scholarly journals Heparin Forms Polymers with Cell-free DNA Which Elongate Under Shear in Flowing Blood

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joost C. de Vries ◽  
Arjan D. Barendrecht ◽  
Chantal C. Clark ◽  
Rolf T. Urbanus ◽  
Peter Boross ◽  
...  
Keyword(s):  
Whole Blood ◽  
Binding Proteins ◽  
Factor Xa ◽  
Heparin Binding ◽  
Cell Free Dna ◽  
Coagulation Assays ◽  
Fibrin Formation ◽  
Free Dna ◽  
Flowing Blood ◽  
Nucleic Acid Stain

AbstractHeparin is a widely used anticoagulant which inhibits factor Xa and thrombin through potentiation of antithrombin. We recently identified that the nucleic acid stain SYTOX reacts with platelet polyphosphate due to molecular similarities, some of which are shared by heparin. We attempted to study heparin in flowing blood by live-cell fluorescence microscopy, using SYTOX for heparin visualisation. Immunostaining was performed with monoclonal antibodies directed against various heparin-binding proteins. In addition, we studied modulation of heparin activity in coagulation assays, as well its effects on fibrin formation under flow in recalcified whole blood. We found that SYTOX-positive polymers appear in heparinised blood under flow. These polymers typically associate with platelet aggregates and their length (reversibly) increases with shear rate. Immunostaining revealed that of the heparin-binding proteins assessed, they only contain histones. In coagulation assays and flow studies on fibrin formation, we found that addition of exogenous histones reverses the anticoagulant effects of heparin. Furthermore, the polymers do not appear in the presence of DNase I, heparinase I/III, or the heparin antidote protamine. These findings suggest that heparin forms polymeric complexes with cell-free DNA in whole blood through a currently unidentified mechanism.

scholarly journals Feasibility of Cell-Free DNA Collection and Clonal Immunoglobulin Sequencing in South African Patients With HIV-Associated Lymphoma

2021 ◽  
pp. 611-621
Author(s):  
Samantha L. Vogt ◽  
Moosa Patel ◽  
Atul Lakha ◽  
Vinitha Philip ◽  
Tanvier Omar ◽  
...  
Keyword(s):  
South Africa ◽  
Heavy Chain ◽  
Whole Blood ◽  
B Cell Lymphoma ◽  
Immunoglobulin Heavy Chain ◽  
Newly Diagnosed ◽  
Hiv Patients ◽  
Cell Free Dna ◽  
Low Resource ◽  
Free Dna

PURPOSE Diagnosis of AIDS lymphoma in low-resource settings, like South Africa, is often delayed, leaving patients with limited treatment options. In tuberculosis (TB) endemic regions, overlapping signs and symptoms often lead to diagnostic delays. Assessment of plasma cell-free DNA (cfDNA) by next-generation sequencing (NGS) may expedite the diagnosis of lymphoma but requires high-quality cfDNA. METHODS People living with HIV with newly diagnosed aggressive B-cell lymphoma and those with newly diagnosed TB seeking care at Chris Hani Baragwanath Academic Hospital and its surrounding clinics, in Soweto, South Africa, were enrolled in this study. Each participant provided a whole blood specimen collected in cell-stabilizing tubes. Quantity and quality of plasma cfDNA were assessed. NGS of the immunoglobulin heavy chain was performed. RESULTS Nine HIV+ patients with untreated lymphoma and eight HIV+ patients with TB, but without lymphoma, were enrolled. All cfDNA quantity and quality metrics were similar between the two groups, except that cfDNA accounted for a larger fraction of recovered plasma DNA in patients with lymphoma. The concentration of cfDNA in plasma also trended higher in patients with lymphoma. NGS of immunoglobulin heavy chain showed robust amplification of DNA, with large amplicons (> 250 bp) being more readily detected in patients with lymphoma. Clonal sequences were detected in five of nine patients with lymphoma, and none of the patients with TB. CONCLUSION This proof-of-principle study demonstrates that whole blood collected for cfDNA in a low-resource setting is suitable for sophisticated sequencing analyses, including clonal immunoglobulin NGS. The detection of clonal sequences in more than half of patients with lymphoma shows promise as a diagnostic marker that may be explored in future studies.

Promyelocytic Extracellular Chromatin Exacerbates Coagulation Disorder in Acute Promyelocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3806-3806
Author(s):  
Muhua Cao ◽  
Ruishuang Ma ◽  
Xiaoming Wu ◽  
Lixiu Wang ◽  
Lu Zhao ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Promyelocytic Leukemia ◽  
Thrombin Generation ◽  
Dnase I ◽  
Promyelocytic Leukemia ◽  
Coagulation Disorder ◽  
Cell Free Dna ◽  
Fibrin Formation ◽  
Free Dna

Abstract Introduction:Despite treatment with all-trans-retinoic acid, the early death rate in unselected acute promyelocytic leukemia (APL) due to hemorrhage still remains unacceptably high. It is attractive to speculate whether other uncovered procoagulants exist which are not attenuated by ATRA. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin (Ma R et al, Cell Death Dis 2016). However, the role of promyelocytic extracellular chromatin in APL-associated coagulation disorder remains unclear. The aims of this study were to identify the novel role of extracellular chromatin in induction of the hypercoagulable state in APL, and to evaluate its interaction with fibrin and endothelial cells (ECs). Methods:Twenty-two newly diagnosed APL patients were included. Fresh APL blasts from bone marrow specimens were treated with 1 μM ATRA or phosphate buffered saline (PBS). ETosis was distinguished by rounded cells whose nuclei stained with PI and whose nuclear contents diffused throughout the cell. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Elastase-DNA complexes and TAT (thrombin-antithrombin) complexes were detected by ELISA. ECs were incubated in growth media containing 20% pooled serum obtained from healthy donors in the presence or absence of 20-fold concentrated extracellular chromatin. Procoagulant activity (PCA) of ECs and APL cells was evaluated by one-stage recalcification time assay, pro-thrombinase assay and fibrin formation assay. DNase I or anti-TF were included in the inhibition assays. Results: ATRA treatment induced markedly increased cf-DNA release in a time-dependent manner compared with no ATRA group. Furthermore, ETosis was the major cell death pattern in the ATRA-treated group while apoptosis was predominant in the no-treatment group until the third day, indicating that the increased cell-free DNA triggered by ATRA was mainly from ETosis. NE-DNA, defined as marker of ETosis, peaked on day 3 and showed no significant elevation to day 5, indicating that increased part of cf-DNA from day 3 to day 5 was mainly from apoptosis. Additionally, thrombin generation was found to parallel the change in the releasing of promyelocytic extracellular chromatin induced by ATRA. Pretreatment with DNase I inhibited thrombin generation by 47%, diminished PCA by 35%, prolonged coagulation time, and attenuated fibrin formation by 50%, while neutralizing anti-TF antibody produced no effect. Confocal microscopy showed that fibrin was preferentially deposited on promyelocytic chromatin from ETosis or apoptosis and exposed PS. Lastly, we found that extracellular chromatin from the ATRA group significantly triggered PS exposure on ECs, converting them to a pro-coagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C (APC) by 31% indicating that DNA scaffold and histones were both necessary for the cytotoxic effect of extracellular chromatin. Conclusions:ATRA promotes procoagulant promyelocytic extracellular chromatin mainly through ETosis. Extracellular chromatin fosters excess thrombin generation, increases fibrin deposition, and causes endothelium damage. To improve the remaining coagulation disturbance in APL patients of high risks during ATRA administration, therapeutic strategies focusing on combined application of DNase I and APC to accelerate the degradation of overwhelmed promyelocytic extracellular chromatin would be of great interest in the future. Disclosures No relevant conflicts of interest to declare.

The APTT Response of Pregnant Plasma to Unfractionated Heparin

2002 ◽  
Vol 87 (01) ◽  
pp. 92-97 ◽  
Author(s):  
E. Young ◽  
M.A. Johnston ◽  
C. Robertson ◽  
I. Naguit ◽  
P. Stevens ◽  
...  
Keyword(s):  
Factor Viii ◽  
Pregnant Women ◽  
Unfractionated Heparin ◽  
Therapeutic Range ◽  
Binding Proteins ◽  
Regression Line ◽  
Factor Xa ◽  
Heparin Binding ◽  
Third Trimester

SummaryPregnancy is associated with a physiological increase in coagulation factors and heparin binding proteins; both can affect the activated partial thromboplastin time (APTT) in response to unfractionated heparin (UFH) invalidating the use of a non-pregnant APTT therapeutic range. We compared the anticoagulant response of UFH added in vitro to the plasma of 13 pregnant (third trimester) and 15 nonpregnant women to determine whether the measured APTT and antifactor Xa activities are lower in pregnancy. Increasing concentrations of UFH were added to platelet-poor plasma from each subject and the APTT and anti-factor Xa activity were measured. The amount of UFH which was reversibly bound and neutralised by plasma heparin binding proteins was assessed by comparing the anti-factor Xa activity before and after addition of low affinity heparin (LAH). Fibrinogen, von Willebrand factor antigen (vWF Ag) and factor VIII levels, were also measured. The APTT response, assessed by the slope of the regression line of log APTT versus added heparin concentration, was attenuated in pregnant plasma (0.76 s/U/mL versus 1.2 s/U/mL, p = 0.005) and was highly correlated to increased non-specific plasma protein binding (47% versus 35% p <0.01) and increased fibrinogen (5.1g/L versus 2.8 g/L, p <0.01) and factor VIII activity (2.7 U/mL versus 1.2 U/mL, p <0.01). Thus, to achieve the same heparin level, pregnant women require higher daily doses of UFH than non-pregnant women. However, if UFH dose adjustments during the third trimester are based upon a non-pregnant APTT therapeutic range, systematic overdosing of pregnant women will result, possibly increasing the risk of bleeding and osteoporosis.

scholarly journals The Role of “Liquid Biopsy” Repositories in Cancer Care in Low to Middle–Income Countries

2018 ◽  
Vol 4 (Supplement 2) ◽  
pp. 244s-244s
Author(s):  
M. Kohli
Keyword(s):  
Cancer Patients ◽  
Whole Blood ◽  
Cancer Care ◽  
Liquid Biopsy ◽  
Healthcare Delivery ◽  
Cancer Center ◽  
Cell Free Dna ◽  
Middle Income ◽  
Middle Income Countries ◽  
Free Dna

Background and context: Translation of underlying individual genomic heterogeneity in cancer into precision medicine practice requires annotated cancer biorepositories. The potential for practice of precise medicine is also coupled to saving vital resources in low to middle–income countries. An overview of experience and outcomes from a tertiary level cancer center in a high-income country for liquid biobank established since 2009 is presented. Aim: To understand the challenges of building economically viable biorepositories that can be used for molecular diagnostics while delivering cancer care. Strategy/Tactics: An institutional ethics–approved prospective liquid biorepository was established in September of 2009 for advanced cancer patients. Informed consent–approved collection of 29.5 mL blood/urine was performed serially on enrolled patients and clinical annotation was obtained during follow-up including previous, current and future treatments and their outcomes. All specimens were processed using a uniform protocol in which extraction of germline DNA from buffy coats; serum for proteomics; platelet-poor and platelet-rich plasma (in citrate and EDTA anticoagulants) for microRNA and cell-free DNA extractions; and extraction of PAXgene RNA/DNA from whole blood was performed. Processing was done within 45 minutes of sample acquisition and storage in −80°C freezers with no freeze–thaw cycles. Program/Policy process: Biobanking for cancer care. Outcomes: Between September of 2009 and January of 2015, 535 advanced-stage prostate cancer patients in hormone-sensitive and castrate-resistant stage; 250 advanced kidney cancer patients; 110 testicular cancer patients were enrolled and 1550 collections were performed serially. This generated >60,000 plasma/serum/DNA/RNA aliquots. Nucleic acids (DNA/RNA) from buffy coats and whole blood of 500-1000 ng volume each were also extracted. Cell-free DNA for somatic mutational and copy number analysis; single nucleotide profiling from germline DNA; RNA expression profiling from whole blood and microRNA analysis in plasma has been performed from this cohort along with proteomics using tandem mass spectrometry. By 2017, this has resulted in >35 scientific publications; 5 patents; multiple national and international grant awards and enhanced precision cancer care for patient care. The cost burden for establishing the infrastructure was highly economical. What was learned: In our experience, liquid biopsy repositories can augment clinical cancer globally, but do not find this discussed in low to middle–income nations. Advancing and applying molecular oncology and team science to prospectively collected and retrospectively annotated biobanks can be a cost-efficient resource in a global cancer healthcare delivery system and a useful tool for scientific and economic opportunities and collaborations.

scholarly journals Deep Sequencing of Peripheral Blood Plasma DNA As a Reliable Test for Confirming the Diagnosis of Myelodysplastic Syndrome

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1909-1909
Author(s):  
Ferras Albitar ◽  
Wanlong Ma ◽  
Kevin Diep ◽  
Ivan De Dios ◽  
Sally Agersborg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Deep Sequencing ◽  
Whole Blood ◽  
Sanger Sequencing ◽  
Cell Free Dna ◽  
Molecular Abnormalities ◽  
Free Dna ◽  
Tumor Load ◽  
Peripheral Blood Plasma

Abstract Background: In patients presenting with cytopenia, myelodysplastic syndrome (MDS) should be considered, but confirmation of diagnosis requires bone marrow biopsy and morphologic and cytogenetic evaluation. It is extremely difficult to rely on subjective morphologic features to confirm the diagnosis of MDS, when the karyotype is normal and blasts are not increased. Objective criteria for the diagnosis of MDS are needed in these cases. With recent advances in the characterization of molecular abnormalities in MDS, diagnosis of early MDS is becoming more objective by documenting the presence of MDS-specific molecular abnormalities in cases with appropriate clinical presentation. Since MDS is a disease of excessive apoptosis in bone marrow, DNA resulting from the apoptosis is abundant in circulation. We explored the potential of using cell free DNA in peripheral blood plasma using next generation sequencing (NGS) to confirm the diagnosis of early MDS without the need for marrow biopsy. Methods: Total nucleic acid was extracted from the plasma of 16 patients presenting with cytopenia and confirmed diagnosis of early MDS (blasts <5%) by the presence of mutations in one or more MDS-specific genes in DNA from cells in bone marrow. Plasma samples from 4 age-matched normals were used as negative controls. We performed targeted sequencing of 14 genes (581 amplicons) using Illumina MiSeq platform. This panel included the following genes: ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBL, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. NGS and Sanger sequencing was used for testing. Results of cell free DNA in plasma were compared to that from cells or whole blood. Results: Deep sequencing of cell free DNA in plasma from the 16 patients with early MDS showed at least one or more mutated gene confirming the diagnosis of MDS. Three patients (19%) showed mutation in one gene and the remaining 13 patients (81%) showed mutations in two or more genes. Cell free DNA in plasma from normal controls showed no evidence of mutations. When NGS data of cell free DNA from plasma was compared with Sanger sequencing data of DNA from cells in bone marrow, 10 of the 16 patients (63%) showed mutations in cell free DNA in plasma not detected by Sanger sequencing in DNA from cells in bone marrow. All mutations detected by NGS in cell free DNA in plasma were below the detection level of the Sanger technique and most likely represent subclones. NGS allowed the measurement of relative tumor load in plasma. Tumor load in plasma as detected by NGS was significantly (P=0.008) higher than that detected in cellular DNA, suggesting higher sensitivity of the former in detecting minimal residual disease and a better tool for monitoring therapy. Without exception, all detected mutations showed higher tumor load in plasma as compared with DNA from cells or whole blood, supporting the concept that plasma is enriched in tumor-specific DNA. Conclusion: NGS of cell free DNA in plasma using limited number of MDS-specific genes, when used in patients with cytopenia, presents an objective test for the confirmation of the diagnosis of MDS. Plasma is enriched in tumor-specific DNA in patients with MDS. Furthermore, mutation analysis of cell free DNA in plasma can detect subclones with mutations and can predict the emergence of new clones. Analysis of cell free DNA in plasma using NGS provides important data on tumor load, which can be used to monitor therapy, and predict progression, and also reduces the need for performing bone marrow biopsies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Quantification of Cell-Free DNA in Red Blood Cell Units in Different Whole Blood Processing Methods

2016 ◽  
Vol 2016 ◽  
pp. 1-5 ◽  
Author(s):  
Andrew W. Shih ◽  
Vinai C. Bhagirath ◽  
Nancy M. Heddle ◽  
Jason P. Acker ◽  
Yang Liu ◽  
...  
Keyword(s):  
Patient Outcomes ◽  
Whole Blood ◽  
Regression Models ◽  
Processing Method ◽  
Red Cell ◽  
Storage Duration ◽  
Cell Free Dna ◽  
Free Dna ◽  
And Storage ◽  
Separate Regression

Background. Whole blood donations in Canada are processed by either the red cell filtration (RCF) or whole blood filtration (WBF) methods, where leukoreduction is potentially delayed in WBF. Fresh WBF red blood cells (RBCs) have been associated with increased in-hospital mortality after transfusion. Cell-free DNA (cfDNA) is released by neutrophils prior to leukoreduction, degraded during RBC storage, and is associated with adverse patient outcomes. We explored cfDNA levels in RBCs prepared by RCF and WBF and different storage durations. Methods. Equal numbers of fresh (stored ≤14 days) and older RBCs were sampled. cfDNA was quantified by spectrophotometry and PicoGreen. Separate regression models determined the association with processing method and storage duration and their interaction on cfDNA. Results. cfDNA in 120 RBC units (73 RCF, 47 WBF) were measured. Using PicoGreen, WBF units overall had higher cfDNA than RCF units (p=0.0010); fresh WBF units had higher cfDNA than fresh RCF units (p=0.0093). Using spectrophotometry, fresh RBC units overall had higher cfDNA than older units (p=0.0031); fresh WBF RBCs had higher cfDNA than older RCF RBCs (p=0.024). Conclusion. Higher cfDNA in fresh WBF was observed compared to older RCF blood. Further study is required for association with patient outcomes.

Cellular and Biochemical Properties of Cell-Free DNA: A Prognostic Marker In Severe Sepsis Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2169-2169
Author(s):  
Travis J Gould ◽  
Dhruva Dwivedi ◽  
Laura Pepler ◽  
Laura Swystun ◽  
Zakhar Lysov ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Healthy Volunteers ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Biochemical Properties ◽  
Dnase Activity ◽  
Blood Monocytes ◽  
Cell Free Dna ◽  
Free Dna ◽  
Induced Apoptosis

Abstract Abstract 2169 Background: Sepsis is the leading cause of morbidity and mortality in patients in the non-coronary ICU. However, the heterogeneity of patients with severe sepsis makes the identification of high-risk patients (ie. those who are sickest and thus would require more aggressive and expensive therapies) an ongoing challenge. Previously, we have shown that high levels of cell-free DNA (cfDNA) appear to be a powerful predictor of death in severe sepsis patients. Using Receiver Operating Characteristic curves, we have demonstrated that the area under the curve for cfDNA is 0.96 (95% CI=0.93–1.00). The purpose of this study is to characterize the source and biochemical properties of cfDNA in sepsis. Methods: Toll-like receptor (TLR) cells (which are stimulated by microbial-specific, unmethylated DNA) were used to determine whether the cfDNA found in septic patients is host- or microbe-derived. Whole blood, monocytes, and neutrophils were isolated from healthy volunteers and stimulated with either lipoteichoic acid and peptidoglycan (from Gram-positive bacteria) or lipopolysaccharide (LPS) from Gram-negative bacteria. Monocytes and neutrophils were also incubated with LPS-induced apoptosis inhibitors, cilostamide and cilostazol, prior to stimulation with LPS. DNAse enzymatic activity was quantified using Org 590 DNAse activity ELISA (Orgentec Diagnostica, GmbH), while DNAse protein was quantified by Western blot analyses. Results: Exposure of TLR9-reporter cells to cfDNA from sepsis patients revealed that the cfDNA is host-derived. Stimulation of whole blood, monocytes, neutrophils, and endothelial cells to microbial cell wall components revealed that monocytes and neutrophils are the major sources of cfDNA in sepsis. Of the total amount of nuclear DNA present within monocytes and neutrophils, nearly all was released into the supernatant upon stimulation. Inhibition of LPS-induced apoptosis with cilostamide and cilostazol did not completely prevent the release of cfDNA from monocytes and neutrophils. We also found that cfDNA levels correlated with levels of histones in septic patients and that the cfDNA was degraded to nucleosomal units. Finally, we observed an inverse correlation between levels of DNAse activity/DNAse protein and levels of cfDNA. Conclusions: These studies are the first to characterize the source and biochemical properties of cfDNA in sepsis. A portion of cfDNA release from monocytes and neutrophils appears to occur via an apoptosis-independent mechanism. Plasma levels of DNAse enzyme were reduced in septic patients compared to healthy volunteers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MARCKS ED Inhibits Fibrin Formation By Blocking Coagulation Protein Complex Assembly on Phosphatidylserine

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2272-2272
Author(s):  
Noah Kastelowitz ◽  
Abimbola Jarvis ◽  
Ormacinda R. White ◽  
Peter N. Brown ◽  
Gary Brodsky ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Whole Blood ◽  
Lipid Membranes ◽  
Factor Xa ◽  
Vehicle Control ◽  
Prothrombinase Complex ◽  
Fibrin Formation ◽  
Activated Platelets ◽  
Microfluidic Flow ◽  
Peptide Treatment

Abstract Introduction: Prothrombinase is an essential enzyme of the coagulation cascade that is made up of a heterodimer of coagulation factor Xa and Va. The complex most readily forms in the presence of lipid membranes containing the anionic lipid phosphatidylserine. The phosphatidylserine lipid head groups provide a surface for the prothrombinase complex to assemble and significantly increase the enzyme's turnover number. Here, we investigate how truncation and modification of the naturally occurring myristoylated alanine-rich C-kinase substrate (MARCKS) protein into small peptide and peptidomimetic fragments (MARCKS ED) forms lipid probes that bind to phosphatidylserine, and block the assembly and enzymatic activity of the prothrombinase complex. Compared to known phosphatidylserine binding proteins such as annexin V or lactadherin, the MARCKS ED probes are less than a tenth of the molecular weight and do not require calcium to bind to phosphatidylserine. Methods: Using biophysical assays, we examine how the MARCKS ED probes assemble on lipid membranes containing phosphatidylserine and how this assembly alters the binding of factor Xa to these surfaces. Effects on the enzymatic activity of the prothrombinase complex in the presence of activated platelets and other phosphatidylserine exposing membranes are explored using a modified prothrombinase assay. The binding and anticoagulant activity of the MARCKS ED probes during whole blood coagulation are examined by quantifying fluorescently labeled MARCKS ED probe localization, platelet accumulation, and fibrin formation in microfluidic flow assays. Results: We find that the MARCKS ED probes can antagonize factor Xa from binding phosphatidylserine by measuring reduced association of factor Xa to lipid membranes treated with the MARCKS ED probes. Pre-treatment of activated platelets with the MARCKS ED probes significantly reduced the assembly and enzymatic activity of prothrombinase (100% activity with vehicle control vs. 51% with 1 μM MARCKS ED peptide treatment, P < 0.05). Similar inhibitory activity was also observed when activated platelets were replaced with other phosphatidylserine exposing lipid membranes, such as synthetic liposomes or biologic microparticles. In the whole blood microfluidic flow assay, the MARCKS ED probes colocalized with adherent platelets and inhibited fibrin formation (100% peak fibrin intensity with vehicle control vs. 17% peak fibrin intensity with 1 μM MARCKS ED peptide treatment, P < 0.05). The MARCKS ED probes did not alter platelet surface area coverage. Electron microscopy images of the final clots formed in the microfluidic flow assay show an absence of fibrin formation with MARCKS ED probe treatment. Conclusions: We demonstrate that the MARCKS ED probes can inhibit the assembly and enzymatic activity of prothrombinase, and that this activity translates to significantly reduced fibrin formation in whole blood. The MARCKS ED peptide and peptidomimetic probes may provide a new therapeutic tool for inhibiting the assembly of phosphatidylserine dependent coagulation factors, as well as a research tool for identifying platelet phosphatidylserine exposure. Disclosures Kastelowitz: University of Colorado Boulder: Patents & Royalties. Yin:University of Colorado Boulder: Patents & Royalties.

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