Circulating Exosomal microRNAs as Predictive Biomarkers of Neoadjuvant Chemotherapy Response in Breast Cancer

Background: Neoadjuvant chemotherapy (NACT) is an increasingly used approach for treatment of breast cancer. The pathological complete response (pCR) is considered a good predictor of disease-specific survival. This study investigated whether circulating exosomal microRNAs could predict pCR in breast cancer patients treated with NACT. Method: Plasma samples of 20 breast cancer patients treated with NACT were collected prior to and after the first cycle. RNA sequencing was used to determine microRNA profiling. The Cancer Genome Atlas (TCGA) was used to explore the expression patterns and survivability of the candidate miRNAs, and their potential targets based on the expression levels and copy number variation (CNV) data. Results: Three miRNAs before that NACT (miR-30b, miR-328 and miR-423) predicted pCR in all of the analyzed samples. Upregulation of miR-127 correlated with pCR in triple-negative breast cancer (TNBC). After the first NACT dose, pCR was predicted by exo-miR-141, while miR-34a, exo-miR182, and exo-miR-183 predicted non-pCR. A significant correlation between the candidate miRNAs and the overall survival, subtype, and metastasis in breast cancer, suggesting their potential role as predictive biomarkers of pCR. Conclusions: If the miRNAs identified in this study are validated in a large cohort of patients, they might serve as predictive non-invasive liquid biopsy biomarkers for monitoring pCR to NACT in breast cancer

CD36-fibrin interaction propagates FXI-dependent thrombin generation of human platelets

Thrombin converts fibrinogen to fibrin and activates blood and vascular cells in thrombo-inflammatory diseases. Platelets are amplifiers of thrombin formation when activated by leukocyte- and vascular cell-derived thrombin. CD36 on platelets acts as sensitizer for molecules with damage-associated molecular patterns, thereby increasing platelet reactivity. Here, we investigated the role of CD36 in thrombin-generation on human platelets, including selected patients with advanced chronic kidney disease (CKD). Platelets deficient in CD36 or blocked by anti-CD36 antibody FA6.152 showed impaired thrombin generation triggered by thrombin in calibrated automated thrombography. Using platelets with congenital function defects, blocking antibodies, pharmacological inhibitors, and factor-depleted plasma, CD36-sensitive thrombin generation was dependent on FXI, fibrin, and platelet signaling via GPIbα and SFKs. CD36-deficiency or blocking suppressed thrombin-induced platelet αIIbβ3 activation, granule exocytosis, binding of adhesion proteins and FV, FVIII, FIX, FX, but not anionic phospholipid exposure determined by flow cytometry. CD36 ligated specifically soluble fibrin, which recruited distinct coagulation factors via thiols. Selected patients with CKD showed elevated soluble fibrin plasma levels and enhanced thrombin-induced thrombin generation, which was normalized by CD36 blocking. Thus, CD36 is an important amplifier of platelet-dependent thrombin generation when exposure of anionic phospholipids is limited. This pathway might contribute to hypercoagulability in CKD

Speech Communication

Contains table of contents for Part IV, table of contents for Section 1 and reports on five research projects.Apple Computer, Inc.C.J. Lebel FellowshipNational Institutes of Health (Grant T32-NS07040)National Institutes of Health (Grant R01-NS04332)National Institutes of Health (Grant R01-NS21183)National Institutes of Health (Grant P01-NS23734)U.S. Navy / Naval Electronic Systems Command (Contract N00039-85-C-0254)U.S. Navy - Office of Naval Research (Contract N00014-82-K-0727

Speech Communication

Contains reports on five research projects.C.J. Lebel FellowshipNational Institutes of Health (Grant 5 T32 NSO7040)National Institutes of Health (Grant 5 R01 NS04332)National Institutes of Health (Grant 5 R01 NS21183)National Institutes of Health (Grant 5 P01 NS13126)National Institutes of Health (Grant 1 PO1-NS23734)National Science Foundation (Grant BNS 8418733)U.S. Navy - Naval Electronic Systems Command (Contract N00039-85-C-0254)U.S. Navy - Naval Electronic Systems Command (Contract N00039-85-C-0341)U.S. Navy - Naval Electronic Systems Command (Contract N00039-85-C-0290)National Institutes of Health (Grant RO1-NS21183), subcontract with Boston UniversityNational Institutes of Health (Grant 1 PO1-NS23734), subcontract with the Massachusetts Eye and Ear Infirmar

Rivaroxaban Effects Illustrate the Underestimated Importance of Activated Platelets in Thrombin Generation Assessed by Calibrated Automated Thrombography

Background: The direct oral anticoagulant rivaroxaban inhibiting specifically activated factor X (FXa) causes delayed thrombin generation (TG) as measured by calibrated automated thrombography (CAT). The implications of these changes for assessing bleeding or residual prothrombotic risks of patients are unclear in the absence of a better understanding of the underlying mechanism. Methods: We compared platelet rich plasma (PRP) without or with prior collagen-induced platelet aggregation (agPRP) in the CAT assay to better characterize TG in the presence of rivaroxaban. Results: In the presence of rivaroxaban, TG curves in agPRP showed a distinct profile with a rapidly ascending phase followed with a protracted phase. Inhibition of tissue factor pathway inhibitor amplified the first phase of the curve which was also modulated by procoagulant phospholipids. Inhibition of FXIIa-dependent FXI activation revealed that aggregated platelets influenced the first phase by a combination of extrinsic and intrinsic coagulation pathway initiations. Thrombin-dependent amplification of TG (even prior collagen activation) was responsible for the second phase of the TG curve. Conclusions: AgPRP fully includes platelet ability to support TG and reveal distinct TG phases in the presence of direct FXa inhibitors highlighting its potential use in an anticoagulated setting

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca2+. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets

The Serine/Threonine Protein Phosphatase 2A (PP2A) Regulates Syk Activity in Human Platelets

Distinct membrane receptors activate platelets by Src-family-kinase (SFK)-, immunoreceptor-tyrosine-based-activation-motif (ITAM)-dependent stimulation of spleen tyrosine kinase (Syk). Recently, we reported that platelet activation via glycoprotein (GP) VI or GPIbα stimulated the well-established Syk tyrosine (Y)-phosphorylation, but also stoichiometric, transient protein kinase C (PKC)-mediated Syk serine(S)297 phosphorylation in the regulatory interdomain-B, suggesting possible feedback inhibition. The transient nature of Syk S297 phosphorylation indicated the presence of an unknown Syk pS297 protein phosphatase. In this study, we hypothesize that the S-protein phosphatase 2A (PP2A) is responsible for Syk pS297 dephosphorylation, thereby affecting Syk Y-phosphorylation and activity in human washed platelets. Using immunoblotting, we show that specific inhibition of PP2A by okadaic acid (OA) alone leads to stoichiometric Syk S297 phosphorylation, as analyzed by Zn2+-Phos-tag gels, without affecting Syk Y-phosphorylation. Pharmacological inhibition of Syk by PRT060318 or PKC by GF109203X only minimally reduced OA-induced Syk S297 phosphorylation. PP2A inhibition by OA preceding GPVI-mediated platelet activation induced by convulxin extended Syk S297 phosphorylation but inhibited Syk Y-phosphorylation. Our data demonstrate a novel biochemical and functional link between the S-protein phosphatase PP2A and the Y-protein kinase Syk in human platelets, and suggest that PP2A represents a potential enhancer of GPVI-mediated Syk activity caused by Syk pS297 dephosphorylation

Rivaroxaban Effects Illustrate the Underestimated Importance of Activated Platelets in Thrombin Generation Assessed by Calibrated Automated Thrombography

International audienceBackground: The direct oral anticoagulant rivaroxaban inhibiting specifically activated factor X (FXa) causes delayed thrombin generation (TG) as measured by calibrated automated thrombography (CAT). The implications of these changes for assessing bleeding or residual prothrombotic risks of patients are unclear in the absence of a better understanding of the underlying mechanism. Methods: We compared platelet rich plasma (PRP) without or with prior collagen-induced platelet aggregation (agPRP) in the CAT assay to better characterize TG in the presence of rivaroxaban. Results: In the presence of rivaroxaban, TG curves in agPRP showed a distinct profile with a rapidly ascending phase followed with a protracted phase. Inhibition of tissue factor pathway inhibitor amplified the first phase of the curve which was also modulated by procoagulant phospholipids. Inhibition of FXIIa-dependent FXI activation revealed that aggregated platelets influenced the first phase by a combination of extrinsic and intrinsic coagulation pathway initiations. Thrombin-dependent amplification of TG (even prior collagen activation) was responsible for the second phase of the TG curve. Conclusions: AgPRP fully includes platelet ability to support TG and reveal distinct TG phases in the presence of direct FXa inhibitors highlighting its potential use in an anticoagulated setting