Nigel S. Key

Venous Thromboembolism Prophylaxis and Treatment in Patients With Cancer: American Society of Clinical Oncology Clinical Practice Guideline Update 2014

PURPOSE To provide current recommendations about the prophylaxis and treatment of venous thromboembolism (VTE) in patients with cancer. METHODS PubMed and the Cochrane Library were searched for randomized controlled trials, systematic reviews, meta-analyses, and clinical practice guidelines from November 2012 through July 2014. An update committee reviewed the identified abstracts. RESULTS Of the 53 publications identified and reviewed, none prompted a change in the 2013 recommendations. RECOMMENDATIONS Most hospitalized patients with active cancer require thromboprophylaxis throughout hospitalization. Routine thromboprophylaxis is not recommended for patients with cancer in the outpatient setting. It may be considered for selected high-risk patients. Patients with multiple myeloma receiving antiangiogenesis agents with chemotherapy and/or dexamethasone should receive prophylaxis with either low-molecular weight heparin (LMWH) or low-dose aspirin. Patients undergoing major surgery should receive prophylaxis starting before surgery and continuing for at least 7 to 10 days. Extending prophylaxis up to 4 weeks should be considered in those undergoing major abdominal or pelvic surgery with high-risk features. LMWH is recommended for the initial 5 to 10 days of treatment for deep vein thrombosis and pulmonary embolism as well as for long-term secondary prophylaxis (at least 6 months). Use of novel oral anticoagulants is not currently recommended for patients with malignancy and VTE because of limited data in patients with cancer. Anticoagulation should not be used to extend survival of patients with cancer in the absence of other indications. Patients with cancer should be periodically assessed for VTE risk. Oncology professionals should educate patients about the signs and symptoms of VTE.

Guidelines for the management of hemophilia.

Hemophilia is a rare disorder that is complex to diagnose and to manage. These evidence‐based guidelines offer practical recommendations on the diagnosis and general management of hemophilia, as well as the management of complications including musculoskeletal issues, inhibitors, and transfusion‐transmitted infections. By compiling these guidelines, the World Federation of Hemophilia aims to assist healthcare providers seeking to initiate and/or maintain hemophilia care programs, encourage practice harmonization around the world and, where recommendations lack adequate evidence, stimulate appropriate studies.

Role of the Extrinsic Pathway of Blood Coagulation in Hemostasis and Thrombosis

Hemostasis requires both platelets and the coagulation system. At sites of vessel injury, bleeding is minimized by the formation of a hemostatic plug consisting of platelets and fibrin. The traditional view of the regulation of blood coagulation is that the initiation phase is triggered by the extrinsic pathway, whereas amplification requires the intrinsic pathway. The extrinsic pathway consists of the transmembrane receptor tissue factor (TF) and plasma factor VII/VIIa (FVII/FVIIa), and the intrinsic pathway consists of plasma FXI, FIX, and FVIII. Under physiological conditions, TF is constitutively expressed by adventitial cells surrounding blood vessels and initiates clotting. In addition so-called blood-borne TF in the form of cell-derived microparticles (MPs) and TF expression within platelets suggests that TF may play a role in the amplification phase of the coagulation cascade. Under pathologic conditions, TF is expressed by monocytes, neutrophils, endothelial cells, and platelets, which results in an elevation of the levels of circulating TF-positive MPs. TF expression within the vasculature likely contributes to thrombosis in a variety of diseases. Understanding how the extrinsic pathway of blood coagulation contributes to hemostasis and thrombosis may lead to the development of safe and effective hemostatic agents and antithrombotic drugs.

Measuring circulating cell-derived microparticles

Cell-derived microparticles (MPs) are receiving increasing attention in recent years, both as a diagnostic aid and investigative tool [1–4]. Because they carry markers of the parent cell, including those induced by activation or apoptosis, endothelial MPs (EMPs) can provide valuable information on the status of the parent cell, obtainable in no other way. In addition, there is a growing belief that MPs can function as important diffusible vectors of specific adhesins and cytokines promoting cellular interactions and signal transmission [2]. ThusMP analysis constitutes a new avenue for investigation of pathologies in various diseases. Although still considered investigational [1–4], recent results from several laboratories suggest that MP analysis may be poised to enter the mainstream of clinical testing. However, a major impediment to that end is the wide variety ofmethodologies used by different laboratories in this field, few of which can be directly compared to the others, and results from which are sometimes inconsistent or conflicting. As a first step in addressing that problem, the Editor has organized this Forum article, consisting of a brief description of the preferred methods and rationality from each of six active laboratories in the field, including our own [5–10]. Table 1 lists some key features of the six methodological approaches. It is seen that major differences exist in the preparation of the MP samples (such as centrifugation), whether or not they are first sedimented and resuspended, means of generic MP detection (4 of 6 use annexin V), and cell lineage-specific antigenic markers. These differences probably account for some of the different findings among the groups.

Andrographolide attenuates inflammation by inhibition of NF-kappa B activation through covalent modification of reduced cysteine 62 of p50.

NF-κB is a central transcriptional factor and a pleiotropic regulator of many genes involved in immunological responses. During the screening of a plant extract library of traditional Chinese herbal medicines, we found that NF-κB activity was potently inhibited by andrographolide (Andro), an abundant component of the plant Andrographis that has been commonly used as a folk remedy for alleviation of inflammatory disorders in Asia for millennia. Mechanistically, it formed a covalent adduct with reduced cysteine (62) of p50, thus blocking the binding of NF-κB oligonucleotide to nuclear proteins. Andro suppressed the activation of NF-κB in stimulated endothelial cells, which reduced the expression of cell adhesion molecule E-selectin and prevented E-selectin-mediated leukocyte adhesion under flow. It also abrogated the cytokine- and endotoxin-induced peritoneal deposition of neutrophils, attenuated septic shock, and prevented allergic lung inflammation in vivo. Notably, it had no suppressive effect on IκBα degradation, p50 and p65 nuclear translocation, or cell growth rates. Our results thus reveal a unique pharmacological mechanism of Andro’s protective anti-inflammatory actions.

Standardization of platelet‐derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop

Microparticles (MPs) are sub-micrometer sized vesicles released from cell membranes in response to activation or apoptosis [1]. MPs originating from several cell sources have been described in human plasma. Among them, plateletderivedMPs (PMPs) are believed to account for themajority of circulating MPs in healthy subjects [2]. Their levels are increased in several prothrombotic and inflammatory disorders [1]. In these clinical settings, PMP counts may be useful for identifying patients at risk for vascular disorders and for monitoring response to treatment [3]. However, their clinical use is not fully established, because standardized methodologies for PMP counting are lacking. A previous International Society on Thrombosis and Haemostasis (ISTH) Vascular Biology Subcommittee survey indicated that approximately 75% of laboratories use flow cytometry (FCM) to enumerate MPs in clinical samples. However, a wide variety of preanalytic variables and analytic variables have been reported in the literature, resulting in a wide range of PMP values in platelet-free plasma (PFP) of healthy subjects (100–4000 PMPs lL). This lack of consensus stresses the need for standardization [4]. Three ISTH Scientific and Standardization Subcommittees (SSC Vascular Biology, DIC, and Haemostasis &Malignancy) have initiated a project aimed at standardizing the enumeration of cellularMPs by FCM.Afirstcollaborative workshopwas set up, to: first, establish the resolution and the level of background noise of the flow cytometers currently used in laboratories with respect to the strategy requirements; and second, define the interinstrument reproducibility of PMP enumeration in human plasma. This strategy was based on the use of fluorescent calibrated sub-micrometer beads (Megamix beads; BioCytex, Marseille, France), which allow the window of MP analysis to be reproducibly set [5]. The study included 40 laboratories accounting for 59 flow cytometers, and was performed in two stages (Fig. S1): in stage A, participating laboratories received Megamix beads and were asked to set up the FCM protocol and to validate an instrument protocol adapted from a previously described method [5]. On the basis of forward scatter (FS)/FS channeling (FSC) resolution and background characteristics, stage A results led to acceptance or rejection of the tested instruments, with some time being allowed for technical intervention in order to improve any deficient performance. In stage B, selected laboratories received PFP samples prepared as frozen aliquots by the core laboratory, and were asked to analyze them with the previously validated instrument(s), common reagents, and the FCM protocol established in stage A. A detailed description of the methodology is available in the Supporting Information (Data S1). The purpose of this initial phase was to check whether the instrument to be used to enumerate PMPs demonstrated the required performance with a blend of fluorescent beads with well-known sizes and relative amounts. The instruments were validated on the basis of their capacity to discriminate between 0.5-lm and 0.9-lm Megamix beads using the FS/FSC parameter, as well as their background noise. Instruments detecting < 0.1% of fluorescent bead events among total events were rejected, because such a level of background may impede the electronics functions and induce amajor loss of events owing to coincidences and electronic aborts (Fig. S2A,B). Analysis of the results demonstrated that instruments were heterogeneous with respect to FS/FSC resolution and background noise. Furthermore, the level of performance could vary over time (Fig. S2C). Some of the parameters affecting FS/FSC resolution were identified with Megamix beads. Among them, FS/ FSC gain, FS/FSCmode, neutral density (intensity scavenging) Correspondence: Francoise Dignat-George, UMR-S608, 27 Bd Jean Moulin, 13005 Marseille, France. Tel.: +33 491 385600; fax: +33 491 385602. E-mail: francoise.dignat-george@univmed.fr Journal of Thrombosis and Haemostasis, 8: 2571–2574 DOI: 10.1111/j.1538-7836.2010.04047.x

D-dimer antigen: current concepts and future prospects.

The D-dimer antigen is a unique marker of fibrin degradation that is formed by the sequential action of 3 enzymes: thrombin, factor XIIIa, and plasmin. First, thrombin cleaves fibrinogen producing fibrin monomers, which polymerize and serve as a template for factor XIIIa and plasmin formation. Second, thrombin activates plasma factor XIII bound to fibrin polymers to produce the active transglutaminase, factor XIIIa. Factor XIIIa catalyzes the formation of covalent bonds between D-domains in the polymerized fibrin. Finally, plasmin degrades the crosslinked fibrin to release fibrin degradation products and expose the D-dimer antigen. D-dimer antigen can exist on fibrin degradation products derived from soluble fibrin before its incorporation into a fibrin gel, or after the fibrin clot has been degraded by plasmin. The clinical utility of D-dimer measurement has been established in some scenarios, most notably for the exclusion of VTE. This article consists of 2 sections: in the first, the dynamics of D-dimer antigen formation is discussed and an overview of commercially available D-dimer assays is provided. The second section reviews available evidence for the clinical utilization of D-dimer antigen measurement in VTE, as well as emerging areas of D-dimer utilization as a marker of coagulation activation in other clinical settings.

Blood Coagulation: Hemostasis and Thrombin Regulation

Perioperative bleeding is a major challenge particularly because of increasing clinical use of potent antithrombotic drugs. Understanding current concepts of coagulation is important in determining the preoperative bleeding risk of patients, and in managing hemostatic therapy perioperatively. The serine protease thrombin plays pivotal roles in the activation of additional serine protease zymogens (inactive enzymatic precursors), cofactors, and cell-surface receptors. Thrombin generation is closely regulated to locally achieve rapid hemostasis after injury without causing uncontrolled systemic thrombosis. During surgery, there are major disturbances in coagulation and inflammatory systems because of hemorrhage/hemodilution, blood transfusion, and surgical stresses. Postoperative bleeding often requires allogeneic blood transfusions, which support thrombin generation and hemostasis. However, procoagulant activity and inflammation are increased postoperatively; thus, antithrombotic therapy may be required to prevent perioperative thrombotic complications. There have been significant advances in the management of perioperative hemostasis and thrombosis because of the introduction of novel hemostatic and antithrombotic drugs. However, a limitation of current treatment is that conventional clotting tests do not reflect the entire physiological processes of coagulation making optimal pharmacologic therapy difficult. Understanding the in vivo regulatory mechanisms and pharmacologic modulation of thrombin generation may help control bleeding without potentially increasing prothrombotic risks. In this review, we focus on the regulatory mechanisms of hemostasis and thrombin generation using multiple, simplified models of coagulation.

Tissue factor expression by endothelial cells in sickle cell anemia.

The role of the vascular endothelium in activation of the coagulation system, a fundamental homeostatic mechanism of mammalian biology, is uncertain because there is little evidence indicating that endothelial cells in vivo express tissue factor (TF), the systems triggering mechanism. As a surrogate for vessel wall endothelium, we examined circulating endothelial cells (CEC) from normals and patients with sickle cell anemia, a disease associated with activation of coagulation. We find that sickle CEC abnormally express TF antigen (expressed as percent CEC that are TF-positive), with 66+/-13% positive in sickle patients in steady-state, 83+/-19% positive in sickle patients presenting with acute vasoocclusive episodes, and only 10+/-13% positive in normal controls. Repeated samplings confirmed this impression that TF expression is greater when sickle patients develop acute vasoocclusive episodes. Sickle CEC are also positive for TF mRNA, with excellent concurrence between antigen and mRNA expression. The TF expressed on the antigen-positive CEC is functional, as demonstrated by a binding assay for Factor VIIa and a chromogenic assay sensitive to generation of Factor Xa. By establishing that endothelial cells in vivo can express TF, these data imply that the vast endothelial surface area does provide an important pathophysiologic trigger for coagulation activation.

Coagulation factor concentrates: past, present, and future

Clotting factor transfusions are vital for people with diseases such as haemophilia. In the 1970s and 1980s, transfusions with pooled plasma led to a devastatingly high number of recipients becoming infected with blood-borne pathogens such as HIV and hepatitis C. This epidemic triggered the development of virus-free factor concentrates through a combination of improved donor selection and screening, effective virucidal technologies, and recombinant protein expression biotechnology. There is now a wide range of recombinant factor concentrates, and an impressive safety record with respect to pathogen transmission. However, remaining therapeutic challenges include the potential threat of transmission of prions and other pathogens, the formation of inhibitory alloantibodies, and the international disparity that exists in product availability due to differences in licensure status as well as prohibitively high costs. In the future, it is likely that bioengineered recombinant proteins that have been modified to enhance pharmacokinetic properties or reduce immunogenicity, or both, will be used increasingly in clinical practice.