Contact System Activation and Cancer: New Insights in the Pathophysiology of Cancer-Associated Thrombosis.

Cancer induces a systemic hypercoagulable state that elevates the baseline thrombotic risk of affected patients. This hypercoagulable state reflects a complex interplay between cancer cells and host cells and the coagulation system as part of the host response to cancer. Although the tissue factor (TF)/factor VIIa pathway is proposed to be the principal initiator of fibrin formation in cancer patients, clinical studies have not shown a consistent relationship between circulating TF levels (often measured as plasma microvesicle-associated TF) and the risk of thrombosis. A renewed interest in the role of the contact pathway in thrombosis has evolved over the past decade, raising the question of its role in the pathogenesis of thrombotic complications in cancer. Recent observations have documented the presence of activation of the contact system in gastrointestinal, lung, breast and prostate cancers. Although the assays used to measure contact activation differ, and despite the absence of standardization of methodologies, it is clear that both the intrinsic and extrinsic pathways may be activated in cancer. This review will focus on recent findings concerning the role of activation of the contact system in cancer-associated hypercoagulability and thrombosis. An improved understanding of the pathophysiology of these mechanisms may lead to personalized antithrombotic protocols with improved efficacy and safety compared with currently available therapies.

Perioperative management of rare coagulation factor deficiency states in cardiac surgery.

Rare bleeding disorders (RBDs) include the hereditary deficiency of fibrinogen, factor (F)II, FV, FV + FVIII, FVII, FX, FXI or FXIII. RBDs do not confer a protective effect against atheromatous plaque formation, and thus the need for cardiovascular (CV) surgery in RBD patients is expected to increase with improved healthcare access (diagnosis and management) and longevity of the population. Clinical data regarding the management of RBDs in this setting are sparse, but the perioperative care team is obliged to gain a better understanding on available biological and pharmacological hemostatic agents. Perioperative management of RBDs in CV surgery is further complicated by heparin anticoagulation, haemodilution, and consumption of procoagulant and anticoagulant proteins associated with cardiopulmonary bypass (CPB). The aims of this review are to summarize pathophysiology of RBDs and laboratory monitoring pertinent to CV surgery, available factor replacement agents, and to provide the framework for perioperative coagulation management of RBD patients.

Global assays of fibrinolysis.

Fibrinolysis is an important and integral part of the hemostatic system. Acting as a balance to blood coagulation, the fibrinolytic system protects the body from unwanted thrombus formation and occlusion of blood vessels. As long as blood coagulation and fibrinolysis remain in equilibrium, response to injury, such as vessel damage, is appropriately regulated. However, alterations in this balance may lead to thrombosis or bleeding. A variety of methods have been proposed to assess fibrinolytic activity in blood or its components, but due to the complexity of the system, the design of a "gold standard" assay that reflects overall fibrinolysis has remained an elusive goal. In this review, we describe the most commonly used methods that have been described, such as thromboelastography (TEG and ROTEM), global fibrinolytic capacity in plasma and whole blood, plasma turbidity methods, simultaneous thrombin and plasmin generation assays, euglobulin clot lysis time and fibrin plate methods. All of these assays have strengths and limitations. We suggest that some methods may be preferable for detecting hypofibrinolytic conditions, whereas others may be better for detecting hyperfibrinolytic states.

Recombinant B-domain-deleted porcine sequence factor VIII (r-pFVIII) for the treatment of bleeding in patients with congenital haemophilia A and inhibitors.

INTRODUCTION: Development of inhibitors to human FVIII (hFVIII) significantly complicates the control of bleeding events in patients with haemophilia A. AIM: This prospective, multicentre, open-label, non-comparative, Phase II study evaluated the haemostatic activity of a recombinant B-domain-deleted porcine FVIII (r-pFVIII), in the treatment of non-life/non-limb-threatening bleeding in individuals with haemophilia A and FVIII inhibitors. METHODS: Acute bleeding episodes in patients with pFVIII inhibitor titres <0.8 BU mL-1 were treated with 50 U kg-1 body weight r-pFVIII. Those with pFVIII inhibitor titres of >0.8 BU mL-1 received an initial calculated r-pFVIII loading dose followed by 50 U kg-1 treatment dose. Treatment continued at 6-hourly intervals until bleeding was determined, controlled or till a maximum of eight doses was reached. RESULTS: All 25 bleeding episodes in nine patients (mean age: 23.7 years; range: 14-34 years) were controlled successfully with eight or fewer injections of r-pFVIII. The median time from bleeding onset to the administration of r-pFVIII was 5.7 h (range: 1.5-20.0 h). Twenty of the bleeding episodes (80%) were controlled with one treatment dose of r-pFVIII (with or without a loading dose, median dose: 200.8 U kg-1 ; range: 50-576 U kg-1 ) regardless of pFVIII level. r-pFVIII was well tolerated and no treatment-emergent serious adverse events were considered by the investigator to be related to r-pFVIII administration. CONCLUSION: The results suggest that FVIII replacement therapy with r-pFVIII could be a viable alternative to bypassing agents for the treatment of bleeding episodes in individuals with haemophilia A and FVIII inhibitors.

Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop.

Essentials The clinical enumeration of microparticles (MPs) is hampered by a lack of standardization. A new strategy to standardize MP counts by flow cytometry was evaluated in a multicenter study. No difference was found between instruments using forward or side scatter as the trigger parameter. This study demonstrated that beads can be used as a standardization tool for MPs. Click to hear the ISTH Academy's webinar on microvesicles SUMMARY: Background Microparticles (MPs) are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter (forward (FSC) or side scatter (SSC)) provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC-optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) of instruments. All instruments correctly ranked the platelet MP (PMP) levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side-scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to demonstrate a real potential of bead-based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MPs' clinical relevance at a multicenter level.

Thrombin generation and cell-dependent hypercoagulability in sickle cell disease.

Essentials Sickle cell disease is increasingly being recognized as a chronic hypercoagulable state. Thrombin generation is elevated in the whole blood, but not the plasma of sickle cell patients. Whole blood thrombin generation inversely correlates to erythrocyte phosphatidylserine exposure. Acquired protein S deficiency is likely explained by binding of protein S to sickle red cells. Click to hear Dr Hillery discuss coagulation and vascular pathologies in mouse models of sickle cell disease. SUMMARY: Introduction Sickle cell disease (SCD) is a hypercoagulable state with chronic activation of coagulation and an increased incidence of thromboembolic events. However, although plasma pre-thrombotic markers such as thrombin-anithrombin complexes and D-dimer are elevated, there is no consensus on whether global assays of thrombin generation in plasma are abnormal in patients with SCD. Based on our recent observation that normal red blood cells (RBCs) contribute to thrombin generation in whole blood, we hypothesized that the cellular components in blood (notably phosphatidylserine-expressing erythrocytes) contribute to enhanced thrombin generation in SCD. Methods Whole blood and plasma thrombin generation assays were performed on blood samples from 25 SCD patients in a non-crisis 'steady state' and 25 healthy race-matched controls. Results Whole blood thrombin generation was significantly elevated in SCD, whereas plasma thrombin generation was paradoxically reduced compared with controls. Surprisingly, whole blood and plasma thrombin generation were both negatively correlated with phosphatidylserine exposure on RBCs. Plasma thrombin generation in the presence of exogenous activated protein C or soluble thrombomodulin revealed deficiencies in the protein C/S anticoagulant pathway in SCD. These global changes were associated with significantly lower plasma protein S activity in SCD that correlated inversely with RBC phosphatidylserine exposure. Conclusion Increased RBC phosphatidylserine exposure in SCD is associated with acquired protein S deficiency. In addition, these data suggest a cellular contribution to thrombin generation in SCD (other than RBC phosphatidylserine exposure) that explains the elevated thrombin generation in whole blood.

NHF-McMaster Guideline on Care Models for Haemophilia Management.

This guideline was developed to identify evidence-based best practices in haemophilia care delivery, and discuss the range of care providers and services that are most important to optimize outcomes for persons with haemophilia (PWH) across the United States. The guideline was developed following specific methods described in detail in this supplement and based on the GRADE (Grading of Recommendations, Assessment, Development and Evaluation approach). Direct evidence from published literature and the haemophilia community, as well as indirect evidence from other chronic diseases, were reviewed, synthesized and applied to create evidence-based recommendations. The Guideline panel suggests that the integrated care model be used over non-integrated care models for PWH (conditional recommendation, moderate certainty in the evidence). For PWH with inhibitors and those at high risk for inhibitor development, the same recommendation was graded as strong, with moderate certainty in the evidence. The panel suggests that a haematologist, a specialized haemophilia nurse, a physical therapist, a social worker and round-the-clock access to a specialized coagulation laboratory be part of the integrated care team, over an integrated care team that does not include all of these components (conditional recommendation, very low certainty in the evidence). Based on available evidence, the integrated model of care in its current structure, is suggested for optimal care of PWH. There is a need for further appropriately designed studies that address unanswered questions about specific outcomes and the optimal structure of the integrated care delivery model in haemophilia.

Care models in the management of haemophilia: a systematic review.

BACKGROUND: Haemophilia care is commonly provided via multidisciplinary specialized management. To date, there has been no systematic assessment of the impact of haemophilia care delivery models on patient-important outcomes. OBJECTIVE: To conduct a systematic review of published studies assessing the effects of the integrated care model for persons with haemophilia (PWH). SEARCH METHODS: We searched MEDLINE, EMBASE and CINAHL up to April 22, 2015, contacted experts in the field, and reviewed reference lists. SELECTION CRITERIA: Randomized and non-randomized studies of PWH or carriers, focusing mainly on the assessment of care models on delivery. DATA COLLECTION AND ANALYSIS: Two investigators independently screened title, abstract, and full text of retrieved articles for inclusion. Risk of bias and overall quality of evidence was assessed using Cochrane's ACROBAT-NRSI tool and GRADE respectively. Relative risks, mean differences, proportions, and means and their variability were calculated as appropriate. RESULTS: 27 non-randomized studies were included: eight comparative and 19 non-comparative studies. We found low- to very low-quality evidence that in comparison to other models of care, integrated care may reduce mortality, hospitalizations and emergency room visits, may lead to fewer missed days of school and work, and may increase knowledge seeking. CONCLUSION: Our comprehensive review found low- to very low-quality evidence from a limited number of non-randomized studies assessing the impact of haemophilia care models on some patient-important outcomes. While the available evidence suggests that adoption of the integrated care model may provide benefit to PWH, further high-quality research in the field is needed.

OC-04 - Tissue factor positive microvesicles activate platelets in vitro and in vivo and enhance thrombosis in mice.

INTRODUCTION: Cancer patients have a 4- to 7- fold increased risk of venous thromboembolism (VTE) compared with general population. Most tumor cells express tissue factor (TF) and constitutively release small membrane microvesicles called tumor microvesicles (TMVs). Clinical studies have shown that circulating MP-TF activity is associated with VTE in pancreatic cancer but not in other types of cancer. Thrombin is a potent platelet agonist and activates platelets via protease activated receptors (PARs). AIM: To determine the contribution of the TF+ TMV-thrombin-platelet pathway to cancer-associated thrombosis. MATERIALS AND METHODS: A human pancreatic adenocarcinoma cell line expressing high levels of TF (BxPc-3) was selected to study the effect of TF+ TMVs on platelet activation and thrombosis. RESULTS: TF+ TMVs induced platelet activation in vitro in a thrombin-dependent manner. The presence of orthotopically grown BxPc-3 tumors in mice was associated with increased levels of thrombin-antithrombin III complexes (TATc) and larger thrombi in an inferior vena cava stenosis model compared with control mice. Furthermore, injection of BxPc-3 TF+ TMVs into mice triggered platelet activation and enhanced venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF+ TMV-enhanced thrombosis was reduced in Par4-deficient mice and wild-type mice treated with the platelet inhibitor clopidogrel, suggesting that platelet activation was required for the enhanced thrombosis. CONCLUSIONS: These studies suggest that platelet inhibitors may reduce thrombosis in cancer patients with elevated levels of TF+ TMVs.

Procoagulant microparticles promote coagulation in a factor XI-dependent manner in human endotoxemia.

UNLABELLED: Essentials The procoagulant effects of microparticles (MPs) on coagulation in endotoxemia are not known. MPs from endotoxemia volunteers were evaluated for procoagulant activity in a plasma milieu. MPs from endotoxemia volunteers shortened clotting times and enhanced thrombin generation. MP procoagulant effects were mediated in a factor XI-dependent manner. SUMMARY: Background Human endotoxemia is characterized by acute inflammation and activation of coagulation, as well as increased numbers of circulating microparticles (MPs). Whether these MPs directly promote coagulation and through which pathway their actions are mediated, however, has not been fully explored. Objectives In this study, we aimed to further characterize endotoxin-induced MPs and their procoagulant properties using several approaches. Methods Enumeration and characterization of MPs were performed using a new-generation flow cytometer. Relative contributions of the extrinsic and intrinsic pathways in MP-mediated procoagulant activity were assessed using plasmas deficient in factor (F) VII or FXI or with blocking antibodies to tissue factor (TF) or FXIa. Results Total MPs and platelet MPs were significantly elevated in plasma at 6 h after infusion of endotoxin in healthy human subjects. MPs isolated from plasma following endotoxin infusion also demonstrated increased TF activity in a reconstituted buffer system. When added to recalcified platelet-poor plasma, these MPs also promoted coagulation, as judged by a decreased clotting time with shortening of the lag time and time to peak thrombin using calibrated automated thrombography (CAT). However, the use of FVII-deficient plasma or blocking antibody to TF did not inhibit these procoagulant effects. In contrast, plasma clotting time was prolonged in FXI-deficient plasma and a blocking antibody to FXIa inhibited all MP-mediated parameters in the CAT assay. Conclusions The initiation of coagulation by cellular TF in endotoxemia is in contrast to (and presumably complemented by) the intrinsic pathway-mediated procoagulant effects of circulating MPs.

Tissue factor-positive tumor microvesicles activate platelets and enhance thrombosis in mice.

UNLABELLED: ESSENTIALS: Cancer patients have a high rate of venous thrombosis (VT) but the underlying mechanisms are unknown. Tumor-derived, tissue factor-positive microvesicles in platelet activation in vitro and in vivo were studied. Tumor-derived, tissue factor-positive microvesicles enhanced VT in mice. Platelets may contribute to VT in some cancer patients, and this could be prevented with antiplatelet drugs. BACKGROUND: Cancer patients have an approximately 4-fold increased risk of venous thromboembolism (VTE) compared with the general population, and cancer patients with VTE have reduced survival. Tumor cells constitutively release small membrane vesicles called microvesicles (MVs) that may contribute to thrombosis in cancer patients. Clinical studies have shown that levels of circulating tumor-derived, tissue factor-positive (TF(+) ) MVs in pancreatic cancer patients are associated with VTE. Objectives We tested the hypothesis that TF(+) tumor-derived MVs (TMVs) activate platelets in vitro and in mice. MATERIALS AND METHODS: We selected two human pancreatic adenocarcinoma cell lines expressing high (BxPc-3) and low (L3.6pl) levels of TF as models to study the effect of TF(+) TMVs on platelets and thrombosis. RESULTS AND CONCLUSIONS: We found that both types of TF(+) TMVs activated human platelets and induced aggregation in vitro in a TF and thrombin-dependent manner. Further, injection of BxPc-3 TF(+) TMVs triggered platelet activation in vivo and enhanced thrombosis in two mouse models of venous thrombosis in a TF-dependent manner. Importantly, BxPc-3 TF(+) TMV-enhanced thrombosis was reduced in Par4-deficient mice and in wild-type mice treated with clopidogrel, suggesting that platelet activation was required for enhanced thrombosis. These studies suggest that TF(+) TMV-induced platelet activation contributes to thrombosis in cancer patients.

Current laboratory practices in the diagnosis and management of haemophilia: a global assessment.

Haemophilia management is complicated by the extreme variability in laboratory practices. Lack of consistency or comparability in testing makes it difficult to establish diagnostic criteria or disease severity, and complicates response assessment. A global survey was conducted to document current practices. A 35-min survey was completed by 30 laboratory scientists in each of seven countries (France, Germany, Italy, Japan, Spain, UK, USA; 210 in total); results were weighted by average country testing volume in haemophilia. Eighty-three per cent of participants reported participation in a Quality Assurance scheme. Ninety per cent reported using clotting tests in haemophilia A and 88% in haemophilia B (55% and 53% frequent use respectively). Sixty-eight per cent reported chromogenic assays were used in haemophilia A, with only 23% reporting frequent use, compared to only 11% reporting any use in haemophilia B. Twenty-nine separate activated partial thromboplastin time (aPTT) reagents were reported for haemophilia A and 27 aPTT reagents were reported for haemophilia B, with one-quarter or less obtaining reagents or kits from any single manufacturer. Fifty-four per cent run a calibration curve with every factor VIII (FVIII) assay. The mean number of plasma dilutions varied from 2 to 4 for FVIII assays and from 1 to 3 for FIX assays. Results indicate very low consistency in materials and practices used to test for factor activity in haemophilia. A number of responses suggest that some laboratory scientists' understanding of best practices or guidelines in haemophilia could be improved. More education and broader understanding is recommended regarding assay types, assay components, test material and instrument features and capabilities.

Prospective study of sickle cell trait and venous thromboembolism incidence.

BACKGROUND: Sickle cell trait may increase risk of venous thromboembolism, but this is not fully established. OBJECTIVES: We sought to determine the association of sickle cell trait with deep vein thrombosis and pulmonary embolism. METHODS: Middle-aged African Americans participating in a prospective, population-based cohort investigation, the Atherosclerosis Risk in Communities Study, were followed from 1987 through 2011 for incident hospitalized pulmonary embolism (n = 111) or isolated deep vein thrombosis (n = 138), verified by physician review of medical records. Sickle cell trait (heterozygosity for hemoglobin S, n = 268) was compared with no sickle cell trait (n = 3748). RESULTS: Over a median of 22 years of follow-up, 249 participants had an incident venous thromboembolism. The hazard ratio of venous thromboembolism was 1.50 (95% confidence interval [CI] 0.96-2.36) for participants with vs. without sickle cell trait, after adjustment for age, sex, ancestry, hormone replacement therapy (women), body mass index, diabetes, and estimated glomerular filtration rate. This hazard ratio was 2.05 (95% CI 1.12-3.76) for pulmonary embolism and 1.15 (95% CI 0.58-2.27) for deep vein thrombosis without pulmonary embolism. CONCLUSIONS: Sickle cell trait in African Americans carries a 2-fold increased risk of pulmonary embolism but does not elevate deep vein thrombosis risk. Because neonatal screening for sickle hemoglobin is being conducted in the United States, consideration should be paid to the increased pulmonary embolism risk of individuals with sickle cell trait.

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations.