Coagulation is a physiological process necessary to achieve homeostasis. Many pathologies lead to spontaneous activation of the coagulation pathways and increase the risk of venous thrombosis (e.g., atrial fibrillation, orthopaedic surgery, cancer). Therefore, a lot of patients need anticoagulant drugs as preventive or curative treatment. In general, older molecules (unfractionated heparin, low-molecular-weight heparins, vitamin K antagonists) have good efficacy. Still, their adverse reactions, increased risk of bleeding, or difficult administration led to low adherence to treatment and had even limited their use. Recently, new molecules were authorised to improve patient adherence to treatment, mainly formulated for oral administration (e.g., dabigatran, rivaroxaban, apixaban, etc.). This therapeutic approach has a low risk of bleeding and does not require special monitoring by laboratory tests. Also, new anticoagulants for patients with heparin-induced thrombocytopenia (e.g., argatroban, lepirudin, bivalirudin, etc.) were obtained. Moreover, reversal agents for the new anticoagulant molecules used in overdoses or in situations where immediate cessation of the anticoagulant effect is required (e.g., emergency surgery) were studied, some of them being authorised on the pharmaceutical market. This narrative review aims to provide a pharmacological and therapeutic overview of anticoagulant drugs, underlining their implementation and limitations.

Synthetic analogues of mitochondrial DNA (mtDNA) have been reported as potent complement system activators. This study investigated the impact of endogenous mtDNA on complement activation. mtDNA and nuclear DNA (nDNA) were extracted from human placental tissue and complement activation was determined using ELISA. When incubated in lepirudin-anticoagulated human blood and plasma, increasing concentrations of mtDNA, but not nDNA, resulted in dose- and time-dependent increases in C3bc, C3bBbP and soluble C5b-9 (sC5b-9). In a clinical context, mtDNA, nDNA, and complement levels of 55 resuscitated out-of-hospital cardiac arrest (OHCA) patients were determined using qPCR and ELISA. C3bc and sC5b-9 correlated significantly with mtDNA and nDNA in OHCA patients. We conclude that mtDNA, but not nDNA, triggers in vitro complement activation, which holds significance in a clinical context in OHCA patients. This novel mode of human complement activation might explain pathophysiological mechanisms of sterile inflammation and could be relevant for innovative therapeutic strategies.

Type II heparin-induced thrombocytopenia (HIT) is an immune-mediated reaction that usually develops 5-10 days after heparin administration and is mediated by antibodies against a neoantigen of heparin and platelet factor 4 complex. The condition is strongly associated with thrombosis, such as pulmonary embolism. Diagnosis of HIT in patients who underwent liver resection is a challenge compared to other surgical patients. We present the case of a chronic carrier of hepatitis B who presented with a single hepatocellular carcinoma (HCC) in the right lobe. After undergoing right lobectomy without blood transfusion, he was provisioned with low-molecular-weight heparin (LMWH), which was tolerated well. However, after a sudden decrease in platelet count and the onset of tachycardia and shortness of breath, the patient was found to have a partially occluded right pulmonary artery due to pulmonary embolism. After initiating lepirudin, it was discontinued due to unwanted side effects. Thus, fondaparinux was administered, which proved to be effective in increasing the platelet number and resolving the pulmonary embolus. A daily monitoring of anti-factor Xa time was applied using RECALMIX (Amax-Accuclott Heptest, Trinity Biotech, Jamestown, New York, United States). During the first week of fondaparinux administration, initial platelet count values began returning to normal values with resolution of the pulmonary embolus. The patient was discharged in stable condition. This is the first report that shows effective maintenance anticoagulation with fondaparinux of a patient with cirrhosis and post-hepatectomy HIT syndrome complicated by pulmonary embolism. Further studies are required to establish fondaparinux as a new alternative anticoagulant in surgical patients with HIT.

Complement activation plays a critical role in the inflammatory response to Escherichia coli and Aspergillus fumigatus conidia. However, the specific contributions of complement components, including anaphylatoxin receptors, remain unclear. Using an ex vivo lepirudin whole blood model, we examined the activation of all three complement pathways (C4c, C3bc, and sC5b-9) induced by these microbes. We also assessed granulocyte and monocyte receptor expression of CD11b, CD64, C3aR, C5aR1, and C5aR2, along with phagocytosis, leukocyte activation (MPO), and cytokine release. Additionally, we investigated selective inhibition of complement components FD, C3, C5, and C5aR1. Both microbes increased complement activation products (C3bc and sC5-9), CD11b and CD64 expression, MPO release, and proinflammatory cytokines (IL-1β, IL-6, IL-8, and TNF), while decreasing C3aR, C5aR1, and C5aR2 expression. Complement inhibition reduced CD11b and CD64 expression and partially restored C3aR and C5aR1 levels, with minimal effects on C5aR2. FD, C3, and C5 inhibition reduced downstream complement markers, with FD and C3 inhibition also reducing phagocytosis, and only C3 inhibition reducing MPO release. The cytokine response varied by microbe: E. coli triggered higher proinflammatory cytokines, and FD and C3 inhibition generally reduced cytokine release, while C5 inhibition was less effective. Interestingly, A. fumigatus-induced cytokines significantly increased with C5aR1 inhibition, highlighting immune response differences related to C5aR1 signalling in bacterial versus fungal infections. In conclusion, regulation of inflammation through FD, C3, C5, and C5aR1 underscores the immunoregulatory role of the complement system in anti-microbial immune responses.

Complement analysis necessitates strict control of pre-analytical blood handling, including time, temperature, and additives. Here, we compared complement function and activation status across five different serum preparations and two plasma preparations. Serum was collected from ten healthy volunteers using glass tubes without additives, tubes with a silica clot activator (with or without a gel separator), and tubes containing thrombin (with or without a gel separator). Plasma was collected in the presence of EDTA or the thrombin inhibitor lepirudin. Serum and plasma aliquots were snap-frozen in liquid nitrogen and stored at -80 °C. Complement functional analysis was performed using Wieslab and Hycult Biotech pathway-specific assays. Complement activation was determined by quantifying specific activation markers: C1s/C1-INH, MASP-1/C1-INH, C3bc, C3bBbP, and sC5b-9. All serum samples exhibited increased complement activation compared to EDTA and lepirudin plasma, with serum tubes containing thrombin and gel separators showing the highest levels of complement activation. However, normal complement function was observed in all serum preparations, indicating that the complement activation and consumption that occurred did not affect complement functional analysis. While all tested serum tubes provided accurate functional activity, the type of tube and the presence of additives like thrombin and gel separators significantly influenced the degree of complement activation. We recommend preparing functionally active serum either in glass tubes or in silica clot activator tubes, and avoiding gel separators. For complement activation studies, lepirudin plasma is preferable over serum due to its complement functional capacity, low level of complement activation, and lack of excessive hemostatic activation.

The currently available point-of-care hemostasis tests are burdened by criticisms concerning the use of different activators and inhibitors and the lack of dynamic flow. These operating conditions may constitute an impediment to the determination of the patient's hemostatic condition. Hence, the diffusion of these tests in clinical practice is still limited to specific scenarios. In this work, we present a new method for analyzing the patient's global hemostasis based on the visualization of the main components of the coagulation process and its computerized quantitative image analysis. The automated "Smart Clot" point-of-care system presents a micro-fluidic chamber in which whole blood flows, without the addition of any activator or inhibitor. In this micro-channel, platelet adhesion, activation and aggregation to the type I collagen-coated surface take place (primary hemostasis), leading to the production of endogenous thrombin on the surface of platelet aggregates and the consequent fibrin mesh and thrombus formation (secondary hemostasis). These observations are verified by inhibiting primary hemostasis with the antiplatelet drugs Indomethacin (-70% on platelet aggregation, -60% on fibrin(ogen) formation) and Tirofiban (complete inhibition of platelet aggregation and fibrin(ogen) formation) and secondary hemostasis with the antithrombin drugs Heparin (-70% on platelet aggregation, -80% on fibrin(ogen) formation) and Lepirudin (-80% on platelet aggregation, -90% on fibrin(ogen) formation). Smart Clot, through a single test, provides quantitative results concerning platelet aggregation and fibrin formation and is suitable for undergoing comparative studies with other coagulation point-of-care devices.

Intravenous direct thrombin inhibitors (DTIs) are used for thromboembolic disorders. This systematic review aims to characterize intravenous DTI agents, dosing, monitoring strategies (or use), bleeding, and mortality, in pediatric patients with acute venous thromboembolism (VTE) or heparin-induced thrombocytopenia with thrombosis (HITT). MEDLINE, Embase, and Cochrane's CENTRAL were searched from inception through July 2023. Case series, retrospective studies, and prospective studies providing per-patient or summary data for patients < 18 years of age with VTE or HITT treated with an intravenous DTI were included. Selection and data extraction were conducted independently by two reviewers. Sixteen studies (7 case reports, 1 case series, 5 retrospective studies, 3 prospective studies) with 85 patients were included. Target conditions included acute VTE in 54 (64%) and HITT in 31 (36%) patients. Bivalirudin, argatroban, and lepirudin were used in 52 (61%), 27 (32%), and 6 (7%) patients, respectively. Fifty-two (61%) patients received a bolus dose, and weighted mean infusion rates for bivalirudin, argatroban, and lepirudin were 0.2 mg/kg/hr, 1.2 mcg/kg/min, and 0.15 mg/kg/hr, respectively. The activated partial thromboplastin time was utilized for monitoring in 82 (96%) patients. Complete or partial thrombus resolution was reported in 53 (62%) patients, mortality in 6 (7%) patients, and bleeding complications in 14 (16%) patients. In this systematic review involving 85 pediatric patients treated with an intravenous DTI for acute VTE or HITT, bivalirudin was the most commonly utilized agent, with a rate of resolution over 60% despite a high acuity in the population studied. Prospective collaborative studies are warranted to establish optimal dosing and further characterize VTE and bleeding outcomes.

Current treatment options for acute ischemic stroke, including intravenous thrombolysis (IVT) and mechanical thrombectomy, have undoubtedly revolutionized stroke care. The need for additional treatment options has brought into the light direct thrombin inhibitors (DTIs) and, specifically, argatroban as a promising candidate. However, there is uncertainty regarding the safety of adding argatroban to IVT, mainly due to the increased hemorrhagic risk. In this study, we performed a systematic review and meta-analysis examining the safety and efficacy of argatroban as an add-on treatment for IVT. The following databases were searched from inception until the 14th of May 2023: Pubmed/MEDLINE, ClinicalTrials.gov, the EU Clinical Trials Register, EMBASE/Scopus, and the Cochrane Library. Only randomized clinical trials (RCTs) enrolling patients with acute ischemic stroke who underwent IVT evaluating the add-on use of any DTIs were selected for the systematic review and further meta-analysis. The PRISMA guidelines were followed at all stages. Four studies with argatroban were included in the final analysis. Analysis of risk ratio and relative risk shows that the add-on therapy with argatroban seems to be effective and favors a good clinical outcome (mRS 0-2) at 90 days, similar to that of alteplase. All studies showed a low pooled incidence of symptomatic intracerebral hemorrhage (5%), parenchymal hematoma (3%), and other major bleeding (1%). Argatroban as an add-on treatment to IVT seems not to be associated with excessive bleeding risk; however, its efficacy remains unproven. According to this synopsis of the currently available evidence, it is premature to use argatroban as an add-on to IVT treatment outside the current clinical trial setting.

The lepirudin-based human whole blood model is a well-established ex vivo system to characterize inflammatory responses. However, the contribution of individual cell populations to cytokine release has not been investigated. Thus, we modified the model by selectively removing leukocyte subpopulations to elucidate their contribution to the inflammatory response. Lepirudin-anticoagulated whole blood was depleted from monocytes or granulocytes using StraightFrom Whole Blood MicroBeads. Reconstituted blood was incubated with Escherichia coli (108/mL) for 2 hours at 37 °C. CD11b, CD62P, and CD63 were detected by flow cytometry. Complement (C3bc, sC5b-9) and platelet activation (platelet factor 4, NAP-2) were measured by enzyme-linked immunosorbent assay. Cytokines were quantified by multiplex assay. A significant (P < 0.05) specific depletion of the monocyte (mean = 86%; 95% confidence interval = 71%-92%) and granulocyte (mean = 97%; 95% confidence interval = 96%-98%) population was obtained. Background activation induced by the depletion protocol was negligible for complement (C3bc and sC5b-9), leukocytes (CD11b), and platelets (NAP-2). Upon Escherichia coli incubation, release of 10 of the 24 cytokines was solely dependent on monocytes (interleukin [IL]-1β, IL-2, IL-4, IL-5, IL-17A, interferon-γ, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-1α, and fibroblast growth factor-basic), whereas 8 were dependent on both monocytes and granulocytes (IL-1ra, IL-6, IL-8, IL-9, IL-10, macrophage inflammatory protein-1β, tumor necrosis factor, and eotaxin). Six cytokines were not monocyte or granulocyte dependent, of which platelet-derived growth factor and RANTES were mainly platelet dependent. We document an effective model for selective depletion of leukocyte subpopulations from whole blood, without causing background activation, allowing in-depth cellular characterization. The results are in accordance with monocytes playing a major role in cytokine release and expand our knowledge of the significant role of granulocytes in the response to E. coli.

Although intravenous (IV) direct thrombin inhibitors (DTI) have gained interest in pediatric extracorporeal membrane oxygenation (ECMO), dosing and safety information is limited. The objective of this systematic review was to characterize DTI types, dosing, monitoring, and outcomes (bleeding and thromboembolic) in pediatric ECMO patients managed with IV DTIs. We conducted searches of MEDLINE (Ovid) and Embase (Elsevier) from inception through December 2022. Case reports, retrospective studies, and prospective studies providing per-patients or summary data for patient(s) <18 years of age receiving IV DTI for ECMO anticoagulation were included. Study selection and data extraction were conducted independently by two reviewers. A total of 28 studies: 14 case reports, 13 retrospective studies, and 1 prospective study were included, totaling 329 patients. Bivalirudin was utilized in 318 (96.7%), argatroban in 9 (2.7%), and lepirudin in 2 (0.6%) patients. Infusion dosing included: bivalirudin 0.14 ± 0.37 mg/kg/h, argatroban 0.69 ± 0.73 µg/kg/min, lepirudin 0.14 ± 0.02 mg/kg/h. Laboratory monitoring tests utilized were the activated clotting time, activated partial thromboplastin time (aPTT), diluted thrombin time, and thromboelastography measures. The aPTT was utilized in most patients (95%). Thromboembolism, bleeding, or death were observed in 17%, 17%, and 23% of bivalirudin, argatroban, and lepirudin patients, respectively. Bivalirudin appears to be the most frequently used DTI in pediatric ECMO. Dosing and laboratory monitoring varied, and bleeding and thromboembolic events were reported in 17% of patients. Prospective studies are warranted to establish dosing, monitoring, safety, and efficacy of bivalirudin and other IV DTI in pediatric ECMO.

Lepirudin is no longer marketed in the United States. Limited information indicates that lepirudin in doses up to 100 mg daily produce very low levels in milk. Because of its large molecular weight, it would not be expected to be absorbed from breastmilk by the infant. Lepirudin would not be expected to cause any adverse effects in breastfed infants, especially if the infant is older than 2 months.[1]

Heparin-induced thrombocytopenia type II (HIT II), as stated in the literature, occurs in about 3% of all patients and in 0.1-5% of surgical patients. Thrombosis develops in 20-64% of patients with HIT. The mortality rate in HIT II has not decreased using non-heparin treatment with anticoagulants such as argatroban and lepirudin. An improved understanding of the pathophysiology of HIT may help identify targeted therapies to prevent thrombosis without subjecting patients to the risk of intense anticoagulation. The review will summarize the current knowledge about the pathogenesis of HIT II, potential new therapeutic targets related to it, and new treatments being developed. HIT II pathogenesis involves multi-step immune-mediated pathways dependent on the ratio of PF4/heparin and platelet, monocyte, neutrophil, and endothelium activation. For years, only platelets were known to take part in HIT II development. A few years ago, specific receptors and signal-induced pathways in monocytes, neutrophils and endothelium were revealed. It had been shown that the cells that had become active realised different newly formed compounds (platelet-released TF, TNFα, NAP2, CXCL-7, ENA-78, platelet-derived microparticles; monocytes-TF-MPs; neutrophils-NETs), leading to additional cell activation and consequently thrombin generation, resulting in thrombosis. Knowledge about FcγIIa receptors on platelets, monocytes, neutrophils and FcγIIIa on endothelium, chemokine (CXCR-2), and PSGL-1 receptors on neutrophils could allow for the development of a new non-anticoagulant treatment for HIT II. IgG degradation, Syk kinase and NETosis inhibition are in the field of developing new treatment possibilities too. Accordingly, IdeS and DNases-related pathways should be investigated for better understanding of HIT pathogenesis and the possibilities of being the HIT II treatment targets.

Iron oxide nanoparticles (IONPs) are widely used in diagnostic and therapeutic settings. Upon systemic administration, however, they are rapidly recognized by components of innate immunity, which limit their therapeutic capacity and can potentially lead to adverse side effects. IONPs were previously found to induce the inflammatory response in human whole blood, including activation of the complement system and increased secretion of cytokines. Here, we investigated the thromboinflammatory response of 10-30 nm IONPs in lepirudin anticoagulated whole blood in interplay with endothelial cells and evaluated the therapeutic effect of applying complement inhibitors to limit adverse effects related to thromboinflammation. We found that IONPs induced complement activation, primarily at the C3-level, in whole blood incubated for up to four hours at 37°C with and without human microvascular endothelial cells. Furthermore, IONPs mediated a strong thromboinflammatory response, as seen by the significantly increased release of 21 of the 27 analyzed cytokines (p<0.05). IONPs also significantly increased cell-activation markers of endothelial cells [ICAM-1 (p<0.0001), P/E-selectin (p<0.05)], monocytes, and granulocytes [CD11b (p<0.001)], and platelets [CD62P (p<0.05), CD63 (p<0.05), NAP-2 (p<0.01), PF4 (p<0.05)], and showed cytotoxic effects, as seen by increased LDH (p<0.001) and heme (p<0.0001) levels. We found that inflammation and endothelial cell activation were partly complement-dependent and inhibition of complement at the level of C3 by compstatin Cp40 significantly attenuated expression of ICAM-1 (p<0.01) and selectins (p<0.05). We show that complement activation plays an important role in the IONPs-induced thromboinflammatory response and that complement inhibition is promising in improving IONPs biocompatibility.