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.

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.

Abstract Appropriate and diverse representation of minority populations in clinical research studies improves long-term outcomes for all demographics and requires targeted solutions to recruitment challenges. During a two year project at Boston Medical Center (BMC), 191 newly diagnosed, treatment-naïve cancer patients and 76 non-cancer control donors were consented to study relationships between cancer and thrombosis biomarkers. 56% of the cancer patients self-identified as minorities (32% Black, 17% Hispanic, 3% Asian, 4% other or multiple), which reflect the overall demographics of patients undergoing surgery at BMC. However, control donor recruitment for the study was only 36% minorities during the first 6 months (n=9 of 25). These control subjects were younger and white, in part because the study was only advertised on the Boston University Medical Campus. Recognizing potential bias and lack of appropriate representation, two initiatives were developed and executed to balance the cancer and control populations with respect to ethnicity and age. First, active recruitment for older, minority-identifying control subjects expanded into the surrounding community of largely African American and Hispanic populations, by hosting donor recruitment days, with one of the days targeted to older males (≥ 50 years) to better reflect the ongoing cancer patient cohort. Second, consent forms were translated into Haitian Creole and Spanish to enable improved communications with these patients who frequent the hospital. To encourage participation from individuals with a negative perception of research, study coordinators received training in consenting and applied it to provide enhanced patient education. This resulted in a significant improvement in the control’s minority percentage to 64% over the project timeline (n=49, 44% Black, 11% Hispanic, 9% Asian). This approach also helped to balance age distributions in the cancer and control populations. The majority of cancer patients were in their 50s and older (81%, mean 59 ± 12 years; n=155). Control subjects in their fifth decade or above was only 32% in the first 6 months, but improved to 55% (mean 46+ 14 years, n=42) by the end of the project. The study was not designed to match comorbidities, but human immunodeficiency virus (HIV) infection, and hepatitis which are known to affect coagulation are highly prevalent in the BMC population. Of the consented cancer patients, 5% had hepatitis and 2% reported active HIV infection, while in the control population 6% had hepatitis and 5% had HIV. Patient demographic, clinical data, analysis results, follow-up data on treatment and thrombotic events, as well as plasma and cell pellets from this study are freely available to the research community from: cssi.cancer.gov/cancer-thrombosis. Overall, frequent data monitoring and adjusting recruitment strategies to emphasize community outreach contributed to balancing ethnic and age distributions in the study populations during this two year study. Citation Format: Morgan P Thompson, Elizabeth R Duffy, Jasmin H Bavarva, Cheryl Spencer, DJ Stearns-Kurosawa, Rachana Agarwal, Michelle A Berny-Lang, Chris Andry. Consenting challenges toward age, ethnicity, and co-morbidity matching of cancer patient and control populations at a minority-majority safety net hospital [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A091.

Abstract The link between cancer and thrombosis, especially venous thromboembolism, is well established and thrombotic risk is exacerbated by cancer treatments, such as surgery and chemotherapy. This ongoing study aims to determine the impact of pre-analytic variables (PAVs) on thrombosis biomarkers in a diverse cancer patient and non-cancer subject population at an urban safety net hospital. Citrated blood from newly diagnosed, treatment-naïve patients of 11 cancer types or from non-cancer controls was processed to examine these variables: time to fractionation (2 and 4 hrs), plasma freeze-thaw cycles (2 and 3 cycles), and plasma delay to testing (24 or 72 hrs at 4oC). Regular processing (< 1 hour to centrifuge or 1 freeze-thaw cycle) served as the control sample for the variables. Current interim data presents biomarker data for D-dimer (DDE), Factor VIII activity (FVIII), soluble P-selectin (sP-Sel), prothrombin fragment 1+2 (F1+2), plasma DNA (DNA) and myeloperoxidase (MPO). Assays are performed in the hospital clinical lab (DDE, FVIII) or in our research lab following 30 detailed standard operating procedures (SOPs). Cancer patient demographics are 60% male, 40% Black/African-American, 38% Caucasian, 18% Hispanic, 3% Native American, and 1% Asian with an age range of 38-86 years. Non-cancer controls are 52% male, 59% Caucasian, 15% Black/African-American, 19% Asian, and 7% Hispanic with an age range of 23-64 years. Interim project data shows increased thrombosis biomarker levels in cancer subjects, except for sP-Sel and F1+2. Biomarker levels in cancer patients (n=9-52) were increased by approximately 300ng/ml DNA, 100ng/ml DDE, and 5ng/mL MPO with a trending increase in FVIII (~30%) when compared to non-cancer controls (n=17-22). Freeze-thaw of plasma had no effect, while a 2hr time to fractionation resulted in significantly increased MPO (~10ng/mL), FVIII (~14%) and F1+2 (~310 pg/mL). Delay to testing done for DNA and DDE showed with no apparent effect on biomarker levels after 24 or 72 hrs at 4oC. Current data show biomarker levels are impacted by presence of cancer rather than ethnicity of the patient. Donor recruitment is ongoing with shifting strategies to meet recruitment goals of non-cancer donors for greater diversity and older age to more closely reflect our cancer patient population. Rigorous control of sample handling and assay performance using SOPs that are compatible with a hospital setting contribute to identifying PAVs that matter for design of generalizable procedures. Citation Format: Morgan P. Thompson, Elizabeth R. Duffy, DJ Stearns-Kurosawa, Jasmin Bavarva, Shinichiro Kurosawa, Jiyoun Kim, Cheryl Spencer, Daniel Remick, Mark Sloan, Joel Henderson, Kerrie P. Nelson, Joseph Y. Tashjian, Yibing Wei, Rachana Agarwal, Michelle A. Berny-Lang, Chris Andry. Effect of preanalytic variables on established and emerging thrombosis-related biomarkers in an ethnically and racially diverse population of cancer patients and healthy subjects at a safety net hospital [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4529.

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.

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 PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

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.

Summary Introduction Early and accurate identification of acute coronary syndrome ( ACS ) vs . noncardiac chest pain in patients presenting to the emergency department ( ED ) is problematic and new diagnostic markers are needed. Previous studies reported that elevated mean platelet volume ( MPV ) is associated with ACS and predictive of cardiovascular risk. MPV is closely related to the immature platelet fraction ( IPF ), and recent studies have suggested that IPF may be a more sensitive marker of ACS than MPV . The objective of the present study was to determine whether the measurement of IPF assists in the diagnosis of ACS in patients presenting to the ED with chest pain. Methods In this single‐center, prospective, cross‐sectional study, adult patients presenting to the ED with chest pain and/or suspected ACS were considered for enrollment. Blood samples from 236 ACS ‐negative and 44 ACS ‐positive patients were analyzed in a S ysmex XE ‐2100 for platelet count, MPV , IPF , and the absolute count of immature platelets ( IPC ). Results Total platelet counts, MPV , IPF , and IPC were not statistically different between ACS ‐negative and ACS ‐positive patients. The IPF was 4.6 ± 2.7% and 5.0 ± 2.8% (mean ± SD , P = 0.24), and the IPC was 10.0 ± 4.6 and 11.5 ± 7.5 × 10 3 / μ L ( P = 0.27) for ACS ‐negative and ACS ‐positive patients, respectively. Conclusion In 280 patients presenting to the ED with chest pain and/or suspected ACS , no differences in IPF , IPC or MPV were observed in ACS ‐negative vs . ACS ‐positive patients, suggesting that these parameters do not assist in the diagnosis of ACS .

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 P1,P4-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.

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.

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 αIIbβ3 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 αIIbβ3. Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin αIIbβ3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in αIIbβ3 (Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial αIIbβ3 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 αIIbβ3.

Abstract In 2011, the NCI Director, Dr. Harold Varmus, created the Provocative Questions (PQ) initiative to encourage imaginative, bold approaches designed to tackle perplexing and previously neglected areas of cancer research. Based upon workshops and discussions with the extramural cancer research community and NCI program directors, a set of 24 questions and accompanying requests for applications (RFAs) were developed, addressing diverse topics in cancer risk and prevention, tumor development, and cancer detection, diagnosis, and treatment. In order to stimulate innovative hypotheses and approaches, the “power of the ideas” was heavily weighted with a de-emphasis on preliminary data. In the first year of the initiative, the NCI awarded 56 unique proposals, totaling $21.5 million of research support via the R01 and R21 mechanisms. Building upon the 2011 RFAs and adjusting to changes in the research landscape, 24 revised questions were developed for the 2012 RFAs with a commitment of up to $30 million in support. The NCI released the 2013 RFAs in September 2013 with a revised set of 20 questions, including new questions related to clinical effectiveness. Several PQ-supported research projects funded in the first year of the initiative will be highlighted in order to demonstrate the breadth of topics and high level of innovation supported by the initiative. Projects will be selected from the four main PQ thematic areas to provide a sense of the many diverse approaches being used to answer the selected PQ. In addition, trends and criteria from awarded proposals will be presented to provide insight to potential applicants regarding the outcomes of the program to date. Through engagement of the cancer research community, the PQ initiative seeks to continually leverage scientific advances to address currently unsolved, understudied questions in cancer, which may influence the future directions of NCI-sponsored research. Citation Format: Michelle A. Berny-Lang, Emily J. Greenspan. NCI's Provocative Questions Initiative. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3500. doi:10.1158/1538-7445.AM2014-3500

Background New antiplatelet agents that provide greater, more consistent inhibition of the platelet ADP receptor P2Y 12 may be used in combination with glycoprotein ( GP ) II b‐ III a 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 P2Y 12 and GPII b‐ III a 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.