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Background Statins have long been suspected to have pleiotropic effects via thrombotic factors. Randomized controlled trials are too limited to be definitive. We examined the associations of genetically mimicking effects of statins, PCSK9 inhibitors, and alternative lipid targets (in genes LDLR, APOC3, and LPL) on key indicators of coagulation system function, i.e., prothrombin time (PT) and activated partial thromboplastin time (aPTT). Methods We assessed the effect of established genetic mimics of effects of lipid modifiers and alternative lipid treatment targets on PT (n = 58,110) and aPTT (n = 37,767), all transformed to z-scores, using Mendelian randomization taking advantage of Biobank Japan. Ischemic heart disease (IHD) was a control outcome. Results Genetically mimicked effects of statins increased PT by 0.31 standard deviation (SD) per SD increase in low-density lipoprotein (95% confidence interval [CI]: 0.10–0.51) based on rs12916 but did not affect aPTT. Genetically mimicking effects of targeting LDLR increased PT based on rs688 (0.33 SD per SD increase in triglyceride, 95% CI: 0.03–0.63) but did not affect aPTT. Genetically mimicking effects of PCSK9 inhibitors or targeting APOC3 or LPL had no effect on PT or aPTT. Genetically mimicking effects of statins, PCSK9 inhibitors, and alternative lipid targets reduced risk of IHD in Biobank Japan. Conclusion Statins, and possibly targeting LDLR, may also act via a coagulation cascade factor, likely specific to the extrinsic or common pathway. Further elucidation of the mechanistic pathway may facilitate development of new interventions and inform use of statins particularly in relation to use of other anticoagulants.

In the life sciences, including hemostasis and thrombosis, methods of structural biology have become indispensable tools for shedding light on underlying mechanisms that govern complex biological processes. Advancements of the relatively young field of computational biology have matured to a point where it is increasingly recognized as trustworthy and useful, in part due to their high space–time resolution that is unparalleled by most experimental techniques to date. In concert with biochemical and biophysical approaches, computational studies have therefore proven time and again in recent years to be key assets in building or suggesting structural models for membrane-bound forms of coagulation factors and their supramolecular complexes on membrane surfaces where they are activated. Such endeavors and the proposed models arising from them are of fundamental importance in describing and understanding the molecular basis of hemostasis under both health and disease conditions. We summarize the body of work done in this important area of research to drive forward both experimental and computational studies toward new discoveries and potential future therapeutic strategies.

Hepatectomy-induced coagulation disturbances have been well studied over the past decade. Cumulative evidence supports the superiority of global coagulation analysis compared with conventional coagulation tests (i.e., prothrombin time or activated partial thromboplastin time) for clinical decision making. Cancer, however, represents an acquired prothrombotic state and liver resection for cancer deserves a more thorough investigation. This prospective observational study was conducted to assess the perioperative coagulation status of patients undergoing major hepatectomies for primary or metastatic hepatic malignancy. Patients were followed up to the 10th post-operative day by serial measurements of conventional coagulation tests, plasma levels of coagulation factors, and thrombin generation assay parameters. An abnormal coagulation profile was detected at presentation and included elevated FVIII levels, decreased levels of antithrombin, and lag time prolongation in thrombin generation. Serial hematological data demonstrated increased Von Willebrand factor, FVIII, D-dimer, fibrinogen and decreased levels of natural anticoagulant proteins in the early post-operative period predisposing to a hyper-coagulable state. The ratio of the anticoagulant protein C to the procoagulant FVIII was low at baseline and further declined post-operatively, indicating a prothrombotic state. Though no bleeding complications were reported, one patient experienced pulmonary embolism while under thromboprophylaxis. Overall, patients with hepatic carcinoma presenting for elective major hepatectomy may have baseline malignancy-associated coagulation disturbances, aggravating the hyper-coagulable state documented in the early post-operative period.

Background Pro- and anticoagulant drugs are commonly used in pediatric liver transplantation to prevent and treat thrombotic and bleeding complications. However, the combination of baseline hemostatic changes in children with liver disease and additional changes induced by transplantation makes this very challenging. This study aimed to analyze the efficacy of clinically available pro- and anticoagulant drugs in plasma from children undergoing liver transplantation. Methods In vitro effects of pro- and anticoagulant drugs on thrombin generation capacity were tested in plasma samples of 20 children (≤ 16 years) with end-stage liver disease undergoing liver transplantation, and compared with 30 age-matched healthy controls. Results Addition of pooled normal plasma had no effect in patients or controls, while 4-factor prothrombin complex concentrate increased thrombin generation in both patients and controls, with enhanced activity in patients. At start of transplantation, dabigatran and unfractionated heparin had a higher anticoagulant potency in patients, whereas 30 days after transplantation low molecular weight heparin was slightly less effective in patients. Effects of rivaroxaban were comparable between patients and controls. Conclusion This study revealed important differences in efficacy of commonly used pro- and anticoagulant drugs in children with end-stage liver disease undergoing liver transplantation. Therefore, dose adjustments of these drugs may be required. The results of this study may be helpful in the development of urgently needed protocols for strategies to prevent and treat bleeding and thrombotic complications in pediatric liver transplantation.

Cardiovascular disease, including stroke, myocardial infarction, and venous thromboembolism, is one of the leading causes of morbidity and mortality worldwide. Excessive coagulation may cause vascular occlusion in arteries and veins eventually leading to thrombotic diseases. Studies in recent years suggest that coagulation factors are involved in these pathological mechanisms. Factors XIa (FXIa), XIIa (FXIIa), and plasma kallikrein (PKa) of the contact system of coagulation appear to contribute to thrombosis while playing a limited role in hemostasis. Contact activation is initiated upon autoactivation of FXII on negatively charged surfaces. FXIIa activates plasma prekallikrein (PK) to PKa, which in turn activates FXII and initiates the kallikrein–kinin pathway. FXI is also activated by FXIIa, leading to activation of FIX and finally to thrombin formation, which in turn activates FXI in an amplification loop. Animal studies have shown that arterial and venous thrombosis can be reduced by the inhibition of FXI(a) or PKa. Furthermore, data from human studies suggest that these enzymes may be valuable targets to reduce thrombosis risk. In this review, we discuss the structure and function of FXI(a) and PK(a), their involvement in the development of venous and arterial thrombosis in animal models and human studies, and current therapeutic strategies.

Background Intracranial hemorrhage (ICH) is one of the major devastating complications of anticoagulation. Matrix metalloproteinase (MMP) inhibition has been proposed as a novel pharmacological approach for ICH treatment. Objectives We evaluated the effects of CM-352 (MMP-fibrinolysis inhibitor) in an experimental ICH model associated with oral anticoagulants as compared with clinically used prothrombin complex concentrate (PCC). Methods ICH was induced by collagenase injection into the striatum of wild type (C57BL/6J) anticoagulated mice (warfarin or rivaroxaban) and Mmp10 −/− mice. Hematoma volume and neurological deficits were measured 24 hours later by diaminobenzidine staining and different behavioral tests. Circulating plasminogen activator inhibitor-1 (PAI-1) activity and interleukin-6 (IL-6) were measured in plasma samples and local inflammation was assessed by neutrophil infiltration. Finally, fibrinolytic effects of MMP-10 and rivaroxaban were evaluated by thromboelastometry and thrombin-activatable fibrinolysis inhibitor (TAFI) activation assays. Results Only PCC reduced hemorrhage volume and improved functional outcome in warfarin-ICH, but both PCC and CM-352 treatments diminished hemorrhage volume (46%, p Conclusion CM-352 treatment, by diminishing MMPs and rivaroxaban-associated fibrinolytic effects, might be a novel antihemorrhagic strategy for rivaroxaban-associated ICH.

Background Warfarin-associated major hemorrhage is frequently treated with prothrombin complex concentrates to correct international normalized ratio (INR). Objective This article aims to investigate the efficacy of activated prothrombin complex concentrate (aPCC) versus 4-factor prothrombin complex concentrate (4PCC) for vitamin K antagonist reversal in patients with warfarin-associated major hemorrhage. Materials and Methods This was a multicenter, retrospective cohort study. Patients included were age ≥ 18 years with pretreatment INR of > 1.5. Exclusion criteria were patients treated for urgent procedures without hemorrhage, treated but not taking warfarin, unavailable INR values, and pregnant patients. Patients were stratified into two groups: aPCC or 4PCC. The primary outcome was achievement of INR ≤ 1.5 at the posttreatment INR sampling. Secondary outcomes focused on thrombotic events and mortality. Results Of 342 patients, 237 patients received aPCC and 105 patients received 4PCC. After 1:1 propensity score matching, 86 patients remained in each group. In the matched cohort, the proportion of patients who achieved target INR ≤ 1.5 was greater with 4PCC (aPCC = 61 [70.9%] vs. 4PCC = 76 [88.4%]; 95% confidence interval [CI] –29.2% to –5.7%) and groups had comparable in-hospital thrombotic events and mortality. In the unmatched cohort, achievement of target INR ≤ 1.5 was greater with 4PCC (aPCC = 151 [63.7%] vs. 4PCC = 92 [87.6%]; 95% CI –32.7% to –15.1%). Conclusion In the treatment of warfarin-associated major hemorrhage, 4PCC compared with aPCC was associated with greater achievement of INR ≤ 1.5 with comparable thrombotic events and mortality. Further controlled studies are needed to confirm these findings and determine the optimal dosing strategy that maximizes efficacy and safety.

Background: Cholesteryl ester transfer protein (CETP) plays an important role in lipoprotein metabolism. Previous studies have suggested that the CETP TaqI B1/B2 allele is associated with the risk of venous thrombosis (VT).Aim: To investigate the associations between genetically determined CETP concentrations and 22 hemostatic factors in healthy individuals, and the risk of a first VT event, in a large VT case-control study.Methods: Analyses were performed in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) case-control study. CETP unweighted/weighted genetic risk scores (GRSs) were derived from three single-nucleotide polymorphisms that were identified from a recent genome-wide association study on serum CETP concentrations. The associations between CETP GRSs and 22 hemostatic factors (procoagulant/anticoagulant and fibrinolytic factors) were assessed by linear regression from an additive model in controls (n = 2813). The associations between CETP GRSs and the risk of a first VT were assessed by logistic regression analyses in 3950 VT cases and 4765 controls.Results: In the controls (median age, 49 years; 53% women), both unweighted and weighted GRSs showed that factor VII activity was negatively associated with the genetically determined CETP concentration (weighted GRS β -3.08 IU/dL per μg/mL genetically determined CETP, 95% confidence interval -5.73 to -0.42). No association was observed with the risk of a first VT.Conclusions: Genetically determined CETP concentrations only showed a weak negative association with factor VII activity. However, this did not lead to an association with the risk of a first VT.

Development of ascites is the most common form of decompensation of cirrhosis. We aimed to investigate the coagulation system in ascitic fluid and plasma of patients with cirrhosis. We determined coagulation parameters and performed clotting and fibrinolysis experiments in ascitic fluid and plasma of thoroughly characterized patients with cirrhosis and ascites (n = 25) and in plasma of patients with cirrhosis but without ascites (n = 25), matched for severity of portal hypertension. We also investigated plasma D-dimer levels in an independent cohort of patients (n = 317) with clinically significant portal hypertension (HVPG ≥ 10 mmHg), grouped according to ascites severity. Ascitic fluid was procoagulant in a clotting assay. The procoagulant potential of ascitic fluid was abolished by depletion of extracellular vesicles from ascitic fluid by filtration or by addition of a tissue factor-neutralizing antibody. Compared with plasma, extracellular vesicle-associated tissue factor activity was high in ascitic fluid, while activities of other coagulation factors were low. The extracellular vesicle-depleted fraction of ascitic fluid induced fibrinolysis, which was prevented by aprotinin, indicating the presence of plasmin in ascitic fluid. Plasma peak thrombin generation and parameters reflecting fibrinolysis were independently associated with the presence of ascites. Finally, plasma D-dimer levels were independently linked to ascites severity in our second cohort comprising 317 patients. In conclusion, coagulation and fibrinolysis become activated in ascites of patients with cirrhosis. While tissue factor-exposing extracellular vesicles in ascitic fluid seem unable to pass the peritoneal membrane, fibrinolytic enzymes get activated in ascitic fluid and may re-enter the systemic circulation and induce systemic fibrinolysis.

von Willebrand disease (VWD) is a quantitative or qualitative defect in von Willebrand factor (VWF) resulting in mucocutaneous bleeding symptoms and hemorrhage following hemostatic challenges, such as trauma or surgery. VWD-specific therapy, DDAVP (1-desamino-8-D-arginine vasopressin) and VWF concentrates, is necessary periprocedurally to ensure adequate hemostasis. The aging VWD patient may complicate this matter. The plasma concentration of many coagulation proteins, including VWF, increases with age. While it has been established that VWF levels increase with age in a healthy population, emerging research demonstrates this occurs in certain subtypes of VWD, too. Thus, the management of periprocedural VWD-specific therapy in the aging VWD patient is problematic when VWF levels increase over time to normal, and hematologists are left with uncertainty regarding whether or not periprocedural VWD-specific therapy is still necessary. In this article, we will review the current state of the literature regarding the effect of age on VWF levels in the healthy population and VWD while exploring possible etiologies for this phenomenon. Further, we will detail how this affects bleeding symptoms and highlight what research remains to be done to optimize care in this patient population.

Oral factor Xa inhibitors are increasingly used for anticoagulation, but there is no approved reversal agent. Prothrombin complex concentrate (PCC) for the management of Xa-inhibitor–associated bleeding has been described in small case series and one cohort study. Patients on apixaban or rivaroxaban, suffering a major bleed, were treated at nine Canadian hospitals as per existing hospital protocol with a fixed dose of PCC 2,000 units and subsequently recruited for a 30-day follow-up. The treating physician evaluated the haemostatic effectiveness as observed during the first day as good, moderate or poor/none, using an assessment guide. Safety outcomes were thromboembolism or death. We recruited 66 patients with major bleeding who were treated with PCC and who were receiving rivaroxaban (56%) or apixaban (44%). The effectiveness was assessed as good in 65% (95% confidence interval [CI], 53–77), moderate in 20% (95% CI, 10–30) and poor/none in 15% (95% CI, 6–24). For the 36 patients with intracranial haemorrhage, the corresponding ratings were 67, 17 and 17%, and for 16 patients with gastrointestinal bleeding they were 69, 12 and 19%, respectively. There were nine deaths (14%) by 30 days, and five (8%) major thromboembolic events. In a post hoc analysis, according to International Society on Thrombosis and Haemostasis criteria, reversal was effective in 68% and ineffective in 32%. For major bleeding associated with oral Xa inhibitors, PCC may have a beneficial effect. The risk of thromboembolism after reversal of anticoagulation in patients with a prothrombotic background has to be taken into account.

Etiopathogenesis of myelodysplastic syndrome (MDS) might cause per se an anomalous haemostasis that can be even more deteriorated by thrombocytopaenia. So, evaluation of haemostasis in patients with MDS rises as a necessity.This work aimed to characterize haemostasis in non-bleeder MDS patients with a platelet count similar to healthy controls to establish differences between the two groups not related to thrombocytopaenia.Thromboelastometry in samples from MDS patients suggested the existence of at least two antagonistic processes: one of them giving a hypocoagulable pattern (prolonged clotting time and lower α angle) and another conferring a procoagulant profile (decreased fibrinolysis). Hypocoagulable state might be due to a decreased ability of platelets to be stimulated and to the presence in plasma of a factor/s that prolonged the time to initiate thrombin generation. This factor/s might be antibodies as this effect was observed in samples from MDS patients with an associated autoimmune-inflammatory condition.Otherwise, hypercoagulable state seemed to rely on an increased presence of red cell- and monocyte-derived microparticles and to the increased exposure of phosphatidylserine that served as scaffold for binding of coagulation factors.We concluded that haemostasis in MDS patients is a complex process influenced by more factors than platelet count.

SummaryTecarfarin is a vitamin K antagonist (VKA) with reduced propensity for drug interactions. To evaluate the pharmacokinetic (PK), pharmacodynamic (PD), and safety of tecarfarin, we performed single ascending dose (SAD) (n=66), multiple ascending dose (MAD) (n=43), and tecarfarin versus warfarin (n=28) studies in human volunteers. In the SAD, tecarfarin was administered to 5 of 6 subjects (1 received placebo) in each of 11 cohorts. AUC0-∞ exhibited linearity and dose proportionality. Elimination T1/2 ranged from 87–136 hours (h) across all doses. In the MAD, tecarfarin was administered to 5 of 6 volunteers in each of 7 cohorts. The starting dose was continued until the subject’s INR reached the target range (TR) of 1.7 to 2.0. Dosing was down-titrated if the TR was achieved. Elimination T1/2 ranged from 107–140 h. Doses Supplementary Material to this article is available online at www.thrombosis-online.com.

Background The use of short and uniform centrifugation schemes contributes significantly to the successful automation of laboratory procedures. It is however unclear if this is applicable to the hemostasis laboratory. Objectives This article assesses the accuracy of measurements obtained with a rapid, high-speed centrifugation scheme in a large set of hemostasis tests, covering the full spectrum of values obtained in clinical practice, and using meaningful statistical measures. Methods Two citrated plasma samples were obtained from consecutive patients of a tertiary hospital with suspected abnormal hemostasis tests and processed with two centrifugation schemes in parallel: 1,500 × g for 10 minutes and 3,137 × g for 7 minutes. The following tests were conducted: prothrombin time (n = 125), international normalized ratio (n = 146), activated partial thromboplastin time (n = 119), thrombin time (n = 105), fibrinogen (n = 125), factor (F)II (n = 69), FV (n = 64), FVII (n = 64), FX (n = 67), FVIII (n = 55), FIX (n = 37), FXI (n = 35), and FXIII (n = 20), D-dimer (n = 34), antithrombin (n = 31), anti-Xa activity (n = 30), von Willebrand antigen (n = 25), and von Willebrand activity (VWF:GPIbM; n = 27). Results A wide range of results were obtained in all tests. Spearman's rank correlation coefficient was at least 0.95 for all tests except FV, FIX, and FXI. The coverage probability π at a given deviation index κ of 15% was above 0.9 for all tests except FV, FVII, FX, FVIII, FIX, FXI, and VWF:GPIbM, suggesting a lack of agreement. Conclusion Our results suggest that high-speed centrifugation is applicable to the majority of routine hemostasis parameters. The coverage probability was more sensitive than Spearman's rank correlation to detect disagreement among centrifugation schemes.

SummaryClinical use of non-vitamin K antagonist oral anticoagulants is increasingly well established. However, specific agents for reversal of these drugs are not currently available. It was to objective of this study to investigate the impact of activated prothrombin complex concentrate (aPCC) on the anticoagulant effects of dabigatran in a randomised, controlled, porcine trauma model. Twenty-one pigs received oral and intravenous dabigatran, resulting in supratherapeutic plasma concentrations. Twelve minutes after injury (standardised bilateral femur fractures and blunt liver injury), animals (n=7/group) received 25 or 50 U/kg aPCC (aPCC25 and aPCC50) or placebo (control) and were followed for 5 hours. The primary endpoint was total volume of blood loss (BL). Haemodynamic and coagulation variables (prothrombin time [PT], activated partial thromboplastin time, diluted thrombin time, thrombin–antithrombin complexes, thromboelastometry, thrombin generation and D-dimers) were measured. Twelve minutes post-injury, BL was similar between groups. Compared with control (total BL: 3807 ± 570 ml) and aPCC25 (3690 ± 454 ml; p=0.77 vs control), a significant reduction in total BL (1639 ± 276 ml; p< 0.0001) and improved survival (p< 0.05) was observed with aPCC50. Dabigatran’s anticoagulant effects were effectively treated in the aPCC50 group, as measured by several parameters including EXTEM clotting time (CT) and PT. In contrast, with aPCC25, laboratory values were initially corrected but subsequently deteriorated due to ongoing blood loss. Thromboembolic or bleeding effects were not detected. In conclusion, blood loss following trauma in dabigatran-anticoagulated pigs was successfully reduced by 50 U/kg aPCC. Optimal methodology for measuring amelioration of dabigatran anticoagulation by aPCC is yet to be determined.

SummaryThe treatment of haemophilia has made significant progress in recent decades, and patients are now being treated safely with great clotting products. However, inhibitor development remains the largest problem, particularly in children. Consequently, the haemostasis that was obtained with traditional clotting factors is not being achieved. Moreover, inhibitor complications translate into adult life and there are an increasing number of situations where adult patients with an inhibitor require major surgery but the clinician is faced with the knowledge that required haemostasis levels are difficult to achieve. Therefore, it is of upmost importance to consider factors relating to inhibitor development, and to determine how inhibitors can be prevented and/or eliminated. Of the various factors at play with regard to inhibitor development, it is important to consider the immunogenicity of factor VIII (FVIII) products, and this topic is the focus of the current paper.

SummaryProphylaxis with the blood clotting factor, factor VIII (FVIII) is ineffective for individuals with haemophilia A and high-titre inhibitors to FVIII. Prophylaxis with the FVIII bypassing agents activated prothrombin complex concentrates (aPCC; FEIBA® Baxalta) or recombinant activated factor VII (rFVIIa; Novo-Seven®, Novo Nordisk) may be an effective alternative. It was our aim to develop evidence -and expert opinion- based guidelines for prophylactic therapy for patients with high-titre inhibitors to FVIII. A panel of nine Spanish haematologists undertook a systematic review of the literature to develop consensusbased guidance. Particular consideration was given to prophylaxis in patients prior to undergoing immune tolerance induction (ITI) (a process of continued exposure to FVIII that can restore sensitivity for some patients), during the ITI period and for those not undergoing ITI or for whom ITI had failed. These guidelines offer guidance for clinicians in deciding which patients might benefit from prophylaxis with FVIII bypassing agents, the most appropriate agents in various clinical settings related to ITI, doses and dosing regimens and how best to monitor the efficacy of prophylaxis. The paper includes recommendations on when to interrupt or stop prophylaxis and special safety concerns during prophylaxis. These consensus guidelines offer the most comprehensive evaluation of the clinical evidence base to date and should be of considerable benefit to clinicians facing the challenge of managing patients with severe haemophilia A with high-titre FVIII inhibitors.

Patients with haemophilia and inhibitors to factors VIII or IX require bypassing therapy. The primary safety concern of bypassing agents is thromboembolism; however, the incidence of thromboembolic adverse events (TAEs) in haemophilia patients with inhibitors remains poorly characterized. The aim of this study was to assess the incidence of TAEs following exposure to bypassing agents in patients with haemophilia. Using U.S. Medicaid database (2000–2010), we identified a cohort of 719 males (mean age: 10 years at cohort entry) with haemophilia A or B and use of either recombinant factor VIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC). Patients were followed up until loss of insurance eligibility, end of study period, or the first occurrence of TAE. Exposure was assessed on as-treated basis, and outcomes were adjudicated through review of healthcare claims. During the observation of a total of 2,823 person-years (py; mean follow-up: 3.9 years), 22 TAEs were identified, leading to incidence rates of 4.2 events (95% confidence interval [CI]: 1.8–8.3) per 1,000 unexposed py; 15.4 events (95% CI: 6.7–30.3) per 1,000 aPCC-exposed py; 18.2 events (95% CI: 8.3–34.6) per 1,000 rFVIIa-exposed py; and 29.7 events (95% CI: 6.1–86.7) per 1,000 py of concomitant exposure to both agents. The results were consistent across sensitivity analyses. In conclusion, we found no difference in the rate of TAEs across agents, but the data are compatible with a possibly increased rate associated with a combination therapy, highlighting the need for continuing safety monitoring through prospective registries or observational data.

SummaryFor centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

SummaryThe use of plasma-derived and recombinant coagulation factors for the treatment of haemophilia A and B is well established and permits patients to live a relatively normal life. In order to improve treatment options, several products are in development, which have a prolonged duration of action, thus enabling less frequent prophylactic dosing and aiming to reduce the burden of treatment. Several innovative approaches are being pursued to extend the half-life of factor VIIa, factor VIII and factor IX, utilising technologies such as Fc fusion, recombinant albumin fusion and addition of polyethyleneglycol (PEG) (PEG ylation). These methods prolong the time in the circulation by reducing degradation and elimination. This review summarises the technologies and products in development and their stages of development, and also discusses their pros and cons.

SummaryThe main purpose of anticoagulants is to diminish fibrin formation, thereby decreasing the risk of venous or arterial thrombosis. Vitamin K antagonist have been used for many decades in order to achieve reduced thrombotic risk, despite major drawbacks of this class of drugs such as cumbersome dossing and monitoring of anticoagulant status. To overcome these drawbacks of VKA, new classes of anticoagulants have been developed including oral anticoagulants for direct inhibition of either thrombin or factor Xa, which can be administrated in a fixed dose without monitoring. Coagulation factors can activate cellular protease-activated receptors, thereby inducing cellular processes as inflammation, apoptosis, migration, and fibrosis. Therefore, inhibition of coagulation proteases not only attenuates fibrin formation, but may also influence pathophysiological processes like vascular calcification and atherosclerosis. Animal models revealed that VKA therapy induced both intima and media calcification and accelerated plaque vulnerability, whereas specific and direct inhibition of thrombin or factor Xa attenuated atherosclerosis. In this review we provide an overview of old and new oral anticoagulants, as well discuss potential pleiotropic effects with regard to calcification and atherosclerosis. Although translation from animal model to clinical patients seems difficult at first sight, effort should be made to fully understand the clinical implications of long-term oral anticoagulant therapy on vascular side effects.

SummaryProthrombin complex concentrates (PCC) can rapidly normalise prolonged prothrombin time, induced by vitamin K antagonists (VKA). We conducted a multicentre retrospective study to investigate whether reversal of VKA coagulopathy with 4-factor PCC improves the survival of patients with VKA-related intracerebral haemorrhage as compared to plasma. We included 135 consecutive patients with VKA-related intracerebral haemorrhage treated either with plasma (mainly in Canada) or 4-factor PCC (The Netherlands and Sweden) for the reversal of VKA. Data on characteristics of the patients and the haemorrhage were collected. The volume of intracerebral haematoma was calculated from the first computed tomography (CT) scan. The unadjusted and adjusted odds ratio (OR) for 30-day all-cause mortality in both treatment groups was compared using logistic regression. Patients who received plasma (n=35, median 4 units) more often had diabetes, antiplatelet therapy, and intraventricular haemorrhage on the initial CT scans than patients who received PCC (n=100, median 22.5 IU/kg [interquartile range 20–26 IU], median of total dose 1,700 IU). The volume of intracerebral haematoma was larger in the plasma-treated group compared to the PCC-treated group (haematoma, mean 64.5 vs 36.0 cm3; p=0.021). The unadjusted OR for all-cause 30-day mortality in the PCC group was 0.40 (95% confidence interval, 0.18–0.87; p=0.021) compared to the plasma group. After adjusting for the haematoma volume, bleeding localisation and age, the effect of PCC on mortality became non-significant. In conclusion, treatment with 4-factor PCC for VKA reversal in patients with intracerebral haemorrhage does not seem to reduce the 30-day all-cause mortality compared to plasma.

SummaryNewer oral anticoagulants offer several advantages over traditional agents (e.g. warfarin), but they are still associated with a bleeding risk and currently there is no validated reversal treatment for them. While there is little support for the use of fresh frozen plasma, and limited data available on the effects of activated recombinant factor VII, preclinical data suggest that prothrombin complex concentrates (PCCs) may have potential in this setting. PCCs are currently used to successfully reverse warfarin-induced anticoagulation; however, clinical evidence for their use with new oral anticoagulants is lacking, with most of the available data coming from preclinical animal studies. Furthermore, there appears to be variation in the ability of different PCCs to reverse the coagulopathy induced by the new anticoagulants, and a lack of correlation between the reversal of laboratory test results and the reversal of anticoagulant-induced bleeding. Although there have been encouraging results, care must be taken in generalising findings from animal models and nonbleeding human subjects to the situation in bleeding patients. Ultimately, more evidence supporting anticoagulation reversal for new anticoagulants is needed, particularly regarding the treatment of bleeding in human patients in a clinical setting. According to the current evidence, use of PCCs may be considered a reasonable approach in dire clinical situations; however, a consensus has not yet been reached regarding PCC use or dosing, due to lack of clinical data.

SummaryThe new direct-acting anticoagulants such as dabigatran and rivaroxaban are usually not monitored but may be associated with haemorrhage, particularly where renal impairment occurs. They have no effective “antidotes”. We studied 17 patients receiving dabigatran 150 mg twice daily for non-valvular atrial fibrillation and 15 patients receiving rivaroxaban 10 mg daily for the prevention of deep venous thrombosis after hip or knee replacement surgery. We assessed the effect of these drugs on commonly used laboratory tests and Calibrated Automated Thrombogram (CAT) using plasma samples. We also assessed effects in fresh whole blood citrated patient samples using thromboelastography on the TEG and the ROTEM. The efficacy of nonspecific haemostatic agents prothrombin complex concentrate (PCC), Factor VIII Inhibitor By-passing Activity (FEIBA) and recombinant activated factor VII (rVIIa) were tested by reversal of abnormal thrombin generation using the CAT. Concentrations added ex vivo were chosen to reflect doses normally given in vivo. Dabigatran significantly increased the dynamic parameters of the TEG and ROTEM and the lag time of the CAT. It significantly reduced the endogenous thrombin potential (ETP) and reduced the peak height of the CAT. Rivaroxaban did not affect the TEG and ROTEM parameters but did increase the lag time and reduce ETP and peak height of the CAT. For both drugs, these parameters were significantly and meaningfully corrected by PCC and FEIBA and to a lesser but still significant extent by rFVIIa. These results may be useful in devising a reversal strategy in patients but clinical experience will be needed to verify them.

SummaryAnticoagulants are effective at preventing and treating thrombosis, but can cause bleeding. For decades, vitamin K antagonists (VKAs) have been the only available oral anticoagulants. The development of non-VKA oral anticoagulants (NOACs), which inhibit either factor Xa or thrombin stoichiometrically, has provided alternatives to VKAs for several indications. The results of recent large-scale randomised controlled trials comparing NOACs with VKAs for the prevention of stroke in patients with non-valvular atrial fibrillation (AF) have produced some unexpected results. As a group, NOACs showed similar efficacy as warfarin, but a reduced risk of major bleeding. The reduction in bleeding with NOACs was greatest with intracranial hemorrhage. In contrast, the relative risk of gastro-intestinal bleeding was increased with some NOACs. In this review, we explore the potential mechanisms as well as the implications of these organ-specific bleeding patterns.

SummaryGlobal coagulation assays display variable effects at different concentrations of rivaroxaban. The aim of this study is to quantify the ex vivo effects of low-dose rivaroxaban on thrombophilia screening assays and coagulation factor activities based on the administration time, and to show how to mask possible interferences. Plasma samples from 40 patients receiving rivaroxaban 10 mg daily were investigated to measure activities of clotting factor II, V, VII, VIII, IX, XI, XII and XIII; protein C- and protein S-levels; lupus anticoagulants; anticardiolipin IgG and IgM; D-dimer, heparin-platelet factor 4 (HPF4) antibodies and screening tests for von Willebrand disease (VWD). Two hours after rivaroxaban administration, the activities of clotting factors were significantly decreased to different extents, except for factor XIII. Dilution of plasma samples resulted in neutralisation of these interferences. The chromogenic protein C activity assay was not affected by rivaroxaban. Depending on the timing of tablet intake in relation to blood sampling protein S activity was measured falsely high when a clotting assay was used. False-positive results for lupus anticoagulants were observed depending on the assay system used and the administration time of rivaroxaban. ELISA-based assays such as anticardiolipin IgG and IgM, D-dimer, HPF4-antibodies and the turbidimetric assays for VWD were not affected by rivaroxaban. Specific haemostasis clotting tests should be performed directly prior to rivaroxaban intake. Assay optimisation in the presence of rivaroxaban can be achieved by plasma dilution. Immunologic assays are not influenced by rivaroxaban, while chromogenic assays can be used, when they do not depend on factor Xa.

SummaryUrgent reversal of warfarin is required for patients who experience major bleeding or require urgent surgery. Treatment options include the combination of vitamin K and coagulation factor replacement with either prothrombin complex concentrate (PCC) or fresh frozen plasma (FFP). However, the optimal reversal strategy is unclear based on clinically relevant outcomes. We searched in MEDLINE, EMBASE and Cochrane library to December 2015. Thirteen studies (5 randomised studies and 8 observational studies) were included. PCC use was associated with a significant reduction in all-cause mortality compared to FFP (OR= 0.56, 95 % CI; 0.37–0.84, p=0.006). A higher proportion of patients receiving PCC achieved haemostasis compared to those receiving FFP, but this was not statistically significant (OR 2.00, 95 % CI; 0.85–4.68). PCC use was more likely to achieve normalisation of international normalised ratio (INR) (OR 10.80, 95 % CI; 6.12–19.07) and resulted in a shorter time to INR correction (mean difference –6.50 hours, 95 %CI; –9.75 to –3.24). Red blood cell transfusion was not statistically different between the two groups (OR 0.88, 95 % CI: 0.53–1.43). Patients receiving PCC had a lower risk of post-transfusion volume overload compared to FFP (OR 0.27, 95 % CI; 0.13–0.58). There was no statistically significant difference in the risk of thromboembolism following administration of PCC or FFP (OR 0.91, 95 % CI; 0.44–1.89). In conclusion, as compared to FFP, the use of PCC for warfarin reversal was associated with a significant reduction in all-cause mortality, more rapid INR reduction, and less volume overload without an increased risk of thromboembolic events.Supplementary Material to this article is available online at www.thrombosis-online.com.

SummaryCardiac surgery with cardiopulmonary bypass (CPB) is associated with blood loss and post-surgery thrombotic complications. The process of thrombin generation is disturbed during surgery with CPB because of haemodilution, coagulation factor consumption and heparin administration. We aimed to investigate the changes in thrombin generation during cardiac surgery and its underlying pro- and anticoagulant processes, and to explore the clinical consequences of these changes using in silico experimentation. Plasma was obtained from 29 patients undergoing surgery with CPB before heparinisation, after heparinisation, after haemodilution, and after protamine administration. Thrombin generation was measured and prothrombin conversion and thrombin inactivation were quantified. In silico experimentation was used to investigate the reaction of patients to the administration of procoagulant factors and/or anticoagulant factors. Surgery with CPB causes significant coagulation factor consumption and a reduction of thrombin generation. The total amount of prothrombin converted and the rate of prothrombin conversion decreased during surgery. As the surgery progressed, the relative contribution of α2-macroglobulin-dependent thrombin inhibition increased, at the expense of antithrombin-dependent inhibition. In silico restoration of post-surgical prothrombin conversion to pre-surgical levels increased thrombin generation excessively, whereas co-administration of antithrombin resulted in the normalisation of post-surgical thrombin generation. Thrombin generation is reduced during surgery with cardiopulmonary bypass because of a balance shift between prothrombin conversion and thrombin inactivation. According to in silico predictions of thrombin generation, this new balance increases the risk of thrombotic complications with prothrombin complex concentrate administration, but not if antithrombin is co-administered.

SummaryPaediatric obesity, like adulthood obesity, is associated with an increase of fibrinolysis inhibitors. No study, however, has evaluated the impact of these changes on plasma fibrinolytic capacity. We investigated plasma fibrinolysis and the role therein of the fibrinolytic changes associated with obesity in 59 obese children (body mass index > 95th percentile) and 40 matched controls. Fibrinolysis was investigated by measuring 1) the plasma levels of relevant fibrinolytic factors; 2) the in vitro fibrinolytic capacity under different conditions, using a microplate plasma clot lysis assay; 3) the circulating levels of markers of clotting and fibrinolysis activation. Plasminogen activator inhibitor 1 (PAI-1), total thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen levels were higher in obese children as compared to controls (p

SummarySeveral coagulation and fibrinolytic parameters appear to be affected by thyroid hormone excess;however, the net effect on the haemostatic system remains unclear. We aimed to update our previous review and systematically summarise and meta-analyse the data by assessing the effects of thyrotoxicosis on the coagulation and fibrinolytic system in vivo. Data sources included MEDLINE (2006–2012), EMBASE (2006–2012), and reference lists. The sources were combined with our previous search containing studies from 1980–2006. Eligible studies were all observational or experimental studies. Two investigators independently extracted data and rated study quality. Weighted mean proportion and 95% confidence intervals were calculated and pooled using a fixed and a random-effects model. A total of 29 articles consisting of 51 studies were included, as in several articles more than one study was described. We included four intervention (before and after treatment in hyperthyroid patients), five cross-sectional (hyperthyroid subjects and euthyroid controls), and four experimental (before and after use of thyroid hormone in euthyroid subjects) medium/high quality studies for meta-analysis. We found that thyrotoxicosis shifts the haemostatic balance towards a hypercoagulable and hypofibrinolytic state with a rise in factors VIII and IX, fibrinogen, von Willebrand factor, and plasminogen activator inhibitor-1. This was observed in endogenous and exogenous thyrotoxicosis, and in subclinical as well as overt hyperthyroidism. We conclude that both subclinical and overt hyperthyroidism induce a prothrombotic state, which is therefore likely to be a risk factor for venous thrombosis.

SummaryEdoxaban, an oral, direct factor Xa inhibitor, has a similar or low incidence of bleeding events compared with other anticoagulants in clinical trials. Therefore, agents to reverse the anticoagulant effects of edoxaban could be desirable in emergency situations. In this study, the reversal effects of haemostatic agents were determined on prothrombin time (PT) prolongation in vitro and bleeding time prolongation in vivo by edoxaban. PT using human plasma was measured in the presence of edoxaban at therapeutic and excess concentrations with the haemostatic agents, prothrombin complex concentrate (PPSB-HT), activated prothrombin complex concentrate (Feiba), and recombinant factor VIIa (rFVIIa). In rats, rFVIIa and Feiba was given during intensive anticoagulation with edoxaban. The haemostatic effect was evaluated in a model of planta template bleeding and a potential prothrombotic effect was evaluated in a venous thrombosis model. PPSB-HT, Feiba, and rFVIIa concentration-dependently shortened PT prolonged by edoxaban. Among these, rFVIIa and Feiba showed potent activities in reversing the PT prolongation by edoxaban. rFVIIa (1 and 3 mg/kg, i.v.) and Feiba (100 U/kg, i.v.) significantly reversed edoxaban (1 mg/kg/h)-induced prolongation of bleeding time in rats. In a rat venous thrombosis model, no potentiation of thrombus formation was observed when the highest dose (3 mg/kg) of rFVIIa was added to edoxaban (0.3 and 1 mg/kg/h) compared with the control. The present study indicated that rFVIIa, Feiba, and PPSB-HT have the potential to be reversal agents for edoxaban.

SummaryAs a potent anticoagulant agent, fondaparinux exposes a risk of bleeding. An effective way to reverse its effects is needed. It was the objective to study efficacy and safety of prothrombin complex concentrate (PCC) to reverse the anticoagulant effect of fondaparinux in a rabbit model of bleeding and thrombosis. In anaesthetised and ventilated rabbits, the Folts model was applied: a stenosis (75%) and an injury were carried out on the carotid artery, inducing thrombosis. Blood flow decreased as thrombus size increased until the pressure gradient was such that the thrombus was released and local blood flow was suddenly restored. This is known as a cyclic flow reduction (CFR). After the first CFR, rabbits were randomised into three groups: control (saline and saline after 1 minute), fondaparinux (fondaparinux [3 mg.kg−1] and saline), PCC (fondaparinux and PCC [40 UI.kg−1]). Then CFRs were recorded over 20 minutes. The following were measured: ear immersion bleeding time (BT), haemoglobin blood level (Hb1) and thrombelasto-metric parameters (ROTEM®). Finally, a hepatosplenic section was performed; 15 minutes later, the amount of blood loss was recorded as primary endpoint and Hb2 was measured. Blood loss was increased with fondaparinux and normalised with PCC. Regarding ROTEM® INTEM, fondaparinux increased clotting time and clotting formation time. PCC normalised these parameters. EXTEM and FIBTEM tests were not modified. Regarding safety, PCC did not increase CFRs. PCC reduced bleeding without increasing thrombosis and was effective to reverse the haemorrhagic effect of fondaparinux in this rabbit model.

SummaryProthrombin complex concentrates (PCCs) and fresh-frozen plasma (FFP) have been clinically used for acute warfarin reversal. The recovery of prothrombin time (PT) or international normalised ratio (INR) is often reported as an endpoint, but haemostatic efficacies of PCCs and FFP may not be fully reflected in static clotting test in platelet-poor plasma. Using various in vitro assays, we compared the effects of two PCC preparations (3-factor PCC; Bebulin and 4-factor PCC; Beriplex) and FFP on warfarin reversal under static and flow conditions. First, we added an aliquot of either PCC (0.3 or 0.72 U/ml) or 20% FFP (v/v) to commercial warfarin plasma (INR 3.2, or 10.3), and then measured PT, factor II, factor VII, and thrombin generation. Subsequently, we collected whole blood samples from six consented warfarin-treated patients with mean INR 3.0 ± 0.5 (range 2.5–3.7), and compared clot formation under flow conditions at 280 s-1 before and after addition of either PCC preparation (0.3 and 0.6 U/ml) or 20% of FFP (v/v). PT/INR were restored by either PCC in plasma with INR 3.0, but they were more effectively corrected by 4-factor PCC than 3-factor PCC in plasma with INR 10.3. Effects of FFP were similar to 0.3U/ml of PCCs in terms of PT, but FFP was less efficacious than PCCs in recovering thrombin generation or factor II levels. In flow experiments, the onset of thrombus formation was shortened by either PCC, but not by FFP, contrary to shortened PT values. For warfarin reversal 20% volume replacement with FFP is inferior to PCCs.

SummaryAlthough prothrombin complex concentrate (PCC) is increasingly used for the treatment of trauma-induced coagulopathy, few studies have investigated the impact and safety of PCC for this indication. The present study was performed to assess PCC for treatment of coagulopathy after blunt liver injury under severe hypothermia. Coagulopathy in 14 anaesthetised pigs was induced by haemodilution. Subsequently, standardised blunt liver injury was induced under severe hypothermia (32.8–33.2°C). Animals were randomised to receive either PCC (35 IU kg-1) or saline (control). Coagulation was assessed over the following 2 hours by thromboelastometry and thrombin generation. Internal organs were examined to determine presence of emboli. The administration of PCC showed a significant reduction in blood loss (p=0.002 vs. controls) and a significant increase in the rate of survival (p=0.022 vs. controls). Plasma thrombin generation in the PCC group increased considerably above baseline levels, with significant increases in peak thrombin levels and endogenous thrombin potential versus controls throughout the follow-up period. In addition, PT decreased significantly in the PCC group versus the control group. However, only slight improvements in thromboelastometry variables were observed. Histology showed an equal degree of liver injury in both groups, and no thromboembolism. In severely hypothermic pigs, the application of PCC corrected trauma-induced coagulopathy and reduced blood loss. Thus, the infusion of PCC might be a reasonable approach to reduce the need for blood cell transfusion in trauma. Furthermore, the impact and safety of PCC application can be monitored through thrombin generation and thromboelastometry under hypothermia.Note: This study was performed at the RWTH Aachen University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany.

SummaryThe development of alloantibodies that inhibit or neutralise the function of factor VIII is considered the most serious complication of the treatment of congenital haemophilia A. In order to describe their course without immune tolerance induction (ITI), we documented data on all performed inhibitor tests with dates as well as on clotting factor infusions of all consecutive patients who were treated in our centre between 1993 and 2006. Patients were tested every 7.1 months (95% confidence interval [CI], 6.6–7.8). A ‘sustained negative inhibitor status’ was defined as consistent non-positive inhibitor measurements for two years or longer. A total of 60/486 (12%) patients tested had a positive inhibitor titre in two or more occasions. Most of the patients (56%) with a maximum inhibitor titre of < 5 Bethesda unit (BU)/ml (named “low titre inhibitor”) developed a sustained negative inhibitor status. Among patients with high (5–9.9 BU/ml) and very high (≥ 10 BU/ ml) inhibitor titres, the proportions were 50% and 3%, respectively. Our findings suggest that ITI might not be needed for all patients with non-transient inhibitors, especially when their maximum inhibitor titre is below 10 BU/ml. Further studies in countries where ITI is not available are needed to examine predictors of the natural sustained negative inhibitor status.

Risk factors for suboptimal efficacy of 3-factor prothrombin complex concentrates in emergency VKA anticoagulation reversal

SummaryThe role of ethanol metabolism in possible haemostatic cardioprotective effects has not yet been determined. To this end, we investigated the effect of a moderate dose of ethanol (35 g) and its metabolism, on haemostatic variables over 14 hours (h). Eighteen Caucasian males participated in a placebo-controlled, randomised, cross-over study. Blood was collected prior to alcohol consumption, and at 10 time points for 14 h. Blood ethanol peaked at 1 h and was cleared after 8 h following ethanol consumption, significantly increasing plasma acetate (p=0.0028). Ethanol did not influence the coagulation factors significantly. PAI-1act increased (p

SummaryProlonged standing activates the coagulation cascade by the activation of endothelial cells, and probably the haemoconcentration effect contributes to this "orthostatic hypercoagulability". It was the objective of this study to assess whether rehydration (haemodilution) prevents or attenuates orthostatic induced thrombin formation. Twelve healthy young subjects were studied during two separate visits. Haematocrit (Hct), total plasma protein, coagulation profile tests, including endothelial activation related factors, and protein C global pathway were studied at rest supine, and while standing at 15 and 30 minutes (min). During the second visit the study was repeated after intravenous 1.5 liter 0.9% saline. While in supine posture, intravenous rehydration resulted in Hct reduction of 14.2 ± 2% (haemodilution), a decrease of 11.5 ± 1.3% in total protein, as well as a significant dilutional effect on most of the coagulation parameters. Still standing for 30 min, with and without rehydration caused a comparable increase in tissue factor by 49.83 ± 13.6%, and 35.34 ± 8.55% (p>0.05), respectively and in von Willebrand factor (vWF) 9.5 ± 2.4% and 13.59 ± 2.17% (p>0.05), respectively. At 30 min standing, after intravenous rehydration, factor V and VIII activities, and fibrinogen rose by 22 ± 1.9%, 31.2 ± 6.2%, 9.15 ± 2.64%, (p

SummaryHaemostatic reference intervals are generally based on samples from non-pregnant women. Thus, they may not be relevant to pregnant women, a problem that may hinder accurate diagnosis and treatment of haemostatic disorders during pregnancy. In this study, we establish gestational age-specific reference intervals for coagulation tests during normal pregnancy. Eight hundred one women with expected normal pregnancies were included in the study. Of these women, 391 had no complications during pregnancy, vaginal delivery, or postpartum period. Plasma samples were obtained at gestational weeks 13–20, 21–28, 29–34, 35–42, at active labor, and on postpartum days 1 and 2. Reference intervals for each gestational period using only the uncomplicated pregnancies were calculated in all 391 women for activated partial thromboplastin time (aPTT), fibrinogen, fibrin D-dimer, antithrombin, free protein S, and protein C and in a subgroup of 186 women in addition for prothrombin time (PT), Owren and Quick PT, protein S activity, and total protein S and coagulation factors II, V, VII, VIII, IX, X, XI, and XII. The level of coagulation factors II, V, X, XI, XII and antithrombin, protein C, aPTT, PT remained largely unchanged during pregnancy, delivery, and postpartum and were within non-pregnant reference intervals. However, levels of fibrinogen, D-dimer, and coagulation factors VII, VIII, and IX increased markedly. Protein S activity decreased substantially, while free protein S decreased slightly and total protein S was stable. Gestational age-specific reference values are essential for the accurate interpretation of a subset of haemostatic tests during pregnancy, delivery, and puerperium.

SummaryOver three decades after the generation of the first mouse monoclonal antibodies by Kohler and Milstein, recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. Technology improvements have provided several approaches to manufacturing human antibodies with high affinity for biologically relevant targets. Approximately 300 development programs for therapeutic antibodies have been reported in industrial and academic laboratories, and this clearly demonstrates the expectations towards antibody technology. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as one of the smallest possible format for recombinant antibodies and their use as diagnostic tools and therapeutic agents. We describe the structure, selection and production of single-chain antibodies. Furthermore, we review current applications of antibody fragments focusing on thrombus targeting using fibrin- and platelet-specific single-chain antibodies as well as describing novel noninvasive imaging approaches for the diagnosis of thrombosis and inflammation.

SummaryElevated procoagulant levels have been correlated with increased thrombin generation in vitro and with increased venous thromboembolism (VTE) risk in epidemiological studies. hrombin generation tests are increasingly being employed as a high throughput method to provide a global measure of procoagulant activity in plasma samples. The objective of this study was to distinguish the effects of assay conditions [tissue factor (TF), thrombomodulin, platelets/lipids] and factor levels on thrombin generation parameters, and determine the conditions and parameters with the highest sensitivity and specificity for detecting elevated factor levels. Thrombin generation was measured using calibrated automated thrombography (CAT) in corn trypsin inhibitor (CTI)-treated platelet-free plasma (PFP) and plateletrich plasma (PRP). Statistical analysis was performed using logarithms of observed values with analysis of variance that accounted for experiment and treatment. he relative sensitivity of lag time (LT), time to peak (TTP), peak height and endogenous thrombin potential (ETP) to elevated factors XI, IX,VIII, X, and prothrombin was as follows: PFP initiated with 1 pM TF > PFP initiated with 5 pM TF > PRP initiated with 1 pM TF. For all conditions, inclusion of thrombomodulin prolonged the LT and decreased the peak and ETP; however, addition of thrombomodulin did not increase the ability of CAT to detect elevated levels of individual procoagulant factors. In conclusion, CAT conditions differentially affected the sensitivity of thrombin generation to elevated factor levels. Monitoring the peak height and/ or ETP following initiation of clotting in PFP with 1 pM TF was most likely to detect hypercoagulability due to increased procoagulant factor levels.

SummaryHeparins, either unfractionated or low-molecular-weight (UFH and LMWHs), and vitamin K antagonists (VKAs) are currently the anticoagulants of choice for the prevention of post-operative venous thromboembolism (VTE) and for the treatment of acute venous and arterial thromboembolism. While VKAs are widely used in the US, LMWHs are the standard of care in the EU. Although efficacious, these agents are associated with a number of drawbacks, such as the risk of heparin-induced thrombocytopenia, the need for frequent coagulation monitoring in the case of UFH and VKAs, and the parenteral mode of administration in the case of heparins, which can lead to problems associated with patient compliance. There is a need for new anticoagulants that overcome these limitations. Direct, small-molecule inhibitors of coagulation proteins targeting a single enzyme in the coagulation cascade – particularly thrombin or Factor Xa – have been developed in recent years. Two agents, the direct thrombin inhibitor dabigatran and the direct Factor Xa inhibitor rivaroxaban, have recently been approved in the EU and several other countries for the prevention of VTE after total hip or knee replacement surgery. Here we will review data that suggest that the antithrombin-independent mechanism of action of these agents, particularly that of direct Factor Xa inhibitors, leads to increased efficacy with similar safety profiles compared with the antithrombin-dependent heparins. Although the end of the heparins era is not to be expected, the new anticoagulants presented in this review potentially represent the future of anticoagulation.

SummaryObesity and oral estrogens are independent risk factors for venous thrombosis, and their combined effect is stronger than the sum of the isolated factors. It was the objective of this study to investigate the interaction between obesity and estrogens at the level of venous thrombotic tendency, coagulation and inflammation in a mouse model.Female C57Bl/6J mice were fed a standard fat diet (SFD) or a high fat diet (HFD) to induce nutritional obesity.After 14 weeks, while maintaining their diet, mice were orally treated eight days with 1 µg ethinylestradiol or vehicle (n=25 per group), and subsequently subjected to an inferior caval vein (ICV) thrombosis model.The ICV thrombosis model resulted in an increased thrombus weight in vehicle-treated HFD mice (3.0 ± 0.7 mg) compared to vehicle-treated SFD mice (1.4 ± 0.4 mg; p=0.064). Surprisingly, estrogens reduced thrombus weight, which was significant for the HFD group (0.8 ± 0.5 mg; p=0.013).As compared to SFD feeding, HFD feeding significantly increased plasma levels of coagulation factor VIII, combined factor II/VII/X (p

SummaryA retrospective review was carried out on the methods of obstetric analgesia/anesthesia used in 80 pregnancies amongst 63 women with inherited bleeding disorders (19 factor XI deficiency, 16 carriers of haemophilia, 15 von Willebrand disease, seven platelet function disorders, four factor VII deficiency, one factor VII and XI deficiency and one factor X deficiency). In 72 pregnancies, the woman was seen antenatally in a multidisciplinary clinic to discuss and plan pain relief options. Regional block was performed for 41 pregnancies. The mothers were known to have a bleeding disorder in 35 of these pregnancies. Prophylactic cover was given in 10 pregnancies prior to the insertion of regional block but not required in the remaining 25 pregnancies because the coagulation defects had spontaneously normalised at term. There were six reported adverse effects from regional block similar to that found in the general population: inadequate anesthesia/analgesia (2), bloody tap (2), hypotension and a possible dural puncture which was treated conservatively. There were no reports of long-term complications. The findings show that it is possible to offer women with inherited bleeding disorders the option of regional block provided their coagulation defects have normalised, either spontaneously during pregnancy or following adequate haemostatic cover.

SummaryThe development of inhibitory antibodies to factor VIII is a serious complication of haemophilia. Two haemostatic agents with different bypassing mechanisms have been used in the treatment of patients with inhibitors: activated prothrombin complex concentrate (aPCC) and recombinant factor VIIa (rFVIIa). The objective was to compare cost and outcome of aPCC and rFVIIa in the treatment of joint bleeds. The analyses were based on the FENOC (FEIBA NovoSeven Comparative Study) crossover study where 48 patients used aPCC and rFVIIa to treat two joint bleeds. Incremental cost-effectiveness ratios were calculated for three outcome measures and the variation in cost was analyzed using two alternative regression methods. Results were subjected to sensitivity analyses. Key determinants of cost were prescribed dose, bodyweight and treatment in addition to protocol.The cost of aPCC was on average lower than rFVIIa. At all but one time point, patients rated slightly higher (but not statistically significantly) percentages of treatment efficacy and stopping of the bleed by aPCC. The reported reduction in pain from start of treatment up to 48 hours varied considerably among individuals. The different relative prices in the US, Turkey and Sweden mattered, but did not reverse the main results. In conclusion, the cost per episode was significantly lower for aPCC. The large individual-level variation in reduction of pain supports decisions that consider the individual patient’s experience and that accept trade-offs between cost and reduction in pain rather than focusing on cost only.

SummaryAcute inflammatory and chronic interstitial lung diseases are characterized by excessive and persistent fibrin deposition in the lung.Intraalveolar fibrin accumulation,observed under these conditions, arises from a leakage of plasma proteins (including fibrinogen) into the alveolar space in combination with a disbalance of alveolar haemostasis. Tissue factor in association with factor VIIa and inhibition of urokinase by plasminogen activator inhibitor-1 are major factors that are responsible for the procoagulant and antifibrinolytic state. In addition, in acute respiratory distress syndrome (ARDS) patients, factor VII-activating protease and extracellular RNA, which may be released into the extracellular milieu from damaged cells during lung injury, may contribute to fibrin formation as well. Fibrin itself can increase vascular permeability, influence the expression of inflammatory mediators and alter the migration and proliferation of various cell types. Additionally, fibrin may inactivate pulmonary surfactant and provide a matrix on which fibroblasts can migrate and produce collagen.Furthermore,cellular activities of haemostatic proteases may also contribute to proinflammatory and fibrotic processes in the lung.The application of coagulation inhibitors, like tissue factor pathway inhibitor, active site-inactivated factor VIIa, activated protein C, antithrombin, heparin or hirudin turned out to be beneficial in experimental models of acute and chronic lung injury. However, the ability of anticoagulant and profibrinolytic agents to improve clinical outcome remains to be elucidated. In the current article, the role of the alveolar coagulation and fibrinolysis systems in acute inflammatory and chronic interstitial lung diseases is discussed with regard to pathomechanisms and modalities of intervention.

SummaryAlterations in haemostasis are frequently observed in children with acute lymphoblastic leukemia (ALL). It was the objective of this study to analyse age-related disturbances in coagulation and fibrinolysis parameters during the induction phase of the anti-leukemic treatment. Sixty-four children were classified by age into three groups (1–5, 6–10, 11–16 years), and studied during induction treatment of ALL including four weeks of dexamethasone, followed by two weeks tapering of dexamethasone during which 6,000 IU/m2 native L-Asparaginase (total 4 doses) was administered intravenously twice weekly. Blood samples were collected immediately before each L-Asparaginase infusion to analyze procoagulant (fibrinogen, factor [F] II, FV, FVII, F IX, F X) and anticoagulant factors (antithrombin [AT], protein C, protein S), parameters of thrombin generation (F1+2, TAT) and fibrinolysis (α2-antiplasmin, plasminogen, PA P , D-dimer). Children were in a hypercoagulable state after four weeks of dexamethasone due to upregulation of coagulation parameters. Upregulation was highest in the two youngest age groups. During L-Asparaginase treatment the 11– to 16-year- olds showed lower values in procoagulant and, even more, in anticoagulant factor levels compared to the younger children. Activation markers of thrombin generation and fibrinolysis did not change over time during the study period. Decreased synthesis of α2-antiplasmin and plasminogen during L-Asparaginase treatment resulted in less potential of clot lysis by plasmin in children older than 11 years of age. In conclusion, a more severe decline of anticoagulant and fibrinolytic parameters in children between 11 and 16 years of age underline that these children are at higher risk of thrombosis during ALL induction treatment.