Platelet Factor 4 (PF4) Modulates the Prothrombotic Nature of Neutrophil-Extracellular Traps (NETs): Therapeutic Implications of a NET-Stabilization Strategy

Abstract Sepsis is a dysregulated response to infection leading to life-threating organ damage. Although it remains one of the most common causes of mortality worldwide, it lacks targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens and prevent bacterial dissemination. However, when NETs are degraded by circulating nucleases they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger coagulation, induce complement activation, and cause oxidative tissue damage. We proposed a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4. Binding of PF4 enhances NET DNase-resistance, promotes NDP sequestration and increases bacterial capture, improving survival in murine sepsis. As NETs are considered prothrombotic, we were concerned that NET stabilization may increase the risk of clot formation. We therefore sought to determine the effect of PF4-NET stabilization on the thrombogenicity of NETs to learn if this strategy is safe for clinical application. To that end, we examined the effect of PF4 on the thrombotic potential of DNA and NET fragments at different states of nuclease digestion. High molecular weight (hmw) genomic DNA (hmwDNA, >50kbp) was isolated from human whole blood. hmwDNA was digested with restriction enzymes (EcoRI and AluI) for 15min to generate DNA fragments of ~4kbp and ~250bp, respectively. Neutrophils were also isolated from human blood and stimulated with 100 nM PMA to produce neutrophil-adherent NETs, which were cleaved from cell bodies by treatment with 4U/mL DNase I for 20 minutes, releasing NETs >50kbp (hmwNETs). Additional incubation of hmwNETs with DNase I yielded smaller NET fragments. We assessed in vitro activation of coagulation by DNA and NETs by measuring thrombin generation and fibrin formation in platelet-poor plasma using fluorogenic substrate and turbidity assays. Neutrophil-adherent NETs induced far less thrombin generation and fibrin formation in plasma than hmwDNA and hmwNETs. PF4 significantly increased lag time and reduced peak thrombin formation induced by both hmwDNA and hmwNETs. Binding of PF4 also delayed clot initiation time and reduced the rate of fibrin generation. Digestion of hmwDNA and hmwNETs to smaller fragments markedly enhanced thrombogenicity. We posited that shorter DNA fragments are more thrombogenic because they have a greater proportion of end-fragment DNA that exposes more single-stranded DNA. To test this hypothesis, we subjected hmwDNA and digested DNA to heat denaturation at 95°C and rapid cooling to generate single stranded DNA and found that this accelerated fibrin generation. Although the anti-thrombotic effect of PF4 was most pronounced with longer DNA and NET fragments, it continued to significantly reduce fibrin generation induced by shorter DNA fragments, perhaps by stabilizing the fragments to prevent exposure of single-stranded DNA. In conclusion, although prior studies have shown that NETs increase the risk of thrombosis in sepsis, we propose the counter-intuitive concept that PF4-stabilization decreases the risk of NET-mediated prothrombotic state by (1) inhibiting DNase cleavage of intact NETs and subsequent liberation of prothrombotic cfDNA from non-thrombogenic neutrophil-adherent NETs, and (2) preventing further digestion of circulating cfDNA into shorter and more prothrombotic fragments. Although NETs are a double-edged sword: capable of capturing pathogens but inducing host-tissue damage and thrombosis when degraded, treatment with PF4 tips the balance, limiting the capacity of NETs to induce fibrin generation and thrombosis, while enhancing their ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis and merits further investigation in translational studies. Disclosures No relevant conflicts of interest to declare.

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Promyelocytic Extracellular Chromatin Exacerbates Coagulation Disorder in Acute Promyelocytic Leukemia

Blood ◽

10.1182/blood.v128.22.3806.3806 ◽

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.

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Understanding How Fc-Modification Transforms a Pathogenic Heparin-Induced Thrombocytopenia (HIT)-like Monoclonal Antibody into a Novel Treatment for Sepsis

Blood ◽

10.1182/blood-2019-123132 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 10-10

Author(s):

Kandace Gollomp ◽

Amrita Sarkar ◽

Gowthami Arepally ◽

Douglas B. Cines ◽

M. Anna Kowalska ◽

...

Keyword(s):

Monoclonal Antibody ◽

Degradation Products ◽

Cecal Ligation ◽

Organ Damage ◽

Microfluidic System ◽

Dnase I ◽

Bacterial Toxin ◽

Platelet Factor ◽

Microfluidic Chamber ◽

Positively Charged

Sepsis is a dysregulated response to infection that leads to life-threatening organ damage. This process induces the release of neutrophil extracellular traps (NETs), webs of negatively-charged cell-free DNA (cfDNA) complexed with positively-charged histones that capture pathogens but also damage host tissue. We believe that upon presentation, most patients with sepsis have already released a large amount of NETs so that preventing NET release (NETosis), or accelerating their lysis with DNase, may be ineffective or harmful. Murine studies of NET lysis in sepsis have met with mixed results raising the concern that this strategy may lead to the release of captured bacteria and harmful NET degradation products (NDPs) such as cell free (cf) DNA and histones. We propose NET stabilization as an alternative therapy in sepsis. In a microfluidic system, we have shown that NETs bound the platelet chemokine platelet factor 4 (PF4, CXCL4), a highly-positively charged protein that aggregates polyanionic molecules including DNA. As a result of this aggregating effect, PF4 physically compacts NETs without inducing histone release. These compact PF4-NET complexes are resistant to DNase lysis. Using a microfluidic system, in which channels coated with NETs were infused with labeled Staphylococcus aureus particles, we now show that PF4 compaction of NETs markedly enhances their ability to capture bacteria. Likely, PF4, which is known to bind to the bacterial cell wall, cross aggregates pathogens to NET DNA. Thus, without added PF4, little bacteria is entrapped in the microfluidic chamber (Fig. 1A & 1B) and those captured are readily released by the infusion of DNase I (Fig. 1C & 1D). In contrast, when PF4 is added, microbial entrapment is markedly enhanced (Fig. 1A & 1B) and persists even with the infusion of DNase (Fig. 1C & 1D). When human (h) PF4 binds to polyanions it changes conformation, revealing HIT antigenic sites that bind the HIT-like monoclonal antibody KKO. Bound KKO further enhances PF4-NET complex resistance to DNase I in the microfluidic chamber, preventing the release of NDPs and entrapped bacteria upon exposure to DNases. These in vitro observations led us to hypothesize that infused PF4 and/or KKO may serve as a targeted therapeutic in sepsis. However, unmodified KKO stimulates leukocytes and induces a prothrombotic state. Indeed, infused KKO accelerates mortality in murine models of sepsis in mice that express hPF4 (hPF4+) (Fig. 2C). We, therefore, modified KKO using an IgG-specific endoglycosidase to create deglycosylated KKO (DG-KKO) that retains the ability to bind to PF4-NET complexes, but has little capacity to interact with hematopoietic cell Fc receptors or activate complement. DG-KKO binding also protects hPF4-NET complexes from DNase lysis and does not interfere with bacterial capture (Fig. 1B). In a murine lipopolysaccharide (LPS) endotoxemia model, DG-KKO infusion prevented thrombocytopenia, decreased the release of NDPs, and enhanced survival exclusively in hPF4+ mice (not shown). Although treatment with LPS recapitulates many aspects of sepsis, it relies on treatment with a bacterial toxin rather than live pathogens. Therefore, to determine if hPF4 and/or DG-KKO mitigate bacterial-induced sepsis, we repeated our studies using cecal ligation and puncture (CLP) to induce polymicrobial sepsis. Infused hPF4, but more so, infused DG-KKO decreased the severity of thrombocytopenia, reduced NDP release, and improved survival (Fig. 2A-2C). These murine studies demonstrate that PF4 released from activated platelets stabilizes NETs, making them resistant to DNase lysis and enhancing their ability to capture bacteria. Infused PF4 and DG-KKO further enhance NET stability, decrease the release of NDPs, and enhance bacterial entrapment, leading to better outcomes. These studies provide mechanistic insights into how NETs contribute to end-organ damage in sepsis and offer a targeted and novel therapeutic that minimizes their harmful effects, while enhancing their protective properties. Disclosures Arepally: Biokit: Patents & Royalties; Apotex Pharmaceuticals: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Antibody Stabilization of Neutrophil Extracellular Trap-Platelet Factor 4 Complexes Is Therapeutic in a Murine Model of Endotoxemia

Blood ◽

10.1182/blood-2018-99-111381 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 271-271

Author(s):

Amrita Sarkar ◽

Gowthami Arepally ◽

M. Anna Kowalska ◽

Douglas B. Cines ◽

Lubica Rauova ◽

...

Keyword(s):

Endothelial Cells ◽

Platelet Activation ◽

Degradation Products ◽

Umbilical Vein ◽

Dnase I ◽

Platelet Factor 4 ◽

Neutrophil Extracellular Trap ◽

Clinical Samples ◽

Platelet Factor ◽

Positively Charged

Abstract Sepsis is characterized by life-threatening organ dysfunction caused by a dysregulated response to infection. Neutrophils play a crucial role in sepsis during which they release neutrophil extracellular traps (NETs), webs of negatively charged cell-free DNA (cfDNA) complexed with positively-charged histones that kill pathogens but also damage host tissue. While it has been proposed that NET digestion may be beneficial in the treatment of sepsis, it is also possible that this strategy leads to the release of harmful NET degradation products (NDPs), such as cfDNA, histones, and myeloperoxidase (MPO) that cause endothelial damage. An alternative approach would be treatment strategies that prevent NET lysis and the release of NDPs. We posited that NET-bound platelet factor 4 (PF4, CXCL4) may have this effect. PF4 is a highly-positively charged, platelet-specific chemokine released in high concentrations following platelet activation and aggregates polyanionic molecules like heparin, polyphosphates, and DNA. We have found that PF4 causes physical compaction of NETs without inducing histone release. We studied the effect of this compaction using an in vitro microfluidic assay in which NETs released from neutrophils stimulated by tissue-necrosis factor (TNF) α were infused over a layer of cultured human umbilical vein endothelial cells (HUVECs) ± PF4 (25 μg/ml) and incubated for 12 hours. Channels exposed to PF4 were "heathier" with significantly more residual attached endothelial cells (44±24 vs. 150±29 cells/hpf, n=6, p=0.0002). We have previously shown that mice that overexpress PF4 are protected from mortality in lipopolysaccharide (LPS) endotoxemia. Compared to wildtype animals, PF4 null mice have increased circulating levels of NET markers including cfDNA, citrullinated histones, and MPO. Treatment with exogenous PF4 leads to a decrease in plasma levels of NET components and a reduction in mortality. In a microfluidic assay in which channel-adherent NETs were treated with increasing concentrations of PF4 (0-25 µg/ml) and then infused with DNase I (100 U/ml), we found that compacted NET-PF4 complexes become resistant to DNase I digestion. Prior studies have revealed that when PF4 binds to polyanionic molecules, it exposes HIT-like antigenic sites. We confirmed that the NET-PF4 complexes similarly bind HIT IgG isolated from clinical samples and the HIT-like monoclonal antibody KKO. Interestingly, KKO binding further enhanced DNase I resistance, a phenomenon not seen with a polyclonal anti-PF4 antibody (abcam). We hypothesized that KKO which causes PF4 oligomerization unlike the polyclonal antibody, further stabilized the crosslinked NET-PF4 complexes, providing additional protection from DNase I digestion and preventing the release of NDPs. We then asked whether KKO may serve as a targeted therapy in the treatment of sepsis, enhancing NET-PF4 complex resistance to nuclease digestion and leading to the sequestration of NDPs. As KKO is an IgG2bk antibody, we were unable to use pepsin digestion to remove the Fc region that causes platelet activation. We therefore used an IgG-specific endoglycosidase to develop a deglycosylated version of KKO (DG-KKO) that retains the ability to bind to NET-PF4 complexes (data not shown), but has a reduced capacity to interact with hematopoietic cell Fc receptors. DG-KKO has a markedly decreased ability to activate human platelets compared to KKO in the presence of added PF4 as measured by P-selectin level (3710±140 control vs. 1580±300 treated MFI, n = 2). Compared to control, DG-KKO infusion in the murine endotoxemia model prevented thrombocytopenia (mean platelet count 286±16 control vs. 537±69 treated X103/µl, n=4-5, p=0.016), the release of cfDNA (8.5±0.3 vs. 3.7±0.7 µg/ml, n=4-5, p=0.016), and the emission of MPO-DNA complexes (234±14 vs. 99±5 % increase from non-LPS injected controls, n=4-5, p=0.016) As depicted in Figure 1, we propose that in sepsis, NETosis occurs (Step 1) causing the release of harmful NDPs (Step 2). PF4 expelled from activated platelets stabilizes PF4-NET complexes (Step 3). Infused DG-KKO enhances NET stability and decreases release of NDPs following DNase I digestion (Step 4). These studies provide mechanistic insights into the release of NDPs during endotoxemia and offer a targeted, novel therapeutic to prevent their contribution to inflammatory states such as sepsis. Disclosures Poncz: Incyte Corporation: Consultancy, Research Funding.

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Microparticles Modulate Formation, Structure, and Properties of Fibrin Clots

Blood ◽

10.1182/blood.v124.21.2807.2807 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2807-2807

Author(s):

Rosa M. Nabiullina ◽

Dzhigangir A. Faizullin ◽

Chandrasekaran Nagaswami ◽

Yuriy F. Zuev ◽

Ilshat G. Mustafin ◽

...

Keyword(s):

Confocal Microscopy ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Clot Lysis ◽

Outer Cell ◽

Plasma Samples ◽

Fibrin Polymerization ◽

Fibrin Formation ◽

Fibrin Clots

Abstract Microparticles (MPs) are phospholipid vesicles about 0.1-1 μm in size released into blood from vascular and blood cells upon activation and apoptosis. The mechanism of MP formation by the exocytic budding of the outer cell membrane provides them with procoagulant activity, mainly due to phosphatidylserine exposure and tissue factor expression. MPs are present in the peripheral blood of healthy subjects but their level is elevated in a variety of vascular, infectious, and immune-mediated pathologies. While there are extensive studies on multiple roles of MPs in various diseases, including thrombotic disorders, the question remains open as to whether MPs are involved in the formation and properties of fibrin, the scaffold of hemostatic clots and obstructive thrombi. To investigate this problem, we studied in vitro the effects of MPs on the kinetics of fibrin polymerization, fibrin clot structure and susceptibility to fibrinolysis. Blood was drawn from healthy donors according to an IRB protocol. MPs were removed from citrated platelet-free plasma (PFP) by filtration through 0.1-μm pores, yielding microparticle-depleted plasma (MDP). To restore the phospholipids eliminated, MDP was replenished with cephalin (MDP-C). Fresh samples of PFP, MDP, and MDP-C from the same donor were analyzed in parallel. Concentrations of MPs and expression of platelet-specific CD61 were studied by flow cytometry and confocal microscopy. A thrombin generation test in recalcified defibrinated plasma was performed using a chromogenic substrate. Fibrin polymerization induced by recalcification of PFP, MDP, and MDP-C was studied by dynamic turbidimetry. tPA-induced fibrinolysis was also followed optically as a decrease of a plasma clot turbidity. Fibrin ultrastructure was studied with scanning electron microscopy (SEM) in a FEI Quanta 250 microscope. Each test was performed with plasma samples from at least 3 donors and the results were averaged. Enumeration of MPs in PFP vs. MDP showed that about 90% of total MPs were removed by filtration, including 99.6% of CD61-positive platelet-derived MPs. The rate of thrombin generation was significantly reduced in MDP vs. PFP and was fully restored in MDP-C, confirming an essential role of MPs in the intrinsic coagulation pathway. Based on the lag times and slopes of the turbidimetric curves, fibrin formation in recalcified plasma samples was significantly delayed in the absence of MPs (MDP) and was much faster in their presence (PFP and MDP-C). The maximum optical density was consistently and significantly higher in MDP than in PFP and MDP-C, suggesting that fibrin clots formed in the absence and presence of MPs have fibers of different thickness. This presumption was confirmed by SEM of fibrin clots formed upon recalcification of the plasma samples. Clots formed in PFP were less porous and had thinner fibers (170 ± 38 nm), while clots from MDP had larger pores and were built of thicker fibers (214 ± 53 nm, p<0.01). Addition of phospholipids to MDP (MDP-C) resulted in the formation of densely packed thin fibrin fibers (141 ± 34 nm), similar to the initial clots from PFP. Consistent with the dissimilarity in ultrastructure, the clots displayed a distinct susceptibility to fibrinolysis: the rate of clot lysis was significantly higher in MDP than in PFP and MDP-PL. A novel and important finding from the SEM of fibrin clots is that fibers formed in MDP were quite smooth, while fibers formed in the presence of MPs (PFP and MDP-C) had relatively rough surfaces with multiple sub-micron size particles attached, suggesting that MPs might directly bind fibrin. Consistent with the SEM of the appearance of MPs on the fibers, confocal microscopy of PFP-clots stained for CD61 with FITC-labeled antibodies, unlike MDP-clots, revealed fluorescent spots associated with fibrin, indicating that MPs were adsorbed on fibrin fibers. In conclusion, MPs have profound effects on fibrin formation and on the final structure and characteristics of fibrin clots via at least two mechanisms. One is an indirect kinetic effect based on the MP-dependent rate of thrombin generation. The other is direct binding of MPs to fibrin fibers during and after fibrin assembly. Both mechanisms underlie a previously underappreciated potential role of MPs in hemostasis and thrombosis as modulators of fibrin formation and properties. (Research supported by NIH grant HL090774 and the Program of Competitive Growth of Kazan Federal University) Disclosures No relevant conflicts of interest to declare.

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The Role of Prothrombinase Complex Assembly and in Situ Fibrin Deposition on the Surface of Acute Promyelocytic Leukemia Cells in Hemorrhage

Blood ◽

10.1182/blood-2018-99-113785 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3770-3770

Author(s):

Tao Li ◽

Xiaoyan Yang ◽

Yan Zhang ◽

Baorong LI ◽

Yinmiao Liu ◽

...

Keyword(s):

Acute Promyelocytic Leukemia ◽

Thrombin Generation ◽

Coagulation Factors ◽

Dnase I ◽

Promyelocytic Leukemia ◽

Fibrin Deposition ◽

Complex Assembly ◽

Prothrombinase Complex ◽

Fibrin Formation

Abstract Background: Despite dramatic improvement in treatment for acute promyelocytic leukemia (APL), early death due to hemorrhage remains a major obstacle to achieving a complete cure. In contrast to classical disseminated intravascular coagulation, APL-associated coagulopathy is characterized by rare microvascular fibrin thrombi. Thus, it is attractive to speculate whether other unknown mechanisms depleting coagulation factors and unrecognized fibrin-deposition location exist. Procoagulant activity associated with APL cells plays a direct role in bleeding complicationsin. We have shown that exposed phosphatidylserine (PS) on APL cells supports purified prothrombinase (Zhou J et al, JTH 2010) and fibrin preferentially deposits on promyelocytic chromatin from ETosis or apoptosis (Cao M et al, Blood 2017). However, relatively little is known about the PS-driven prothrombinase complex assembly and in situ fibrin deposition on APL cells. Aims: Our objectives were to determine how APL cells promote thrombin generation and modulate fibrin formation and distribution, as well as to explore the relationship between in situ fibrin deposition and consumptive hemorrhage in APL patients. Methods: Twenty-seven newly diagnosed APL patients were included. Fresh APL blasts were obtained from bone marrow specimens by centrifugation through Ficoll-Hypaque. Lactadherin was used as a probe for PS exposure on the fresh APL blasts and on an immortalized APL cell line (NB4). PS exposure and fluorescein-labeled FV/X binding were evaluated by flow cytometry. Thrombin generation was measured by modifed thrombin generation test. Fibrin production was quantified by turbidity. The distribution of PS, prothrombinase complex and in situ fibrin deposition were imaged by confocal microscopy. For the inhibition assay, APL cells were pre-treated with lactadherin, DNase I or anti-TF antibody for 10 min at 37 °C before incubation with plasma. Results: Thrombin generation and fibrin formation supported by NB4 and APL cells increased approximately 1.5-fold after exposure to daunorubicin and decreased 80% after treatment with all-trans retinoic acid (ATRA) or arsenic trioxide (ATO). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure increased approximately 2.7-fold after treatment with daunorubicin, while ATRA and ATO initially led to a 70% reduction in PS exposure, which rose again on day 3 and 5 (P<0.001), respectively. Levels of externalized PS on APL cells paralleled levels of FV/FX binding, lag time, peak thrombin, endogenous thrombin potential and fibrin formation. Lactadherin inhibited the above parameters by approximately 80%, while anti-tissue factor antibody or DNase I produced no effect. Interestingly, confocal imaging showed that fibrin preferentially deposited on the surface of APL cells, which we defined as "in situ fibrin". Untreated viable APL and NB4 cells displayed discrete or occasionally annular fibrin deposition on the membrane. Moreover, fibrin formation supported by apoptotic APL cells displayed the following progression: (i) patchy deposition, (ii) diffuse rim, (iii) "fibrin coat", and (iv) network. The dynamic changes in fibrin formation paralleled the kinetics of PS exposure and prothrombinase assembly. Furthermore, initial percentage of PS-positive fresh APL cells was negatively correlated with plasma levels of fibrinogen and factor II, V, VIII, X in APL patients on admission (all P<0.01). Conclusion: PS-driven prothrombinase complex assembly and in situ fibrin deposition on the surface of APL cells consume massive coagulation factors, providing a novel explanation for consumptive hemorrhage in APL patients. Blockade of PS might be a novel therapeutic approach for preventing bleeding in APL via inhibiting invisible "in situ coagulation", especially in high-risk APL. Disclosures No relevant conflicts of interest to declare.

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NORMAL FIBRINOPEPTTDE A (FPA) AND ELEVATED FIBRINOGEN DEGRADATION PRODUCTS AFTER LONG DISTANCE RUNNING

10.1055/s-0038-1643132 ◽

1987 ◽

Author(s):

P Bartsch ◽

A Haeberli ◽

P W Straub

Keyword(s):

Physical Exercise ◽

Degradation Products ◽

Time Lag ◽

Thrombin Time ◽

Plasmin Activity ◽

Long Distance ◽

Platelet Factor ◽

Mean Values ◽

Fibrin Formation

Physical exercise leads to a shortening of activated partial thrcnboplastin time (PIT) and euglobulin lysis time (ELT). Whether this activation causes in-vivo thrombin and plasmin action remains controversial however. 19 well trained long distance runners were examined 25 min (range 5-53) after termination of a 100 km race (post-race values) run in 577 min (457-755) and 5 days later after at least one day without physical exercise (control values). FPA, platelet factors and fibrin (ogen) split products (fragment E and BB 15-42) were measured with radioimnunoassays. ELT was assessed before and after venous occlusion (VO). The table gives mean values ±SD:Thrombin time, platelet count, platelet factor 4 and haematocrit did not change significantly. FPA was normal in all post-race samples and did not correlate significantly with the time lag between arrival and blood sampling, indicating that activation of blood coagulation in exhaustive physical exercise of long duration does not lead to in-vivo fibrin formation. Activation of fibrinolysis, however, results in circulating plasmin activity as demonstrated by the elevation of the fibrin (ogen) degradation products fragment E and BB 15-42. Since the polyclonal antibodies used in the latter assay crossreact with BB 1-42, these results alone do not allow to jjdge whether plasmin degrades fibrin or fibrinogen. However, lack of fibrin formation suggests fibrinogenolysis rather than fibrinolysis.

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Novel Plasma Based Microfluidic Assay for Measuring Fibrin Formation,Thrombin Generation and Fibrinolysis Under Flow: Monitoring Patients with Hemophilia on Inhibitors

Blood ◽

10.1182/blood.v128.22.2579.2579 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2579-2579

Author(s):

Abimbola Jarvis ◽

Katherine Ruegg ◽

Linda Jacobson ◽

Brian Branchford ◽

Elizabeth Villalobos-Menuey

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Substrate Surface ◽

Lag Time ◽

Clot Formation ◽

Plasma Samples ◽

Flow Monitoring ◽

Fibrin Formation

Abstract INTRODUCTION The unpredictable clinical response of patients to bypassing therapy and the lack of a proper laboratory tools to measure clot formation and stability renders prophylaxis and surgery on these patients a huge challenge. These patients are at a risk for bleeding or thromboembolic complications. AIMS In this study we introduce a novel plasma based microfluidic assay that can qualitatively and quantitatively measure fibrin deposition, thrombin and plasmin generation, and fibrinolysis under flow. We then examined the dynamics of thrombus formation in patients with hemophilia and their response to replacement and bypass therapies under flow conditions. METHODS Coagulation in the plasma based assay was initiated by spherical 1µm lipidated- Tissue Factor biomimetic silica beads which were patterned into 200µm circles on a substrate surface. Plasma samples were obtained from Hemophilia patients and inhibitor patients, before and after replacement or bypassing treatment and perfused over the tissue factor rich surfaces at a sheer rate of 100 s-1 Fibrinolysis was initiated with the addition of tPAto the plasma samples before perfusion. RESULTS The microfluidic assay was sensitive enough to measure the activation of coagulation triggered by the bypassing agents. Fibrin generation and thrombin generation were measurable both qualitatively and quantitatively using three metrics; lag time, rate of production and maximum quantity produced. Individuals on replacement therapy showed normalized fibrin formation with a 69% increase in fibrin formation, a decrease in lag time and an overall increase in maximum fibrin and thrombin production (See attached Figure). The microfluidic assay was also able to show an increase in overall fibrin generation in certain Individuals that were given more bypassing treatment than needed. Compared to healthy controls the rate of fibrin generation and maximum fibrin was greater, thereby identifying a risk for prothrombotic state. (See attached Figure). Finally, using the microfluidic assay we were able to observe both clot formation and lysis and asses the the stability of the fibrin clot produced when these inhibitor patients were on and off treatments, which reflects a more complete picture of the coagulation process. CONCLUSION We are able to show that individuals, treated by replacement therapies showed normalized clot formation. Individuals with hemophilia treated by bypassing therapies also showed normalized clot formation. Sometimes however, the fibrin production is more than a normal control, which could lead to a risk of prothrombosis. By using microfluidic assay, the treatment can be given in doses and fibrin production observed, to decrease the overall fibrin formation from a hypocoagulable to a hemostatic state, avoiding hypercoagulability. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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