Initiating and Managing Patients with Venous Thromboembolism on Anticoagulant Drugs: A Practical Overview

Several new oral anticoagulants have recently been approved for the treatment of venous thromboembolism (VTE). In this review, we discuss the currently approved drugs and the factors that influence the choice of anticoagulant in a given patient. Once anticoagulation is initiated, periodic monitoring of adequacy of anticoagulation may be necessary depending on the choice of anticoagulant and patient-related factors, such as renal function. Situations that may warrant need for monitoring and the tests available for this purpose are discussed. We review reversal of anticoagulation in urgent/emergent situations as well as perioperative anticoagulation interruption in the elective setting. The data on use of direct oral anticoagulants in patients with compromised renal function, obesity and bariatric surgery, and in the treatment of cancer-associated thrombosis are discussed. The review aims to provide the clinician with the essential information to allow effective and safe use of anticoagulants for the treatment of VTE

RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion.

Plasmodium falciparum invasion of host erythrocytes is essential for the propagation of the blood stage of malaria infection. Additionally, the brief extracellular merozoite stage of P. falciparum represents one of the rare windows during which the parasite is directly exposed to the host immune response. Therefore, efficient invasion of the host erythrocyte is necessary not only for productive host erythrocyte infection, but also for evasion of the immune response. Host traits, such as hemoglobinopathies and differential expression of erythrocyte invasion ligands, can protect individuals from malaria by impeding parasite erythrocyte invasion. Here we combine RBC barcoding with flow cytometry to study P. falciparum invasion. This novel high-throughput method allows for the (i) direct comparison of P. falciparum invasion into different erythrocyte populations and (ii) assessment of the impact of changing erythrocyte population dynamics on P. falciparum invasion

RBC Barcoding Allows for the Study of Erythrocyte Population Dynamics and P. falciparum Merozoite Invasion

Plasmodium falciparum invasion of host erythrocytes is essential for the propagation of the blood stage of malaria infection. Additionally, the brief extracellular merozoite stage of P. falciparum represents one of the rare windows during which the parasite is directly exposed to the host immune response. Therefore, efficient invasion of the host erythrocyte is necessary not only for productive host erythrocyte infection, but also for evasion of the immune response. Host traits, such as hemoglobinopathies and differential expression of erythrocyte invasion ligands, can protect individuals from malaria by impeding parasite erythrocyte invasion. Here we combine RBC barcoding with flow cytometry to study P. falciparum invasion. This novel high-throughput method allows for the (i) direct comparison of P. falciparum invasion into different erythrocyte populations and (ii) assessment of the impact of changing erythrocyte population dynamics on P. falciparum invasion

Host iron status and iron supplementation mediate susceptibility to erythrocytic stage Plasmodium falciparum.

Iron deficiency and malaria have similar global distributions, and frequently co-exist in pregnant women and young children. Where both conditions are prevalent, iron supplementation is complicated by observations that iron deficiency anaemia protects against falciparum malaria, and that iron supplements increase susceptibility to clinically significant malaria, but the mechanisms remain obscure. Here, using an in vitro parasite culture system with erythrocytes from iron-deficient and replete human donors, we demonstrate that Plasmodium falciparum infects iron-deficient erythrocytes less efficiently. In addition, owing to merozoite preference for young erythrocytes, iron supplementation of iron-deficient individuals reverses the protective effects of iron deficiency. Our results provide experimental validation of field observations reporting protective effects of iron deficiency and harmful effects of iron administration on human malaria susceptibility. Because recovery from anaemia requires transient reticulocytosis, our findings imply that in malarious regions iron supplementation should be accompanied by effective measures to prevent falciparum malaria

Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism

AbstractCancer patients exhibit a high rate of thromboembolism (VTE). In this study, we analyzed levels of microparticle (MP) tissue factor (TF) activity in cancer patients with or without VTE. Blood was collected from cancer patients within 24 h of objectively diagnosed VTE (n=53) and from cancer patients without VTE (n=13). MPs were isolated from platelet poor plasma by centrifugation at 20,000g for 15 min. MP TF activity was measured using a two-stage chromogenic assay. Cancer patients with VTE had a significantly higher mean MP TF activity compared with cancer patients without VTE (1.7±3.8 pg/mL vs 0.6±0.4 pg/mL,

Role of Tissue Factor in Cancer

Tissue factor (TF) is a transmembrane glycoprotein that localizes the coagulation serine protease factor VII/VIIa (FVII/VIIa) to the cell surface. The primary function of TF is to activate the clotting cascade. The TF:FVIIa complex also activates cells by cleavage of a G-protein coupled receptor called protease-activated receptor 2 (PAR2). TF is expressed by tumor cells and contributes to a variety of pathologic processes, such as thrombosis, metastasis, tumor growth, and tumor angiogenesis. For instance, tumor cells release TF-positive procoagulant microparticles into the circulation and these may trigger venous thromboembolism in patients with cancer. TF on circulating tumor cells also leads to the coating of the cells with fibrin that traps them within the microvasculature and facilitates hematogenous metastasis. In addition, TF:FVIIa-dependent activation of PAR2 on tumor cells increases tumor growth via an undefined mechanism. One possibility is that PAR2-dependent signaling increases the expression of proangiogenic proteins. Other studies have reported that endothelial cells in the tumor vasculature express TF and this may enhance angiogenesis. These results suggest that inhibition of TF should reduce several pathologic pathways that increase tumor growth and metastasis. This would represent a novel approach to anticancer therapy. Initial studies using inhibitors of the TF:FVIIa complex in mouse tumor models have produced encouraging results. Nevertheless, additional studies are needed to determine if this strategy can be successfully translated to the treatment of cancer patients

RBCs barcoded with CellTrace DDAO and Violet can be combined to directly compare <i>P. falciparum</i> invasion in an invasion assay.

<p>RBCs were labeled with 5 µM of either DDAO (A) or CellTrace Violet (B). Cells were then combined and infected with MACS purified unlabeled pRBCs. Experiments were incubated 18–24 hours. Cells were then stained with DNA dye SYBR Green I, fixed, and examined by brightfield (C) and fluorescence microscopy (D, E) and by flow cytometry (F–H). (A) shows red channel only, (B) shows violet channel only, (C) shows brightfield, (D) shows green channel only and (E) shows merge of red, violet, and green channels. (F) Flow cytometry plot of RBCs stained with CellTrace DDAO (R1) and CellTrace Violet (R4) and non-stained pRBC (R3). (G) Flow cytometry plot shows DDAO negative pRBCs (R5), DDAO negative uninfected RBCs (R7), DDAO positive pRBCs (R6) and DDAO positive uninfected RBCs (R8). (H) Flow cytometry plot shows CellTrace Violet negative pRBCs (R9), CellTrace Violet negative uninfected RBCs (R11), CellTrace Violet positive pRBCs (R10) and CellTrace Violet positive uninfected RBCs (R12).</p

Invasion of <i>P. falciparum</i> strain 3D7 decreases as untreated RBCs are replaced with enzyme treated RBCs.

<p>RBCs were labeled with either CellTrace DDAO or Violet before enzyme treatment (neuraminidase, trypsin, or chymotrypsin). 1.8×10⁷, 1×10⁷, and 2×10⁶ non-enzymatically treated RBCs (RBC^Ø) were combined with 2×10⁶, 1×10⁷, and 1.8×10⁷ of enzyme treated RBCs (RBC^N, RBC^T, or RBC^C) respectively to achieve 10∶1, 1∶1, and 1∶10 combinations of RBC^Ø to either RBC^N, RBC^T, or RBC^C in the barcoded RBC invasion assay. Invasion assays were inoculated with 2×10⁵ of MACS purified trophozoite stage <i>P. falciparum</i> strain 3D7 parasites and invasion assays were performed as previously described. Data is from a single representative experiment of three independent experiments performed in triplicate. (A) Rate of 3D7 invasion into 1∶10, 1∶1, and 10∶1 combinations of RBC^Ø and either RBC^N, RBC^T, or RBC^C. Bars represent the mean invasion rate and error bars represent the SD. Elongated triangles below the X-axis represent the replacement of RBC^Ø (white triangle) with either RBC^N, RBC^T, or RBC^C (gray triangle) in the total RBC population. Student's <i>t</i>-tests were used to compare invasion rates, *p<0.02 and **p<0.0002. (B, C, and D) Data shows the number of invasion events into RBC^Ø (triangles) and either RBC^N, RBC^T, or RBC^C (circles) as the frequency of each RBC type increases from 10% to 90% of the total RBC population. Linear regression was used to determine the best fit line for <i>P. falciparum</i> invasion of RBC^Ø, RBC^N, RBC^T, and RBC^C. ANCOVA was performed to compare the slopes of the lines fit to <i>P. falciparum</i> invasion of RBC^Ø, RBC^N, RBC^T, and RBC^C. The null hypothesis was no difference between RBC^Ø and either RBC^N, RBC^T, or RBC^C (H₀: β_Ø  =  β_enzyme, α = 0.05). ANCOVA performed with GraphPad, Prism, v. 5.04, La Jolla, CA calculated a p<0.0001. (E) RBC^Ø datum from panels B–D were superimposed to compare invasion into RBC^Ø when RBC^Ø was combined with RBC^N (circles) RBC^T (triangles) or RBC^C (diamonds). Linear regression was used to determine the best fit line for RBC^Ø invasion data. ANCOVA was performed to compare the slopes of the lines fit to <i>P. falciparum</i> invasion of RBC^Ø. The null hypothesis was that there would be no difference in the invasion of RBC^Ø in the presence of the other RBC populations (H₀: β_Ø  =  β_enzyme, α = 0.05). ANCOVA performed with GraphPad, Prism, v. 5.04, La Jolla, CA calculated a p<0.2599.</p