Intrinsic Pathway of Coagulation and Thrombosis.

Activation of the intrinsic pathway of coagulation contributes to the pathogenesis of arterial and venous thrombosis. Critical insights into the involvement of intrinsic pathway factors have been derived from the study of gene-specific knockout animals and targeted inhibitors. Importantly, preclinical studies have indicated that targeting components of this pathway, including FXI (factor XI), FXII, and PKK (prekallikrein), reduces thrombosis with no significant effect on protective hemostatic pathways. This review highlights the advances made from studying the intrinsic pathway using gene-specific knockout animals and inhibitors in models of arterial and venous thrombosis. Development of inhibitors of activated FXI and FXII may reduce thrombosis with minimal increases in bleeding compared with current anticoagulant drugs.

Over two decades of orthopaedic surgery in patients with inhibitors-Quantifying the complication of bleeding.

Patients with haemophilia who have developed inhibitors against factor VIII (FVIII) or factor IX present a significant concern to those surgeons who operate on them. The evidence base for bypassing agents such as recombinant factor VIIa and activated prothrombin complex concentrate has amassed over several decades. The literature is open to positive interpretation on the successful use of these agents in the treatment of inhibitor-positive patients. However, there are equally persistent concerns amongst surgeons, in particular orthopaedic surgeons, regarding the high complication rate of bleeding. To explore and quantify this concern, we present a literature review spanning two decades of publications on haemophilia patients with inhibitors undergoing orthopaedic surgery. Irrespective of the progress made with haemostatic protocols, trepidation on embarking on surgery is valid. The high risk of bleeding is a function of the inherent complexity of the disease and rightfully translates into difficulties in its management. Combined with the prospect of orthopaedic surgery, those involved in the care of such patients are justified in their continued anxiety and diligence when considering the benefits in quality of life against the prevalent complications.

Computer Design of Low-Molecular-Weight Inhibitors of Coagulation Factors.

The review discusses main approaches to searching for new low-molecular-weight inhibitors of coagulation factors IIa, Xa, IXa, and XIa and the results of such studies conducted from 2015 to 2018. For each of these factors, several inhibitors with IC50 < 10 nM have been found, some of which are now tested in clinical trials. However, none of the identified inhibitors meets the requirements for an "ideal" anticoagulant, so further studies are required.

Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2.

BACKGROUND/AIMS: The Kunitz Protease Inhibitor (KPI) domain of protease nexin 2 (PN2) potently inhibits coagulation factor XIa. Recombinant KPI has been shown to inhibit thrombosis in mouse models, but its clearance from the murine circulation remains uncharacterized. The present study explored the pharmacokinetic and pharmacodynamic effects of fusing KPI to human serum albumin (HSA) in fusion protein KPIHSA. METHODS: Hexahistidine-tagged KPI (63 amino acids) and KPIHSA (656 amino acids) were expressed in Pichia pastoris yeast and purified by nickel-chelate chromatography. Clearance profiles in mice were determined, as well as the effects of KPI or KPIHSA administration on FeCl3-induced vena cava thrombus size or carotid artery time to occlusion, respectively. RESULTS: Fusion to HSA increased the mean terminal half-life of KPI by 8-fold and eliminated its interaction with the low density lipoprotein receptor-related protein. KPI and KPIHSA similarly reduced thrombus size and occlusion in both venous and arterial thrombosis models when administered at the time of injury, but only KPI was effective when administered one hour before injury. CONCLUSIONS: Albumin fusion deflects KPI from rapid in vivo clearance without impairing its antithrombotic properties and widens its potential therapeutic window.

Strategies for inhibitor treatment and costs in the short and long term: a critical evaluation of recent clinical studies.

One important complication of patients with severe haemophilia A is the formation of inhibitory antibodies to factor VIII (FVIII). Immune tolerance induction (ITI) is the treatment of choice for patients with inhibitors, but this approach is successful in about 60% of patients. Treatment of acute bleeding in patients with inhibitors is one of the greatest challenges in haemophilia management and is costly. Bypassing agents are the mainstay of treatment in these patients. The aims of this study were to review the most recent publications concerning the costs of inhibitor treatment. We conducted a literature review using PubMed which yielded 63 papers analysing the costs of inhibitor management of which 12 were suitable for our study. Four of eight studies supported the use of activated prothrombin complex concentrate (aPCC) with lower costs, but the remaining four studies showed that recombinant factor VIIa (rFVIIa) had a lower average treatment cost. Of four ITI studies, two supported lifelong cost-effectiveness of ITI vs. bypassing agents and the remaining two papers showed a high cost of inhibitor treatment. Dosages, time between onset of bleeding and treatment, patient characteristics and the price of drugs are some of the important issues that should be considered for further studies.

A novel coagulation inhibitor from Schistosoma japonicum.

Little is known about the molecular mechanisms whereby the human blood fluke Schistosoma japonicum is able to survive in the host venous blood system. Protease inhibitors are likely released by the parasite enabling it to avoid attack by host proteolytic enzymes and coagulation factors. Interrogation of the S. japonicum genomic sequence identified a gene, SjKI-1, homologous to that encoding a single domain Kunitz protein (Sjp_0020270) which we expressed in recombinant form in Escherichia coli and purified. SjKI-1 is highly transcribed in adult worms and eggs but its expression was very low in cercariae and schistosomula. In situ immunolocalization with anti-SjKI-1 rabbit antibodies showed the protein was present in eggs trapped in the infected mouse intestinal wall. In functional assays, SjKI-1 inhibited trypsin in the picomolar range and chymotrypsin, neutrophil elastase, FXa and plasma kallikrein in the nanomolar range. Furthermore, SjKI-1, at a concentration of 7·5 µ m, prolonged 2-fold activated partial thromboplastin time of human blood coagulation. We also demonstrate that SjKI-1 has the ability to bind Ca(++). We present, therefore, characterization of the first Kunitz protein from S. japonicum which we show has an anti-coagulant properties. In addition, its inhibition of neutrophil elastase indicates SjKI-1 have an anti-inflammatory role. Having anti-thrombotic properties, SjKI-1 may point the way towards novel treatment for hemostatic disorders.

Complement Interception Across Humoral Incompatibility in Solid Organ Transplantation: A Clinical Perspective.

The humoral barrier in transplant biology is the result of preformed donor-specific antibodies (DSAs), directed either against human leukocyte antigens (HLA) or non-HLA antigens such as blood group (ABO) molecules. The term "sensitization" applies to patients carrying these antibodies. Transplantation is widely accepted as a life-saving opportunity for patients with terminal end-organ disease. However, in sensitized patients, transplant outcome is hampered by antibody-mediated rejection (AMR) as a consequence of DSA exposure. Furthermore, sensitized patients have limited access to "matched" organs from the both living and deceased donor pool.Considering the crucial role of the complement system in the pathophysiology of AMR and the availability of complement intervention therapeutics, there is a growing interest in complement-targeting strategies. This review highlights the emerging importance of monitoring and modulation of the complement system in the context of enabling transplantation across humoral incompatibility in sensitized recipients with preformed anti-HLA or natural anti-ABO antibodies. It also discusses the significance of the complement system in the induction of accommodation and further emphasizes current and future perspectives of novel complement therapeutics.

Acquired functional coagulation inhibitors: review on epidemiology, results of a wet-workshop on laboratory detection, and implications for quality of inhibitor diagnosis.

The accurate detection and quantification of coagulation inhibitors remains a challenging problem for most diagnostic laboratories. Prolonged screening assays and abnormal results of mixing tests with normal plasma may indicate the presence of such inhibitors. Yet, the presence of lupus anticoagulant, heparin, and potential contamination of plasma with therapeutically active antithrombotic drugs has to also be ruled out. This review covers the epidemiology of acquired functional coagulation inhibitors, and reports the results of a wet-workshop, organized by the External Quality Control for Assays and Test (ECAT) Foundation, on laboratory detection of such inhibitors. The aim of the workshop was to investigate, within groups of experts from dispersed professional laboratories, the quality of inhibitor detection and the difficulties encountered during the analytical process. In this workshop 8 samples representing varying milieu were tested by 10 groups of participants from 20 different countries. Workshop participants were asked to report the results of all investigations performed and to provide a likely diagnosis and/or a conclusion of the hemostasis abnormality represented by the test samples. Generally, the sensitivity of inhibitor detection was high but the differential diagnosis of the type of inhibitors identified was unsatisfactory, as many false-positive and false-negative results were observed. The most remarkable observation was the lack of a clear step-by-step analysis of the nature of an inhibitor once a positive mixing test had been detected. The possible consequences of these observations for the appropriate diagnosis and clinical management of patients are outlined. A diagnostic algorithm for the differential diagnosis and confirmation of acquired coagulation inhibitors is presented.

Acquired inhibitors of coagulation factors: part I-acquired hemophilia A.

Acquired hemophilia A (AHA) is a rare, but often severe, bleeding disorder caused by autoantibodies against clotting factor VIII (FVIII). AHA occurs more frequently in the elderly and in association with several conditions, such as malignancies, autoimmune diseases, postpartum, or drug exposure; however, about half of the cases remain idiopathic. At variance with congenital hemophilia, where hemarthroses are the most common bleeding symptoms, hemorrhages in AHA involving soft tissues (muscle, skin) are more frequently reported. AHA is diagnosed in patients: with negative personal or family bleeding history; in which prolonged activated partial thromboplastin time is not corrected after mixing and incubating equal volumes of patient and normal plasma for ~2 hours at 37°C; FVIII levels are reduced; and a specific FVIII-inhibiting activity is detected. Prompt recognition and treatment of AHA are mandatory, as inadequate management and complications of the disease are associated with high mortality rates. Therapeutic approaches should aim to control acute bleeds, eradicate FVIII-autoantibody production, treat associated diseases, and when possible, eliminate them. Present knowledge about this often overlooked and challenging condition has significantly increased following establishment of recent national and international studies, as will also be reviewed in this article.

Acquired inhibitors of coagulation factors: part II.

Acquired coagulation inhibitors are antibodies that bind to coagulation factors and neutralize their activity or accelerate their clearance. Inhibitors occurring in patients with inherited deficiencies of coagulation factors are referred to as "alloantibodies," while those developing spontaneously in individuals with previously normal coagulation factor function are designated as "autoantibodies." The latter category includes inhibitors against coagulation factors I, II, V, VII, VIII (acquired hemophilia A), IX (acquired hemophilia B), X, XI, and XIII. This review will discuss the most important pathogenic, clinical, laboratory, and therapeutic aspects of the inhibitors of coagulation factors other than acquired hemophilia A, as this is reviewed separately within this issue of the journal.

Aptamer-based modulation of blood coagulation.

Nucleic acid based aptamers are single-stranded oligonucleotide ligands isolated from random libraries by an in-vitro selection procedure. Through the formation of unique three-dimensional structures, aptamers are able to selectively interact with a variety of target molecules and are therefore also promising candidates for the development of anticoagulant drugs. While thrombin represents the most prominent enzymatic target in this field, also aptamers directed against other coagulation proteins and proteases have been identified with some currently being tested in clinical trials. In this review, we summarize recent developments in the design and evaluation of aptamers for anticoagulant therapy and research.

Therapeutic recombinant murine activated protein C attenuates pulmonary coagulopathy and improves survival in murine pneumococcal pneumonia.

BACKGROUND: Recombinant human activated protein C (APC) improves survival of patients with severe sepsis; this beneficial effect is especially apparent in patients with pneumococcal pneumonia. The aim of this study was to determine the effect of APC treatment initiated after induction of pneumococcal pneumonia on pulmonary coagulation, inflammation, and survival, with or without concurrent antibiotic therapy. METHODS: Mice were infected intranasally with viable Streptococcus pneumoniae and were treated intraperitoneally after 24 h of infection with vehicle, recombinant mouse (rm) APC (125 µg), ceftriaxone (500 µg), or rm-APC plus ceftriaxone. Treatment with rm-APC or vehicle was repeated every 8 h for a maximum of 96 h. Animals were either killed 48 h after infection or were monitored in a survival study (with an extra dose of ceftriaxone given after 72 h). RESULTS: Rm-APC treatment inhibited pulmonary activation of coagulation, as reflected by lower levels of thrombin-antithrombin complexes and D-dimer. Rm-APC did not affect the pulmonary levels of 55 inflammatory mediators in the context of antibiotic therapy. Rm-APC added to ceftriaxone markedly improved survival, compared with ceftriaxone treatment alone. CONCLUSIONS: Rm-APC inhibits pulmonary activation of coagulation and, when added to antibiotic therapy, improves survival in murine pneumococcal pneumonia.

Inhibitor development in mild haemophilia after a major surgery for periampullary cancer (Whipple's procedure) in an elderly man.

Around the world, with the availability of factor concentrates, patients with haemophilia have undergone major and minor surgeries. Inhibitor development in early postoperative period leading to inadequate factor recovery and ongoing bleeding is a nightmare for both operating surgeon as well as haematologists. We describe a case of an elderly man with mild haemophilia A, who was diagnosed with pancreatic carcinoma and underwent Whipple's procedure. After an uneventful procedure, he developed high-titre inhibitors and bleeding a week after surgery posing major challenges in his management. The case highlights the importance of experienced surgeons, trained haematologists, regular monitoring of factor assay/inhibitors, adequate factor and bypassing-agent support while performing such procedures.

Hematin-derived anticoagulant. Generation in vitro and in vivo.

Prolongation of clotting times produced by hematin was investigated both in vitro and in vivo. Hematin-derived anticoagulant (HDA) was found to be due to a degradative product or derivative of hematin, and was generated in vitro in standing (aging) aqueous solutions of the parent compound. Generation of HDA in vitro was inhibited by antioxidants. The anticoagulant effect of HDA was inhibited by freshly prepared hematin, fresh Sn-protoporphyrin, imidazole, or the iron chelator desferrioxamine. Ferrioxamine did not inhibit HDA, and inhibition by imidazole was reversed with ferric citrate, suggesting a role for iron in the mechanism of HDA activity. HDA activity was dissociated from hematin in plasma by clotting with thrombin. HDA segregated into the clot fraction, whereas hematin remained largely in the serum fraction, suggesting that HDA may preferentially bind to fibrinogen. TLC and HPLC showed a single peak of HDA activity that was not associated with the parent compound. Evidence for HDA generation in vivo was found when rats were injected with fresh (no HDA) hematin. Prolongation of clotting times appeared after hematin appeared in the plasma, and anticoagulant activity persisted after a fall in plasma hematin concentration. Thus, there was a temporal dissociation between hematin and HDA, suggesting that a modification of hematin must occur in vivo before an anticoagulant effect is produced. Generation of HDA in vitro has implications for hematin preparation and administration. Generation of HDA in vivo suggests that similar modifications of endogenous heme or other porphyrins may occur to produce HDA under physiologic or pathophysiologic conditions.

Anticoagulant-active sulfated arabinogalactan from Chaetomorpha linum: Structural characterization and action on coagulation factors.

A sulfated polysaccharide from the green alga Chaetomorpha linum, designated CLS4, was isolated by water extraction, anion-exchange and size-exclusion chromatography. Chemical and spectroscopic analyses demonstrated that CLS4 was a sulfated arabinogalactan, which was constituted by (1→6)-ß-d-galactopyranose and (1→5)-α-l-arabinofuranose residues with sulfate groups at C-2/ C-3 of (1→5)-α-l-arabinofuranose and C-2/C-4 of (1→6)-ß-d-galactopyranose. CLS4 possessed strong anticoagulant activity in vitro or in vivo as evaluated by activated partial thromboplastin time and thrombin time assays. CLS4 largely inhibited the activities of the coagulation factors XII, XI, IX and VIII. CLS4 was a potent thrombin inhibitor mediated by antithrombin III (ATIII) or heparin cofactor II, and it also effectively stimulated the factor Xa inhibition by potentiating ATIII. Moreover, CLS4 had a high thrombolytic activity in vitro as assessed by clot lytic rate assay. The results suggested that CLS4 could be a promising source of anticoagulant agent.

Systemic adenoviral gene delivery to orthotopic murine breast tumors with ablation of coagulation factors, thrombocytes and Kupffer cells.

BACKGROUND: Rapid clearance of adenoviruses from blood by macrophage lineage cells of the liver and spleen, and binding to platelets, hinder their successful systemic use for cancer gene therapy. Vitamin K dependent coagulation factors are important mediators for the adenovirus liver tropism. Here we aim to determine the effects of coagulation factor, thrombocyte and liver macrophage (Kupffer cell) ablation on biodistribution of serotype 5 adenoviruses in mice with orthotopic breast tumors. METHODS: Prior to intravenous injection of adenoviruses, mice bearing orthotopic breast tumors were pretreated with warfarin to inhibit vitamin K dependent coagulation factor synthesis, an anti-platelet antibody causing thrombocytopenia or an inhibitor of the Kupffer cell scavenger receptor or their combination. Virus availability in blood after injection was determined from blood samples and transgene expression in tissues analyzed 72 hours afterwards with In Vivo Imaging and luciferase assays. RESULTS: Warfarin pretreatment reduced gene delivery to liver, spleen and lung. Kupffer cell ablation increased persistence of adenoviruses in blood but didn't affect biodistribution significantly. Depletion of Kupffer cells combined with thrombocytopenia doubled the systemic gene delivery of 5/3 chimeric adenovirus to tumors (p < 0.05). Triple ablation of platelets, Kupffer cells and coagulation factors increased the tumor to liver ratio of systemic adenovirus gene delivery by 81% (p < 0.05). CONCLUSIONS: Depletion of coagulation factors can reduce transduction of liver, spleen and lung. Kupffer cell depletion is the most feasible method of increasing amount adenovirus in systemic blood flow and in combination with ablation of thrombocytes can increase the transduction of adenovirus to tumors.

Single-chain antibodies as diagnostic tools and therapeutic agents.

Over 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.

Correction of coagulopathy in warfarin associated cerebral hemorrhage.

PURPOSE OF REVIEW: Warfarin is the most commonly used oral anticoagulant. Intracranial hemorrhage is the most serious complication of anticoagulation and the anticoagulant effect of warfarin has to be urgently reversed in this situation. Traditional methods of reversal of the anticoagulant effect of warfarin involving the use of vitamin K and fresh frozen plasma are slow and relatively ineffective and there is a need for alternative treatment approaches. RECENT FINDINGS: Agents such as prothrombin complex conjugates and recombinant activated factor VII are being increasingly used to emergently correct warfarin-associated coagulopathy. Over the last decade, several small case series have suggested that these agents may lead to more rapid correction of the INR, however, improved clinical outcome is yet to be proven. A recent small prospective trial has also demonstrated the safety of a prothrombin complex conjugate and its efficacy in rapidly correcting an elevated INR in these patients. SUMMARY: There is a need for well designed randomized clinical trials aimed at evaluating the efficacy of these agents in improving the outcome of patients with anticoagulant associated intracranial hemorrhage.

New anticoagulants.

PURPOSE OF REVIEW: Although current anticoagulants such as unfractionated and low-molecular-weight heparins and the vitamin K antagonists are effective for the prevention and treatment of thrombosis, they have several limitations. The vitamin K antagonists, the only approved oral anticoagulants, have a narrow therapeutic window, thereby requiring regular laboratory monitoring of the international normalized ratio and intermittent adjustments in dose. New anticoagulants have been developed that selectively inhibit thrombin or factor Xa, and have predictable dose-response relationships. RECENT FINDINGS: Fondaparinux is a synthetic pentasaccharide, which binds to antithrombin, thereby indirectly inhibiting factor Xa. Fondaparinux demonstrated efficacy compared with low-molecular-weight heparin in randomized clinical trials and is approved for the prevention and treatment of venous thromboembolism. A number of oral direct factor Xa inhibitors and oral direct thrombin inhibitors are in advanced phases of clinical development for the prevention and treatment of thrombosis. The current status of these anticoagulants will be reviewed along with the challenges faced in designing pivotal clinical trials of these agents in comparison to existing anticoagulants. SUMMARY: Selective inhibitors of specific coagulation factors have the potential to be more effective, safer, and easier to use than existing anticoagulants. Approval of one or more of these agents will lead to an improved drug armamentarium for the prevention and treatment of thrombosis.