood platelets, which have 5-HT and 14-3-3 signaling pathways similar to brain neurons, are abnormal in SIDS. We also studied platelet surface glycoprotein IX (GPIX), a cell adhesion receptor which is physically linked to 14-3-3. In infants dying of SIDS compared to infants dying of known causes, we found significantly higher intra-platelet 5-HT and 14-3-3 and lower platelet surface GPIX. Serum and plasma 5-HT were also elevated in SIDS compared to controls. The presence in SIDS of both platelet and brainstem 5-HT and 14-3-3 abnormalities suggests a global dysregulation of these pathways and the potential for platelets to be used as a model system to study 5-HT and 14-3-3 interactions in SIDS. Platelet and serum biomarkers may aid in the forensic determination of SIDS and have the potential to be predictive of SIDS risk in living infants.

Impact of specific preclinical variables on coagulation biomarkers in cancer-associated thrombosis.

Coagulation biomarkers are being actively studied for their diagnostic and prognostic value in patients with venous thromboembolism and cancer, as well as in the study of pathogenic mechanisms between cancer and thrombosis. For the results of such studies to be accurate and reproducible, attention must be paid to minimize sources of error in all phases of testing. The pre-analytical phase of laboratory testing is known to be fraught with the majority of errors. Coagulation testing is particularly susceptible to conditions during collection, processing, transport and storage of specimens which can lead to clinically significant errors in results. In addition, changes in pre-analytical conditions can impact different biomarkers differently. Therefore, research studies investigating coagulation biomarkers must carefully standardize not just the analytical phase, but also the pre-analytical phase of testing to ensure accuracy and reliability. We briefly review the impact of pre-analytical conditions on coagulation testing in general, and on specific biomarkers in cancer and thrombosis. In addition, we provide recommendations to reduce pre-analytical errors by developing and sharing standard operating procedures that specifically target standardization of methodologies for collecting specimens and measuring current and emerging coagulation biomarkers in cancer studies.

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.

Coated platelets function in platelet-dependent fibrin formation via integrin αIIbß3 and transglutaminase factor XIII.

Coated platelets, formed by collagen and thrombin activation, have been characterized in different ways: i) by the formation of a protein coat of α-granular proteins; ii) by exposure of procoagulant phosphatidylserine; or iii) by high fibrinogen binding. Yet, their functional role has remained unclear. Here we used a novel transglutaminase probe, Rhod-A14, to identify a subpopulation of platelets with a cross-linked protein coat, and compared this with other platelet subpopulations using a panel of functional assays. Platelet stimulation with convulxin/thrombin resulted in initial integrin α(IIb)ß3 activation, the appearance of a platelet population with high fibrinogen binding, (independently of active integrins, but dependent on the presence of thrombin) followed by phosphatidylserine exposure and binding of coagulation factors Va and Xa. A subpopulation of phosphatidylserine-exposing platelets bound Rhod-A14 both in suspension and in thrombi generated on a collagen surface. In suspension, high fibrinogen and Rhod-A14 binding were antagonized by combined inhibition of transglutaminase activity and integrin α(IIb)ß3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)ß3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)ß3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)ß3 activation and high fibrinogen binding, form a subpopulation of phosphatidylserine-exposing platelets, and function in platelet-dependent star-like fibrin fiber formation via transglutaminase factor XIII and integrin α(IIb)ß3.

New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors.

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P(1),P(4)-di(adenosine-5') tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure-activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents.

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 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.

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.

P2Y12 receptor blockade augments glycoprotein IIb-IIIa antagonist inhibition of platelet activation, aggregation, and procoagulant activity.

BACKGROUND: New antiplatelet agents that provide greater, more consistent inhibition of the platelet ADP receptor P2Y12 may be used in combination with glycoprotein (GP) IIb-IIIa antagonists, but their combined effect on platelet function and procoagulant activity is not well studied. Therefore, the objective of this study was to evaluate the independent and complementary effects of P2Y12 and GPIIb-IIIa inhibition on platelet function and procoagulant activity. METHODS AND RESULTS: Healthy donor blood was treated with the active metabolite of prasugrel (R-138727 5 µmol/L), GPIIb-IIIa antagonists (abciximab 3 µg/mL or eptifibatide 0.9 µg/mL), and combinations thereof, exposed to physiologically relevant agonists (collagen and ADP) and then evaluated for markers of platelet activation and procoagulant activity. Significant interactions between R-138727 and GPIIb-IIIa antagonists were observed. R-138727 and the GPIIb-IIIa antagonists had additive inhibitory effects on collagen-stimulated platelet aggregation and on the collagen plus ADP-stimulated level of activated platelet surface GPIIb-IIIa. R-138727 and abciximab each inhibited collagen plus ADP-stimulated platelet phosphatidylserine expression and prothrombin cleavage, and the combination produced greater inhibition than achieved with abciximab alone. In contrast, eptifibatide did not inhibit, but instead enhanced, collagen plus ADP-stimulated prothrombin cleavage. Addition of R-138727 reduced prothrombin cleavage in eptifibatide-treated samples, suggesting a novel mechanism for potential benefit from combined prasugrel and eptifibatide treatment. CONCLUSIONS: The complementary effects of abciximab and R-138727 on platelet activation, aggregation, and procoagulant activity suggest their combined use may, to a greater degree than with either agent alone, reduce thrombus formation in vivo.

Hyaluronan anchored to activated CD44 on central nervous system vascular endothelial cells promotes lymphocyte extravasation in experimental autoimmune encephalomyelitis.

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 µm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Inhibition of Factor XII-Mediated Activation of Factor XI Provides Protection Against Experimental Acute Ischemic Stroke in Mice.

Blood coagulation factor XI (FXI) is an established risk factor for acute ischemic stroke (AIS) and thrombosis, but is also needed for normal hemostasis. Contact factor XII (FXII), an upstream activator of FXI, also contributes to experimental stroke, but is not required for hemostasis. We investigated whether selectively inhibiting FXII-mediated FXI activation, while leaving other FXI and FXII functions intact, could improve the outcome of experimental AIS in mice. Twenty-four hours before induction of AIS by placement of a filament into the internal carotid artery for 60 min, mice were anticoagulated with an antibody directed against the apple 2 domain of FXI. This antibody selectively reduces the prothrombotic activation of FXI by FXIIa but does not affect activated FXI or hemostatic activation of FXI by thrombin, thus leaving hemostasis intact in mice and primates. In this model of AIS/reperfusion injury, mice that received the antibody before AIS displayed less ischemic damage, manifested as reduced cerebral infarction and fibrin deposition (thrombosis), increased cortical reperfusion, and improved neurological behavior. Further, the antibody-anticoagulated mice had no detectable hemostasis impairment. Consistent with the neuroprotective phenotype of FXII-deficient mice, our data suggest that a single molecular event, FXII-mediated FXI activation, contributes to the development of experimental AIS.

Thrombin mutant W215A/E217A treatment improves neurological outcome and reduces cerebral infarct size in a mouse model of ischemic stroke.

BACKGROUND AND PURPOSE: Treatment of ischemic stroke by activation of endogenous plasminogen using tissue plasminogen activator is limited by bleeding side effects. In mice, treatment of experimental ischemic stroke with activated protein C improves outcomes; however, activated protein C also has bleeding side effects. In contrast, activation of endogenous protein C using thrombin mutant W215A/E217A (WE) is antithrombotic without hemostasis impairment in primates. Therefore, we investigated the outcome of WE-treated experimental ischemic stroke in mice. METHODS: The middle cerebral artery was occluded with a filament for 60 minutes to induce ischemic stroke. Vehicle, recombinant WE, or tissue plasminogen activator was administered during middle cerebral artery occlusion or 2 hours after middle cerebral artery occlusion. Neurological performance was scored daily. Intracranial bleeding and cerebral infarct size, defined by 2,3,5-triphenyltetrazolium chloride exclusion, were determined on autopsy. Hemostasis was evaluated using tail bleeding tests. RESULTS: WE improved neurological performance scores, increased laser Doppler flowmetry-monitored post-middle cerebral artery occlusion reperfusion of the parietal cortex, and reduced 2,3,5-triphenyltetrazolium chloride-defined cerebral infarct size versus vehicle controls. However, unlike tissue plasminogen activator, WE did not increase tail bleeding or intracranial hemorrhage. CONCLUSIONS: WE treatment is neuroprotective without hemostasis impairment in experimental acute ischemic stroke in mice and thus may provide an alternative to tissue plasminogen activator for stroke treatment.

Breakup feared after filamin leaves GPIb.

In this issue of Blood, Cranmer and colleagues demonstrate that the linkage between the cytoskeletal protein filamin A and the platelet receptor glycoprotein (GP) Ibα provides structural integrity to the plasma membrane during platelet adhesion to von Willebrand factor (VWF) under high shear.