Pro-Coagulant and Pro-Thrombotic Effects of Paclitaxel Mediated by Red Blood Cells

Background Paclitaxel is one of the most widely used anti-cancer drugs, but numerous case reports of thrombotic events in the cancer patients using paclitaxel raise concern over its pro-thrombotic risk. Materials and Methods We investigated whether paclitaxel can elicit pro-thrombotic properties in red blood cells (RBCs) through phosphatidylserine (PS) exposure and microvesicle (MV) release. Results In freshly isolated human RBCs, paclitaxel induced thrombin generation through PS exposure and MV release, whereas either coagulation factors or platelets were unaffected. Paclitaxel-induced PS exposure in RBC was mediated by scramblase activation which was induced by calcium-independent protein kinase C (PKC)ζ activation. Paclitaxel also increased RBC-endothelial cell adhesion and RBC aggregate formation which can also contribute to thrombosis. Indeed, intravenous administration of paclitaxel to rats induced PS exposure and PKCζ activation in RBCs in vivo which ultimately promoted venous thrombus formation. Conclusion These results demonstrated that paclitaxel may elicit pro-thrombotic properties in RBCs through PS exposure and MV release, which can ultimately promote thrombus formation.

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Dapsone Hydroxylamine, an Active Metabolite of Dapsone, Can Promote the Procoagulant Activity of Red Blood Cells and Thrombosis

Toxicological Sciences ◽

10.1093/toxsci/kfz188 ◽

2019 ◽

Vol 172 (2) ◽

pp. 435-444 ◽

Cited By ~ 2

Author(s):

Yiying Bian ◽

Keunyoung Kim ◽

Gwang-Jin An ◽

Thien Ngo ◽

Ok-Nam Bae ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Thrombus Formation ◽

Procoagulant Activity ◽

Ros Generation ◽

Thrombotic Risk ◽

Repeated Doses ◽

Hydroxylated Metabolite

Abstract Dapsone hydroxylamine (DDS-NHOH), N-hydroxylated metabolite of a sulfonamide antibiotic, dapsone, is responsible for various adverse effects of dapsone that include methemoglobinemia, hemolytic anemia, and thrombosis. However, the mechanism underlying DDS-NHOH-induced thrombosis remains unclear. Here, we demonstrated that DDS-NHOH, but not dapsone, could increase prothrombotic risks through inducing the procoagulant activity of red blood cells (RBCs). In freshly isolated human RBCs in vitro, sub-hemolytic concentrations of DDS-NHOH (10–50 μM) increased phosphatidylserine (PS) exposure and augmented the formation of PS-bearing microvesicles (MV). Reactive oxygen species (ROS) generation and the subsequent dysregulation of enzymes maintaining membrane phospholipid asymmetry were found to induce the procoagulant activity of DDS-NHOH. Dapsone hydroxylamine also accelerated thrombin generation and enhanced RBC self-aggregation and adherence of RBCs to endothelial cells in vitro. Most importantly, both the single dose of 50 or 100 mg/kg (i.p.) DDS-NHOH and repeated doses of 10 mg/kg per day (i.p.) for 4 days increased thrombus formation in rats (six rats per dose) in vivo, substantiating a potential prothrombotic risk of DDS-NHOH. Collectively, these results demonstrated the central role of RBC procoagulant activity induced by DDS-NHOH in the thrombotic risk of dapsone.

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Spatially Organized Structure of Coronary Thrombus in Acute Myocardial Infarction

Blood ◽

10.1182/blood.v128.22.716.716 ◽

2016 ◽

Vol 128 (22) ◽

pp. 716-716

Author(s):

Jan E. Dyr ◽

Tomas Riedel ◽

Jana Stikarova ◽

Jiri Suttnar ◽

Jaroslav Cermak ◽

...

Keyword(s):

Myocardial Infarction ◽

Red Blood Cells ◽

Symptom Onset ◽

Blood Cells ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Freeze Fracture ◽

Ischemic Time ◽

Internal Structures

Abstract Introduction The use of thromboaspiration in primary percutaneous intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) has offered a unique opportunity to study thrombus composition, its dynamic formation, and architecture in vivo. There has been, however, several limitations, not least the fact that the technique has not yet allowed a precise transversal analysis from one side of the artery to the other, as is done in histological analysis. The dynamic process of intracoronary thrombus formation in STEMI patients is thus still not well understood. Ischemic time was hypothesized to be among the strongest independent correlates of thrombus architecture. In time the platelets are decreasing its proportion and fibrin proportion is increasing (J Silvain, J-P Collet, JW Weisel et al, J Am Coll Cardiol 2011; 57:1359). However, no real report on the internal structures of the in vivo formed thrombi has been shown so far. Therefore, we investigated both the surface and the composition of longitudinally freeze-fractured thrombi. Methods Thrombi were collected by PCI from 119 STEMI patients. Out of the patients there were "early comers " (˃12 h from symptom onset; 23 patients) and "late comers" (more than 720 min; 29 patients). The mean age of all patients was 64 years, 70% of patients were males, 51% were smokers, 50% had arterial hypertension, 20% were diabetics and 23% had chronic renal insufficiency. Scanning electron microscopy; collected thrombi obtained by PCI were thoroughly washed in saline solution and stored in 4% formaldehyde prior dehydration. To reveal the internal structures of the thrombi selected samples were longitudinally freeze fractured in liquid nitrogen and coated with platinum. Samples were examined in SEM Vega Plus TS 5135 (Tescan s.r.o., Brno, Czech Republic). Whole areas of the freeze-fractured thrombi were scanned. Results and discussion The thrombus composition of longitudinally freeze-fractured thrombi was compared between groups of "early-comers" and "late-comers. The distribution of the components in the "early comers" thrombi freeze-fracture seemed to be uniform. Platelets were far the main component (about 75 % in proportion) of the "early comers" thrombus, followed by fibrin and other compounds. The amount of red blood cells was negligible (about 2 - 8 %). We did not observe any significant differences between the thrombi in the group of early comers. Thrombi of the "late-comers" group were composed mainly of red blood cells; platelets and fibrin formed only minority of the thrombi. In contrast to the "early comers" the distribution of the main thrombus components in the "late comers" thrombi was dramatically different between individual parts of the thrombus. The number of platelets and red blood cells varied from 0% to almost 99% and vice versa. It was possible to estimate the initiating place of the thrombus as well as the direction of the growth. Each thrombus could be divided into parts formed mainly either by platelets or by red blood cells. It seems that thrombus develops a regional architecture defined by the extent of platelet activation and packing density. It has been reported that in contracted clots and thrombi, erythrocytes are compressed to close-packed polyhedral structures with platelets and fibrin on the surface demonstrating how contracted clots form an impermeable barrier important for hemostasis and wound healing (D Cines, T Lebedeva, J Weisel et al, Blood 2014; 123:1596). Our investigation of the composition of the in vivo formed thrombi supports these results and helps to explain how fibrinolysis is greatly retarded as clots grow and contract. We have found that on the surfaces of late-comers thrombi fibrin thick fibrils were present. It has been shown that the association of soluble fibrinogen with the fibrin clot results in the reduced adhesiveness of such fibrinogen/fibrin matrices toward leukocytes and platelets (VK Lishko, T Burke, T Ugarova, Blood 2007; 109:1541). Fibrinopeptides A are less accessible for thrombin in surface bound fibrinogen which thus provides additional level of protection of thrombi from premature dissolution (T Riedel, L Medved, JE Dyr, Blood 2011; 117:1700). These findings may have great impact on our knowledge of pathophysiology of the thrombus growth and possible therapeutic consequences related to the time of symptom onset. Disclosures No relevant conflicts of interest to declare.

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Infusion of Recombinant Full-Length ADAMTS13 and Carboxyl-Terminal Truncated Variant Alters Composition of Ferric Chloride-Induced Arterial Thrombi in Adamts13−/− mice

Blood ◽

10.1182/blood.v118.21.2313.2313 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2313-2313

Author(s):

Christopher G. Skipwith ◽

Juan (Jenny) Xiao ◽

John W. Weisel ◽

X. Long Zheng

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Full Length ◽

Apparent Difference ◽

Cell Accumulation ◽

Carboxyl Terminal ◽

Normal Hemostasis

Abstract Abstract 2313 Proteolytic cleavage of ultra large von Willebrand factor (ULVWF) released from endothelial cells by ADAMTS13 metalloprotease is critical for maintaining normal hemostasis. However, the effect of infusing ADAMTS13 on thrombus composition remains poorly understood. In this study, we determined the morphology and composition of thrombi formed in carotid arteries after topical application of FeCl3 in Adamts13−/− mice receiving PBS, recombinant human full-length ADAMTS13 (FL) and carboxyl-terminal truncated variant after spacer domain (S), using scanning electron microscopy and quantitative image analysis. We showed that in Adamts13−/− mice 5 min after FeCl3 injury, formed arterial thrombi were comprised ∼39% platelets, ∼26% red blood cells, and ∼35% fibrin. The arterial thrombi in these mice were structurally deformed. An infusion of recombinant FL (final concentration of 10 nM) significantly reduced the accumulation of platelets (∼18%) but increased the fibrin network (57%) without affecting the composition of red blood cells (∼25%) in the arterial thrombi formed at the same time point after FeCl3 injury. Similar effects on the morphology and composition of FeCl3-induced arterial thrombi were observed after infusion of recombinant S (10 nM) into Adamts13−/− mice. Kinetic analysis showed that there was a decrease in platelet accumulation over the time of 30 min during thrombus formation with a slight increase in accumulation of red blood cells and formation of fibrin in Adamts13−/− mice. But, the infusion of recombinant FL and S into Adamts13−/− mice restored the kinetics of platelet/red blood cell accumulation and fibrin formation to those observed in wild-type mice. Our findings, revealing the apparent difference in thrombus composition, provide novel insight into the mechanism of ADAMTS13 function in vivo, which may shed more light on the pathogenesis of thrombotic thrombocytopenic purpura and other arterial thrombotic disorders associated with deficiency of plasma ADAMTS13 activity. Disclosures: No relevant conflicts of interest to declare.

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Additive roles of platelets and fibrinogen in whole-blood fibrin clot formation upon dilution as assessed by thromboelastometry

Thrombosis and Haemostasis ◽

10.1160/th13-06-0493 ◽

2014 ◽

Vol 111 (03) ◽

pp. 447-457 ◽

Cited By ~ 21

Author(s):

Marisa Ninivaggi ◽

Gerhardus Kuiper ◽

Marco Marcus ◽

Hugo ten Cate ◽

Marcus Lancé ◽

...

Keyword(s):

Red Blood Cells ◽

Whole Blood ◽

Blood Cells ◽

Thrombin Generation ◽

Prothrombin Complex Concentrate ◽

Coagulation Factors ◽

Fibrin Clot ◽

Clot Formation

SummaryBlood dilution after transfusion fluids leads to diminished coagulant activity monitored by rotational thromboelastometry, assessing elastic fibrin clot formation, or by thrombin generation testing. We aimed to determine the contributions of blood cells (platelets, red blood cells) and plasma factors (fibrinogen, prothrombin complex concentrate) to fibrin clot formation under conditions of haemodilution in vitro or in vivo. Whole blood or plasma diluted in vitro was supplemented with platelets, red cells, fibrinogen or prothrombin complex concentrate (PCC). Thromboelastometry was measured in whole blood as well as plasma; thrombin generation was determined in parallel. Similar tests were performed with blood from 48 patients, obtained before and after massive fluid infusion during cardiothoracic surgery. Addition of platelets or fibrinogen, in additive and independent ways, reversed the impaired fibrin clot formation (thromboelastometry) in diluted whole blood. In contrast, supplementation of red blood cells or prothrombin complex concentrate was ineffective. Platelets and fibrinogen independently restored clot formation in diluted plasma, resulting in thromboelastometry curves approaching those in whole blood. In whole blood from patients undergoing dilution during surgery, elastic clot formation was determined by both the platelet count and the fibrinogen level. Thrombin generation in diluted (patient) plasma was not changed by fibrinogen, but improved markedly by prothrombin complex concentrate. In conclusion, in dilutional coagulopathy, platelets and fibrinogen, but not red blood cells or vitamin K-dependent coagulation factors, independently determine thromboelastometry parameters measured in whole blood and plasma. Clinical decisions for transfusion based on thromboelastometry should take into account the platelet concentration.

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Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162132 ◽

1990 ◽

Vol 48 (3) ◽

pp. 914-915

Author(s):

D.J.P. Ferguson ◽

A.R. Berendt ◽

J. Tansey ◽

K. Marsh ◽

C.I. Newbold

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Blood Cells ◽

Umbilical Vein ◽

Cell Types ◽

Amelanotic Melanoma ◽

Cytoplasmic Process ◽

Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

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The Ullrastructure of Platelet Participation in Hemostasis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656288 ◽

1965 ◽

Vol 13 (01) ◽

pp. 065-083 ◽

Cited By ~ 7

Author(s):

Shirley A. Johnson ◽

Ronaldo S. Balboa ◽

Harlan J. Pederson ◽

Monica Buckley

Keyword(s):

Platelet Aggregation ◽

Red Blood Cells ◽

Blood Cells ◽

Guinea Pigs ◽

Platelet Factor ◽

Mesenteric Vessels ◽

Platelet Aggregates ◽

Electron Lucent

SummaryThe ultrastructure of platelet aggregation in vivo in response to bleeding brought about by transection of small mesenteric vessels in rats and guinea pigs has been studied. Platelets aggregate, degranulate and separating membranes disappear in parallel with fibrin appearance which is first seen at several loci after 30 seconds of bleeding. About 40 per cent of the electron opaque granules, some of which contain platelet factor 3 have disappeared after one minute of bleeding while the electron lucent granules increase by 70 per cent suggesting that some of them may be empty vesicles. Most of the platelet aggregates of the random type disappear leaving clumped red blood cells entrapped by a network of fibrin fibers which emanate from the remains of platelet aggregates of the rosette type to maintain hemostasis.

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Cytotoxicity of chitosan ultrafine nanoshuttles on MCF-7 cell line as surrogate model for breast cancer

Current Drug Delivery ◽

10.2174/1567201817666200719005440 ◽

2020 ◽

Vol 17 ◽

Author(s):

Tarek Faris ◽

Gamaleldin I. Harisa ◽

Fars K. Alanazi ◽

Mohamed M. Badran ◽

Afraa Mohammad Alotaibi ◽

...

Keyword(s):

Red Blood Cells ◽

Factorial Design ◽

Cell Line ◽

Cell Lines ◽

Blood Cells ◽

Sodium Tripolyphosphate ◽

Physicochemical Characterization ◽

Spherical Shape ◽

Mcf 7

Aim: This study aimed to explore an affordable technique for the fabrication of Chitosan Nanoshuttles (CSNS) at the ultrafine nanoscale less than 100 nm with improved physicochemical properties, and cytotoxicity on the MCF-7 cell line. Background: Despite several studies reported that the antitumor effect of CS and CSNS could achieve intracellular compartment target ability, no enough available about this issue and further studies are required to address this assumption. Objectives: The objective of the current study was to investigate the potential processing variables for the production of ultrafine CSNS (> 100 nm) using Box-Benhken Design factorial design (BBD). This was achieved through a study of the effects of processing factors, such as CS concentration, CS/TPP ratio, and pH of the CS solution, on PS, PDI, and ZP. Moreover, the obtained CSNS was evaluated for physicochemical characteristics, morphology Also, hemocompatibility, and cytotoxicity using Red Blood Cells (RBCs) and MCF-7 cell lines were investigated. Methods: Box-Benhken Design factorial design (BBD) was used in the analysis of different selected variables. The effects of CS concentration, sodium tripolyphosphate (TPP) ratio, and pH on particle size, Polydispersity Index (PDI), and Zeta Potential (ZP) were measured. Subsequently, the prepared CS nanoshuttles were exposed to stability studies, physicochemical characterization, hemocompatibility, and cytotoxicity using red blood cells and MCF-7 cell lines as surrogate models for in vivo study. Result: The present results revealed that the optimized CSNS have ultrafine nanosize, (78.3±0.22 nm), homogenous with PDI (0.131±0.11), and ZP (31.9±0.25 mV). Moreover, CSNS have a spherical shape, amorphous in structure, and physically stable. Also, CSNS has biological safety as indicated by a gentle effect on red blood cell hemolysis, besides, the obtained nanoshuttles decrease MCF-7 viability. Conclusion: The present findings concluded that the developed ultrafine CSNS has unique properties with enhanced cytotoxicity. thus promising for use in intracellular organelles drug delivery.

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Multimodal Diagnostics of Microrheologic Alterations in Blood of Coronary Heart Disease and Diabetic Patients

Diagnostics ◽

10.3390/diagnostics11010076 ◽

2021 ◽

Vol 11 (1) ◽

pp. 76

Author(s):

Anastasia Maslianitsyna ◽

Petr Ermolinskiy ◽

Andrei Lugovtsov ◽

Alexandra Pigurenko ◽

Maria Sasonko ◽

...

Keyword(s):

Coronary Heart Disease ◽

Heart Disease ◽

Red Blood Cells ◽

Blood Cells ◽

Optical Methods ◽

Diabetic Patients ◽

Aggregation Index ◽

Significant Difference

Coronary heart disease (CHD) has serious implications for human health and needs to be diagnosed as early as possible. In this article in vivo and in vitro optical methods are used to study blood properties related to the aggregation of red blood cells in patients with CHD and comorbidities such as type 2 diabetes mellitus (T2DM). The results show not only a significant difference of the aggregation in patients compared to healthy people, but also a correspondence between in vivo and in vitro parameters. Red blood cells aggregate in CHD patients faster and more numerously; in particular the aggregation index increases by 20 ± 7%. The presence of T2DM also significantly elevates aggregation in CHD patients. This work demonstrates multimodal diagnostics and monitoring of patients with socially significant pathologies.

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Role of Calcium in Phosphatidylserine Externalisation in Red Blood Cells from Sickle Cell Patients

Anemia ◽

10.1155/2011/379894 ◽

2011 ◽

Vol 2011 ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

Erwin Weiss ◽

David Charles Rees ◽

John Stanley Gibson

Keyword(s):

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Phosphatidylserine Exposure ◽

Channel Inhibition ◽

Cation Conductance ◽

Gardos Channel ◽

Cation Permeability

Phosphatidylserine exposure occurs in red blood cells (RBCs) from sickle cell disease (SCD) patients and is increased by deoxygenation. The mechanisms responsible remain unclear. RBCs from SCD patients also have elevated cation permeability, and, in particular, a deoxygenation-induced cation conductance which mediates entry, providing an obvious link with phosphatidylserine exposure. The role of was investigated using FITC-labelled annexin. Results confirmed high phosphatidylserine exposure in RBCs from SCD patients increasing upon deoxygenation. When deoxygenated, phosphatidylserine exposure was further elevated as extracellular [] was increased. This effect was inhibited by dipyridamole, intracellular chelation, and Gardos channel inhibition. Phosphatidylserine exposure was reduced in high saline. levels required to elicit phosphatidylserine exposure were in the low micromolar range. Findings are consistent with entry through the deoxygenation-induced pathway (), activating the Gardos channel. [] required for phosphatidylserine scrambling are in the range achievablein vivo.

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Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells

10.1101/2021.10.11.463714 ◽

2021 ◽

Author(s):

Andrew D. Beale ◽

Priya Crosby ◽

Utham K. Valekunja ◽

Rachel S. Edgar ◽

Johanna E. Chesham ◽

...

Keyword(s):

Circadian Rhythms ◽

Red Blood Cells ◽

Blood Cells ◽

Human Subjects ◽

Developmental Regulation ◽

Physiological Role ◽

Cell Types ◽

The Body

AbstractCellular circadian rhythms confer daily temporal organisation upon behaviour and physiology that is fundamental to human health and disease. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body. Being naturally anucleate, RBC circadian rhythms share key elements of post-translational, but not transcriptional, regulation with other cell types. The physiological function and developmental regulation of RBC circadian rhythms is poorly understood, however, partly due to the small number of appropriate techniques available. Here, we extend the RBC circadian toolkit with a novel biochemical assay for haemoglobin oxidation status, termed “Bloody Blotting”. Our approach relies on a redox-sensitive covalent haem-haemoglobin linkage that forms during cell lysis. Formation of this linkage exhibits daily rhythms in vitro, which are unaffected by mutations that affect the timing of circadian rhythms in nucleated cells. In vivo, haemoglobin oxidation rhythms demonstrate daily variation in the oxygen-carrying and nitrite reductase capacity of the blood, and are seen in human subjects under controlled laboratory conditions as well as in freely-behaving humans. These results extend our molecular understanding of RBC circadian rhythms and suggest they serve an important physiological role in gas transport.

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