Pharmacology of Heparin and Related Drugs: An Update.

Heparin has been used extensively as an antithrombotic and anticoagulant for close to 100 years. This anticoagulant activity is attributed mainly to the pentasaccharide sequence, which potentiates the inhibitory action of antithrombin, a major inhibitor of the coagulation cascade. More recently it has been elucidated that heparin exhibits anti-inflammatory effect via interference of the formation of neutrophil extracellular traps and this may also contribute to heparin's antithrombotic activity. This illustrates that heparin interacts with a broad range of biomolecules, exerting both anticoagulant and nonanticoagulant actions. Since our previous review, there has been an increased interest in these nonanticoagulant effects of heparin, with the beneficial role in patients infected with SARS2-coronavirus a highly topical example. This article provides an update on our previous review with more recent developments and observations made for these novel uses of heparin and an overview of the development status of heparin-based drugs. SIGNIFICANCE STATEMENT: This state-of-the-art review covers recent developments in the use of heparin and heparin-like materials as anticoagulant, now including immunothrombosis observations, and as nonanticoagulant including a role in the treatment of SARS-coronavirus and inflammatory conditions.

Multiplex genome editing of mammalian cells for producing recombinant heparin.

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.

Laboratory coagulation tests and recombinant porcine factor VIII: A United Kingdom Haemophilia Centre Doctors' Organisation guideline.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare bleeding disorder caused by development of auto-antibodies to endogenous coagulation factor VIII (FVIII). Recombinant porcine factor VIII (rpFVIII) is currently licensed only for the management of bleeding in patients with AHA. Regular monitoring of rpFVIII is recommended to assess treatment effectiveness. AIM: This guideline from the United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO) examines the current publications in the area and aims to offer advice for the laboratory monitoring of rpFVIII in patients with AHA. METHODS: A review of the current literature was undertaken followed by critical review by the authors. RESULTS/CONCLUSIONS: A validated one-stage clotting FVIII assay is recommended for the measurement and regular monitoring of rpFVIII. Assessment of cross-reacting rpFVIII inhibitors by one-stage porcine Bethesda assay should be performed as part of the initial diagnosis of AHA or prior to treatment with rpFVIII. Available data show that chromogenic FVIII assays underestimate rpFVIII and this should be considered if measurement of rpFVIII is required in patients receiving Emicizumab.

Enabling accurate measurement of activated factor XI (FXIa) in therapeutic immunoglobulin products.

BACKGROUND AND OBJECTIVES: In 2010, an intravenous immunoglobulin (IVIG) product was removed from the market due to an association with serious thromboembolic events. Investigations revealed that factor XIa (FXIa) was present as a process-related impurity. This study investigated the ability of two commercial FXIa assays to measure FXIa in immunoglobulin preparations and conducted a survey of FXIa activity in marketed immunoglobulin products. MATERIALS AND METHODS: Factor XIa assays were modified to include spiking of samples with FXIa before testing. An immunoglobulin product and its excipient were used to assess the ability of the assays to recover the spiked FXIa levels. RESULTS: The Biophen FXIa assay required a high pre-dilution of the sample to obtain statistically valid results and complete FXIa recovery. The ROX FXIa assay was more sensitive, giving statistically valid results at a lower sample pre-dilution and FXIa spike level. This modified ROX FXIa assay was used to assay 17 lots of immunoglobulin products for FXIa. Two product lots had measurable FXIa levels without the need for spiking. A further 3 lots produced detectable but not statistically valid FXIa results when left unspiked. Spiking produced statistically valid assays and recoveries above 100%, demonstrating inherent FXIa. CONCLUSION: This study shows marketed immunoglobulin products can contain detectable levels of FXIa. Spiking brings the FXIa levels into the quantifiable range of the assay, allowing measurement of inherent FXIa. Accurate measurement is important to inform on 'safe' levels of FXIa in these products and allow future safety guidelines to be set.

Activity measurements of dalcinonacog alfa.

INTRODUCTION: Many recombinant and modified FIX products have been, and continue to be, developed with the aim of improving treatment for patients with haemophilia B. One such new product is dalcinonacog alfa, a recombinant FIX with modifications to provide improved features such as subcutaneous administration. AIM: In view of previously observed assay discrepancies with modified FIX therapeutics, the aim of this study was to assess potential discrepancies in potency measurement of dalcinonacog alfa between and within different assay methods. METHODS: Potency of dalcinonacog alfa was measured against the 5th International Standard (IS) for FIX Concentrate and the 4th IS for FIX Plasma by One-Stage Clotting Assay, using 9 different APTT reagents and 2 commercially available FIX chromogenic kits. Plasma-derived concentrate and recombinant FIX samples were also included for comparison in every assay. RESULTS: Substantial discrepancies were observed when assaying dalcinonacog alfa using the one-stage clotting assay against both standards. No statistically valid results were obtained when testing dalcinonacog alfa using either chromogenic kit. Increasing the incubation time with the activation reagent in both chromogenic kits resulted in valid assays and increased the potency to become more in line with potencies by one-stage clotting assays. Increasing the incubation time in the chromogenic kits had no effect on the potencies of the plasma-derived or recombinant samples. However, incubation time influenced in the one-stage clotting assay using Dapttin. CONCLUSIONS: Within and between assay method discrepancy was found when assaying dalcinonacog alfa. Methods for potency labelling and clinical monitoring should be given careful consideration.

Laboratory measurement of factor replacement therapies in the treatment of congenital haemophilia: A United Kingdom Haemophilia Centre Doctors' Organisation guideline.

Assay discrepancies can occur with laboratory monitoring of FVIII and FIX replacement therapy, particularly for the extended half-life products. This guideline collates current published data and provides advice on appropriate choice of assays for laboratory measurement of replacement therapy for patients with Haemophilia A and B without inhibitors. It is recommended that each haemophilia centre should ensure that appropriate laboratory assays are available for FVIII and FIX products in local clinical use. Patient samples should be assayed against calibrators traceable to WHO Plasma International Standards. Assay discrepancies are common especially for the extended half-life FVIII and FIX products, and assays of these products may need to be verified with the specific CFC being used.

Laboratory coagulation tests and emicizumab treatment A United Kingdom Haemophilia Centre Doctors' Organisation guideline.

INTRODUCTION: The factor VIII mimetic emicizumab (Hemlibra, Hoffman-la Roche, Basel, Switzerland) has a novel mode of action that affects the laboratory monitoring of patients receiving this treatment. AIM: This guideline from the United Kingdom Haemophilia Centre Doctors Organisation (UKHCDO) aims to provide advice for clinical and laboratory staff on appropriate use of laboratory assays in patients with Haemophilia A treated with emicizumab. METHODOLOGY: The guideline was prepared by a review of the available literature and discussion and revision by the authors. RESULTS: The guideline describes the effect of emicizumab on commonly used coagulations tests and provides recommendations on the use of assays for measurement of factor VIII and factor VIII inhibitor in the presence of emicizumab. The guideline also provides recommendations on measurement of emicizumab. CONCLUSION: Knowledge of the effect of emicizumab on coagulation tests and factor assays is required to ensure appropriate testing and monitoring of therapy in patients receiving this drug.

Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products.

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

Pharmacology of Heparin and Related Drugs.

Heparin has been recognized as a valuable anticoagulant and antithrombotic for several decades and is still widely used in clinical practice for a variety of indications. The anticoagulant activity of heparin is mainly attributable to the action of a specific pentasaccharide sequence that acts in concert with antithrombin, a plasma coagulation factor inhibitor. This observation has led to the development of synthetic heparin mimetics for clinical use. However, it is increasingly recognized that heparin has many other pharmacological properties, including but not limited to antiviral, anti-inflammatory, and antimetastatic actions. Many of these activities are independent of its anticoagulant activity, although the mechanisms of these other activities are currently less well defined. Nonetheless, heparin is being exploited for clinical uses beyond anticoagulation and developed for a wide range of clinical disorders. This article provides a "state of the art" review of our current understanding of the pharmacology of heparin and related drugs and an overview of the status of development of such drugs.

Potency estimates for recombinant factor IX in the one-stage clotting assay are influenced by more than just the choice of activated partial thromboplastin time reagent.

The activated partial thromboplastin time (APTT) one-stage clotting (OSC) assay is used to potency label factor IX (FIX) therapeutics and to monitor the treatment of deficient patients. Studies have highlighted differences in potency estimates for FIX therapeutics depending on what activator type the APTT reagent contains. During the study to replace the 4th International Standard (IS) for FIX Concentrate, it was noted that for the recombinant FIX (rFIX) candidates, a potency difference of 20% was obtained by some between laboratories using the reagent DAPTTIN. This study was designed to investigate this discrepancy. Two plasma-derived (pdFIX) and 2 rFIX materials were assayed against the 4th IS for FIX concentrate (07/182) using the OSC assay. Three different APTT reagents were used (DAPTTIN, SynthAFax and SynthASil), plus 4 different activation times. The pdFIX samples were not affected by activation time or APTT reagent, with expected potencies recovered in all assays and at all activation times. For rFIX, expected potencies were recovered using SynthASil, but SynthAFax generated potencies around 20% lower. This was consistent across the activation times. For DAPTTIN, potencies for rFIX dropped progressively and by up to 30% as activation time increased from 120 to 300 seconds. This study demonstrates that activation time as well as choice of APTT reagent can result in discrepancies in the potency estimation of rFIX. These factors should be taken into account when assaying rFIX.

Calibration of the 7th British Working Standard for factors II, IX and X, concentrate.

Seven laboratories from 5 different countries participated in the calibration of the 7th British Working Standard (BWS) for blood coagulation factors II, IX and X. The candidate, 15/182, was assayed for Factors II and X potencies against the 4th International Standard (IS) for Factors II and X, Concentrate (11/126) and for Factor IX potency against the 5th IS for Factor IX, Concentrate (14/148). Intra-laboratory GCVs for all 3 factors were less than 10%, with the majority less than 5%. Inter-laboratory GCVs were 3.4%, 3.2% and 2.3% for FII, IX and X respectively. All participants agreed with the value assigned and preparation 15/182 was established by NIBSC in October 2017 as the 7th BWS for FII, IX, X Concentrate with potencies of 6.0 IU/ampoule, 6.7 IU/ampoule and 4.9 IU/ampoule for FII, IX and X respectively.

Biochemical and functional characterization of glycosaminoglycans released from degranulating rat peritoneal mast cells: Insights into the physiological role of endogenous heparin.

The properties of commercially prepared heparin as an anticoagulant and antithrombotic agent in medicine are better understood than is the physiological role of heparin in its native form, where it is uniquely found in the secretory granules of mast cells. In the present study we have isolated and characterised the glycosaminoglycans (GAGs) released from degranulating rat peritoneal mast cells. Analysis of the GAGs by NMR spectroscopy showed the presence of both heparin and the galactosaminoglycan dermatan sulphate; heparinase digestion profiles and measurements of anticoagulant activity were consistent with this finding. The rat peritoneal mast cell GAGs significantly inhibited accumulation of leukocytes in the rat peritoneal cavity in response to IL-1ß (p < 0.05, n = 6/group), and inhibited adhesion and diapedesis of leukocytes in the inflamed rat cremasteric microcirculation in response to LPS (p < 0.001, n = 4/group). FTIR spectra of human umbilical vein endothelial cells (HUVECs) were altered by treatment of the cells with heparin degrading enzymes, and restored by the addition of exogenous heparin. In conclusion, we have shown that rat peritoneal mast cells contain a mixture of GAGs that possess anticoagulant and anti-inflammatory properties.

pH-Responsive, Thermoset Polymer Coatings for Active Protection against Aluminum Corrosion.

This paper describes the synthesis and use of multifunctional methacrylic monomers, which contain basic (amine) functional groups, including an example in which an acid-labile tert-butylcarbamate-protected glycine is used to form a novel methacrylic monomer. The "protected" amino acid-derived functional monomer (BOC-Gly-MA) is copolymerized with an epoxide functional methacrylic monomer (GMA), to deliver novel multifunctional polymers, which are processed into powder coatings and used to study filiform corrosion at the surface of an aluminum substrate. The BOC-Gly-MA-containing copolymers were shown to improve a coating's anticorrosion performance, presenting the lowest average filiform corrosion (FFC) track length, total FFC number, and total corroded surface area (CSA) of the coatings investigated. Further to this, a mode of action for the role of BOC-Gly functional polymers in corrosion protection is proposed, supported by both solution and polymer-aluminum interface studies, delivering new insights into the mode of action of pH-responsive polymer coatings.

Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

Heparin, Heparan Sulphate and Sepsis: Potential New Options for Treatment.

Sepsis is a life-threatening hyperreaction to infection in which excessive inflammatory and immune responses cause damage to host tissues and organs. The glycosaminoglycan heparan sulphate (HS) is a major component of the cell surface glycocalyx. Cell surface HS modulates several of the mechanisms involved in sepsis such as pathogen interactions with the host cell and neutrophil recruitment and is a target for the pro-inflammatory enzyme heparanase. Heparin, a close structural relative of HS, is used in medicine as a powerful anticoagulant and antithrombotic. Many studies have shown that heparin can influence the course of sepsis-related processes as a result of its structural similarity to HS, including its strong negative charge. The anticoagulant activity of heparin, however, limits its potential in treatment of inflammatory conditions by introducing the risk of bleeding and other adverse side-effects. As the anticoagulant potency of heparin is largely determined by a single well-defined structural feature, it has been possible to develop heparin derivatives and mimetic compounds with reduced anticoagulant activity. Such heparin mimetics may have potential for use as therapeutic agents in the context of sepsis.

High Resolution Imaging of the Outer Retina in Type 2 Acute Macular Neuroretinopathy.

PURPOSE: To investigate the outer retinal changes in a patient with type 2 acute macular neuroretinopathy (AMN). METHODS: A 35-year-old Caucasian female complaining of a unilateral blind spot was imaged using various retinal imaging modalities including clinical optical coherence tomography (OCT), OCT-angiography, fundus fluorescein angiography and adaptive optics (AO). RESULTS: Fundus examination revealed multiple paracentral reddish-brown petaloid lesions in the symptomatic left eye, while the other eye was unremarkable. Clinical OCT showed areas of hyper-reflectance at the outer plexiform layer / outer nuclear layer complex with a disrupted inner /outer segment junction, which are characteristic features of type 2 AMN. AO imaging further revealed either shortening or absence of cone outer segments within the AMN lesions attributing to the darker features observed in the enface images from fundus photography and scanning laser ophthalmoscopy. CONCLUSION: The AO findings indicate that the petaloid lesions in type 2 AMN are caused by a combination of the shortening and absence of the outer segment in individual cone photoreceptors.

Foveal cone loss in tamoxifen maculopathy: a case report.

BACKGROUND: Tamoxifen is used in low dose concentrations (20-40 mg per day) as a therapy for breast cancer but is known to have ocular side effects. In this case report, the foveal cone integrity in a tamoxifen-treated patient who complained of a small central scotoma in the left eye while reading was examined using high resolution adaptive optics imaging. CASE PRESENTATION: Both eyes of a 54-year-old Caucasian, non-hispanic female who had been treated with tamoxifen for 1.5 years were examined using various imaging modalities including fundus photography, fundus autofluorescence, fluorescein angiography, spectral-domain optical coherence tomography, and adaptive optics scanning laser ophthalmoscopy. Clinical spectral-domain optical coherence tomography showed a very small disruption to the photoreceptor layer at the fovea in the left eye only. However, adaptive optics scanning laser ophthalmoscopy imaging revealed foveal cone loss in both eyes, but to a lesser extent in the right eye. Inner retinal changes were not observed in either eye. CONCLUSION: The area of cone loss was similar in size to a single newsprint letter when projected onto the retina, matching the patient's description of a scotoma in the left eye. Given the isolated loss of foveal cone photoreceptors with the absence of previously reported inner retinal and vascular changes, our results may indicate the earliest retinal changes associated with tamoxifen retinopathy.

Differences in wild-type- and R338L-tenase complex formation are at the root of R338L-factor IX assay discrepancies.

Adeno-associated virus (AAV) gene therapy has the potential to functionally cure hemophilia B by restoring factor (F)IX concentrations into the normal range. Next-generation AAV therapies express a naturally occurring gain-of-function FIX variant, FIX-Padua (R338L-FIX), that increases FIX activity (FIX:C) by approximately eightfold compared with wild-type FIX (FIX-WT). Previous studies have shown that R338L-FIX activity varies dramatically across different clinical FIX:C assays, which complicates the monitoring and management of patients. To better understand mechanisms that contribute to R338L-FIX assay discrepancies, we characterized the performance of R338L-FIX in 13 1-stage clotting assays (OSAs) and 2 chromogenic substrate assays (CSAs) in a global field study. This study produced the largest R338L-FIX assay dataset to date and confirmed that clinical FIX:C assay results vary over threefold. Both phospholipid and activating reagents play a role in OSA discrepancies. CSA generated the most divergent FIX:C results. Manipulation of FIX:C CSA kits demonstrated that specific activity gains for R338L-FIX were most profound at lower FIX:C concentrations and that these effects were enhanced during the early phases of FXa generation. Supplementing FX into CSA had the effect of dampening FIX-WT activity relative to R338L-FIX activity, suggesting that FX impairs WT tenase formation to a greater extent than R338L-FIX tenase. Our data describe the scale of R338L-FIX assay discrepancies and provide insights into the causative mechanisms that will help establish best practices for the measurement of R338L-FIX activity in patients after gene therapy.

Standardisation of unfractionated and low-molecular-weight heparin.

Unfractionated and low-molecular-weight heparins are complex biologicals. Standardisation and global harmonisation of units and methods of measurement are essential for safety and efficacy of this important class of anticoagulants. This chapter describes the traceability of the international unit and current status of the relationship between the international and pharmacopoeial standards, together with a review on current pharmacopoeial assay methods.