Extrinsic Coagulation Pathway Research Articles

Purification of an Acidic Polysaccharide with Anticoagulant Activity from the Marine Sponge Sarcotragus spinosulus.

Thromboembolic conditions are the most common cause of death in developed countries. Anticoagulant therapy is the treatment of choice, and heparinoids and warfarin are the most adopted drugs. Sulphated polysaccharides extracted from marine organisms have been demonstrated to be effective alternatives, blocking thrombus formation by inhibiting some factors involved in the coagulation cascade. In this study, four acidic glycan fractions from the marine sponge Sarcotragus spinosulus were purified by anion-exchange chromatography, and their anticoagulant properties were investigated through APTT and PT assays and compared with both standard glycosaminoglycans and holothurian sulphated polysaccharides. Moreover, their topographic localization was assessed through histological analysis, and their cytocompatibility was tested on a human fibroblast cell line. A positive correlation between the amount of acid glycans and the inhibitory effect towards both the intrinsic and extrinsic coagulation pathways was observed. The most effective anticoagulant activity was shown by a highly charged fraction, which accounted for almost half (about 40%) of the total hexuronate-containing polysaccharides. Its preliminary structural characterization, performed through infrared spectroscopy and nuclear magnetic resonance, suggested that it may consist of a fucosylated chondroitin sulphate, whose unique structure may be responsible for the anticoagulant activity reported herein for the first time.

Impact of tissue factor expression and administration routes on thrombosis development induced by mesenchymal stem/stromal cell infusions: re-evaluating the dogma.

Hyperactive coagulation might cause dangerous complications such as portal vein thrombosis and pulmonary embolism after mesenchymal stem/stromal cell (MSC) therapy. Tissue factor (TF), an initiator of the extrinsic coagulation pathway, has been suggested as a predictor of this process. The expression of TF and other pro- and anticoagulant genes was analyzed in xeno- and serum-free manufactured MSCs. Furthermore, culture factors affecting its expression in MSCs were investigated. Finally, coagulation tests of fibrinogen, D-dimer, aPPTs, PTs, and TTs were measured in patient serum after umbilical cord (UC)-MSC infusions to challenge a potential connection between TF expression and MSC-induced coagulant activity. RESULTS: Xeno- and serum-free cultured adipose tissue and UC-derived MSCs expressed the highest level of TF, followed by those from dental pulp, and the lowest expression was observed in MSCs of bone marrow origin. Environmental factors such as cell density, hypoxia, and inflammation impact TF expression, so in vitro analysis might fail to reflect their in vivo behaviors. MSCs also expressed heterogeneous levels of the coagulant factor COL1A1 and surface phosphatidylserine and anticoagulant factors TFPI and PTGIR. MSCs of diverse origins induced fibrin clots in healthy plasma that were partially suppressed by an anti-TF inhibitory monoclonal antibody. Furthermore, human umbilical vein endothelial cells exhibited coagulant activity in vitro despite their negative expression of TF and COL1A1. Patients receiving intravenous UC-MSC infusion exhibited a transient increase in D-dimer serum concentration, while this remained stable in the group with intrathecal infusion. There was no correlation between TF expression and D-dimer or other coagulation indicators. The study suggests that TF cannot be used as a solid biomarker to predict MSC-induced hypercoagulation. Local administration, prophylactic intervention with anticoagulation drugs, and monitoring of coagulation indicators are useful to prevent thrombogenic events in patients receiving MSCs. Trial registration NCT05292625. Registered March 23, 2022, retrospectively registered, https://www. gov/ct2/show/NCT05292625?term=NCT05292625&draw=2&rank=1 . NCT04919135. Registered June 9, 2021, https://www. gov/ct2/show/NCT04919135?term=NCT04919135&draw=2&rank=1 .

P146 Latent activation of the extrinsic coagulation pathway in patients with ulcerative colitis

Abstract Background Ulcerative colitis (UC) is associated with an increased risk of venous thromboembolism. We investigated the correlation of UC severity with latent activation of the extrinsic coagulation pathway and colonic stromal tissue factor (TF) expression. Methods Plasma exosomes, microparticles (EM), TF protein, activities of EM-bound TF (EM-TF) and FVIIa, as well as TF mRNA (F3) in primary colonic stromal cells in culture were measured. Disease extent by Montreal classification, medications, UC clinical activity by partial Mayo score (PMS), endoscopic severity by the MS endoscopic component (eMS; loss of response defines as ≥2), quality of life by the short IBD questionnaire- SIBDQ, complete blood count, CRP, ESR and albumin were retrieved. Statistical significance (p) was assessed with the following tests: Student’s t for 2 groups with normally distributed values, Mann-Whitney U if not, Wilcoxon test for paired data. Correlation of continuous against scale variables was assessed with Pearson’s or Spearman’s r and corresponding p values for linear or logarithmic continuous variables, respectively. Results Plasma was obtained from 38 UC patients (31 males, disease duration 151±25 months, 14 with extensive colitis, 5 current smokers) and 28 healthy blood donors (HBD; 20 males) who served as controls. TF mRNA (F3) was measured in primary colonic stromal cells in culture from 12 UC patients (11 males, disease duration 169±53 months, 8 with extensive colitis, 1 current smoker) and from 7 controls undergoing screening colonoscopy. UC patients had 4- and 3.7- times more exosomes and microparticles than controls, respectively (A). TF protein correlated with PMS (r 0.443), albumin (-0.362), ESR (0.353), PLT (0.575) and endoscopic UC severity (0.468; B). EM-TF activity was 1.6-times higher (C) and correlated to disease extent (E3: 1.9 x E2), albumin (-0.624), endoscopic severity (0.422; D) and SIBDQ (-0.64). Downstream, FVIIa activity was 2.2-times higher. Refractory-to-treatment patients had 5.8-, 1.5-, 2.1- times higher TF protein (E), MP-TF, FVIIa activity, respectively. Within responders, the need for steroids or biologics correlated with 2.2-times higher MP-TF activity. Colonic stromal cells from patients with UC maintained a 2.2-times higher F3 (F), which correlated to PMS (0.56), albumin (Spearman’s r -0.543), endoscopic severity (M3: 8.3 x M2). All the aforementioned were statistically significant with a < 0.05. Conclusion The extent of spontaneous activation of the extrinsic coagulation pathway is associated with clinical and endoscopic UC activity and response to treatment. TF in colonic stromal cells mirrors its systemic activity and indicates the possible contribution of such cells to increased TF plasma bioactivity.

Bioactive glass incorporated dressing matrix for rapid hemostatic action with antibacterial activity

Uncontrolled bleeding stands as a leading cause of preventable death in both civilian trauma and military battlefield scenarios. Existing hemostatic dressings like HemCon, Celox, and QuickClot, often lack instant hemostasis at the bleeding site and may have biodegradability and exothermic issues. To address the above in this communication, we have synthesized a hemostatic glass (acronym AlBG, CaO-SiO2 system, incorporated with Al2O3 and ZnO) by sol gel route and carried out detail physicochemical characterizations, e.g., XRD, FESEM, FTIR, BET and particle size analysis etc. The AlBG of particle size range 140–253 nm was incorporated into the nonwoven surgical cotton gauze to obtain AlBGscg. In vitro biological assays including cytocomtability, hemocompatibility assays of the above coated gauze was undertaken using NIH3T3 of which, the later showed optimum hemocompatibility with <6 % lysis of red blood cells. Potent antibacterial action on both gram positive and gram negative strains were obtained with significant zone of inhibition of 17 ± 0.34 mm and 16 ± 0.56 mm, respectively. Importantly, significant reduction of the clotting time (31.81 ± 0.12 % for P time and 33.3 ± 0.23 % for aPTT) compared to the control group, indicated activation of both intrinsic and extrinsic coagulation pathways. Scanning electron microscope (SEM) images exhibited an enhanced in vitro blood clot formation in case of AlBGscg in comparison to the control (uncoated surgical cotton gauze), visually. Additionally, a noticeable reduction of ∼30 % in the time required for the blood clot formation, in case of AlBGscg, was observed. The in vivo hemostatic potential of AlBGscg was evaluated in Wistar rats through femoral artery injury and was compared with commercially available surgical cotton gauze. The results demonstrated substantial reduction in the time required for hemostasis (approximately 50 % reduction), a lower amount of blood loss (7.33 % with AlBGscg compared to 22.31 % with surgical cotton gauze), and importantly, there were no incidents of re-bleeding observed with AlBGscg, highlighting its promise as a candidate for effective hemostasis.

Sulfated polysaccharides from the viscera of Mustelus shark: Characterization and antioxidant, anticoagulant and anti-proliferative activities

In the present study, sulfated polysaccharides (SPs) from smooth hound shark were extracted using different precipitation agents, the cetylpyridinium chloride (CPC) or ethanol, named SHSP-I and SHSP-II, respectively. The UV–visible scan demonstrated the absence of nucleic acids or proteins in the extracted SHSPs. The antioxidant, anticoagulant and anti-proliferative activities were also investigated. Results showed that SHSPs displayed reducing and scavenging capacities, as shown by the oxygen radical absorbance capacity, the total antioxidant capacity and the hydroxyl radical-scavenging activities. Moreover, extracted SPs were able to prolong the activated partial thromboplastin time (aPTT), the prothrombin time (PT) and the thrombin time (TT) and they reduced the fibrinogen level (FL), without inducing erythrocytes hemolysis, which indicated that they inhibited the intrinsic and extrinsic coagulation pathways and the thrombin-mediated fibrin formation. The SHSP-I was more effective on prolonging the blood clotting time, by more than 120 s at 1 mg/ml in the aPTT and about 70 s and 60 s at 2.5 mg/ml in the PT and TT, respectively, compared to the SHSP-II. The anti-proliferative activity of SHSPs was tested against the growth of K562 human myelogenous leukemia cells and Caco-2 colon cancer cells. Data showed that both polysaccharides have a dose-dependent inhibitory effect, where SHSP-I exhibited the strongest activity against the tested cells and the K562 was more sensitive than Caco-2 line cells to the action of SHSPs. The overall data suggested that SPs from shark viscera could be useful as natural nutraceutical agents.

Chitosan nonwoven fabric composited calcium alginate and adenosine diphosphate as a hemostatic bandage for acute bleeding wounds

Acute bleeding following accidental injury is a leading cause of mortality. However, conventional hemostatic bandages impede wound healing by inducing excessive blood loss, dehydration, and adherence to granulation tissue. Strategies such as incorporating active hemostatic agents and implementing chemical modifications can augment the properties of these bandages. Nevertheless, the presence of remote thrombosis and initiators may pose risks to human health. Here, a hemostatic bandage was developed by physically combined chitosan nonwoven fabric, calcium alginate sponge, and adenosine diphosphate. The presented hemostatic bandage not only exhibits active and passive mechanisms for promoting clotting but also demonstrates excellent mechanical properties, breathability, ease of removal without causing damage to the wound bed or surrounding tissues, as well as maintaining an optimal moist environment conducive to wound healing. In vitro evaluation results indicated that the hemostatic bandage possesses favorable cytocompatibility with low levels of hemolysis. Furthermore, it effectively aggregates various blood cells while activating platelets synergistically to promote both extrinsic and intrinsic coagulation pathways. In an in vivo rat model study involving liver laceration and femoral artery injury scenarios, our developed hemostatic bandage demonstrated rapid clot formation capabilities along with reduced blood loss compared to commercially available fabrics.

Marstacimab, an Anti-Tissue Factor Pathway Inhibitor, in Participants with Hemophilia Α or B, with and without Inhibitors: An Integrated Analysis of Safety

Background: Marstacimab (PF-06741086) targets tissue factor pathway inhibitor (TFPI) to restore hemostasis via the extrinsic pathway of blood coagulation. In phase 1/2 studies, long-term subcutaneous (SC) administration of marstacimab reduced bleeding episodes in adults with severe hemophilia A (HA) or hemophilia B (HB) (factor VIII or factor IX [FIX] ˂1%, respectively), with or without inhibitors. BASIS, the pivotal phase 3 study of marstacimab in adolescents and adults with severe HA (&amp;lt;1%) or moderately severe to severe (FIX ≤2%) HB, is ongoing. Given the potential risk for thrombotic and thromboembolic complications with novel non-factor-based agents, integrated safety analysis of studies of such agents are of interest. Wereport an integrated analysis of marstacimab safety in participants with HA or HB. Methods: Data from participants in the phase 1b/2 study (NCT02974855), its long-term follow-up (LTFU; NCT03363321), the pivotal phase 3 BASIS study (NCT03938792), and its long-term extension (LTE; NCT05145127) were included. In the phase 1b/2 study, male participants (N=26) with severe HA or HB, with or without inhibitors, were assigned to escalating weekly SC doses of marstacimab based on inhibitor status (without inhibitors: 300 mg, a single 300-mg loading dose with subsequent 150-mg doses, or 450 mg; with inhibitors: 300 mg). In the BASIS study, male participants (N=163) with severe HA or moderately severe to severe HB, with or without inhibitors, received a single SC 300-mg loading dose followed by once-weekly 150 mg in the 12-month active treatment phase (ATP). Adverse events (AEs), serious adverse events (SAEs), and AEs of special interest (AESI), including thromboembolic events and injection site reactions (ISRs), were monitored. Results: Overall, 144 unique participants received marstacimab and were included in this analysis: 28 in the phase 1b/2 study (median treatment duration, 2.8 [range, 1.2-3.0] months) and its LTFU (15.0 [1.2-16.4] months) and 116 in the BASIS study (12.1 [0.9-13.1] months) and its LTE (6.4 [1.1-16.1] months; overall BASIS + LTE, 17.3 [0.9-28.2] months). Demographics, baseline characteristics, and marstacimab treatment and dosing ( Table 1) and a summary of safety are shown in Table 2. There were no treatment-related SAEs in the phase 1b/2 study and 1 treatment-related SAE (peripheral swelling) in the BASIS study and its LTE. There were no deaths in any study. There were no AEs related to thromboembolism during treatment in participants with HA or HB across the marstacimab clinical program. Adverse drug reactions were consistent across studies. In the BASIS study and its LTE, ISRs (n=13, 11.2%) tended to be transient and mild in severity and did not lead to discontinuation from the study: other reactions were also mild: pruritus (n=4, 3.4%); rash (n=1, 0.9%) and headache (n=8, 6.9%). One participant, with a history of hemorrhoids, in the BASIS ATP had a grade 2 event of thrombosed hemorrhoids considered related to marstacimab (a local inflammation event, not related to any other thromboembolic event; dose was not interrupted or changed due to this event). No clinically important findings in laboratory values were associated with marstacimab. In the BASIS ATP, antidrug antibodies (ADAs) developed in 23/112 participants (20.5% incidence; titers were low and resolved in 22/23 participants by end of study); 6/23 were positive for neutralizing antibodies (NAbs; titers were transient and resolved by the end of study). In the LTE, ADA developed in 1/44 (2.3%) participants; the ADA titer was negative prior to marstacimab but positive at the end of the main study (titer = 2.48), positive at Day 180 in the LTE (titer = 2.41), and the participant tested negative for NAbs at both timepoints. No differences in the marstacimab safety profile, as determined by AEs, SAEs, AESIs, and discontinuations due to AEs, were reported in ADA positive or negative participants. Across the BASIS study and its LTE, 18 participants had a marstacimab dose escalation from 150 mg to 300 mg; 8/18 (44.4%) had AEs, all mild or moderate in severity and there were no AEs or SAEs leading to treatment discontinuation. Conclusion: Once-weekly SC marstacimab was well tolerated in participants with severe HA or moderately severe to severe HB, without inhibitors, with a low rate and mild severity of ISRs, transient ADAs, and no thromboembolic events with continuous treatment up to 28 months in the phase 3 program.

Efficacy and Safety of the Anti-Tissue Factor Pathway Inhibitor Marstacimab in Participants with Severe Hemophilia without Inhibitors: Results from the Phase 3 Basis Trial

Background:Marstacimab (PF-06741086) is a monoclonal antibody targeted to the tissue factor pathway inhibitor protein to improve hemostasis via the extrinsic pathway of blood coagulation. Previous phase 1/2 studies demonstrated the efficacy and safety of long-term administration of marstacimab up to 450 mg weekly for reducing bleeding episodes in adults with severe hemophilia A (HA) or hemophilia B (HB), with or without inhibitors, compared with on-demand (OD) therapy. We evaluated the efficacy and safety of marstacimab in participants with severe HA or moderately severe to severe HB without inhibitors compared with previous factor replacement therapy. Methods: BASIS (NCT03938792) is an open-label, multicenter, pivotal phase 3 study that enrolled male participants aged ≥12 to ˂75 y with severe HA (factor [F] VIII ˂1%) or moderately severe to severe (FIX ≤2%) HB, with or without inhibitors. Following screening, participants entered a 6-month observational phase (OP) and were categorized by factor replacement treatment: (1) OD or (2) routine prophylaxis (RP). Participants who completed the OP crossed over to 12-month active treatment phase (ATP) and received a single subcutaneous loading dose of 300 mg followed by once weekly 150 mg marstacimab. Primary endpoints were annualized bleeding rate (ABR) for treated bleeds and safety outcomes. Secondary endpoints included incidence of various types of breakthrough bleeds and health-related quality of life (HRQoL) measures. Participants who completed the ATP were eligible to enroll in the long-term extension (LTE) study. Informed consent/ethics committee approvals were obtained. Results for participants without inhibitors are presented. Results: Participants (N=128; 108 adults, 20 adolescents) with HA or HB without inhibitors entered the OP (OD: HA n=29, HB n=8; RP: n=72, HB n=19); of these, 116 entered the ATP. The median age was 30 [range, 13-66] y, most participants were White (50.8%) or Asian (47.7%) and predominately from Europe and India (51.6%). At baseline, 89 participants (69.5%; OD: n=36; RP: n=53) had ≥1 target joint. Mean (range) duration of marstacimab treatment was 12.1 (11.5-13.1) months for OD and 11.6 (0.9-12.8) months for RP. Eighty-eight participants entered the LTE (OD: HA n=22, HB n=7; RP: HA n=45; HB n=13). Data were not available by the cutoff date for 1 RP participant and was not included in the LTE safety analysis set. The mean (range) treatment duration in the LTE was 8.0 (1.2-14.5) months for OD and 6.5 (1.1-16.1) months for RP. In the phase 3 study, the OD group reported 12 (36.4%) adverse events (AEs) during ATP vs 9 (24.3%) in OP whereas the RP group reported 62 (74.7%) AEs in ATP vs 20 (22.0%) in OP. Both groups reported more treatment-related AEs during ATP ( Table 1). ADAs developed in 23/112 participants (20.5% incidence), of which titers were low and resolved in 22 participants by end of study. One RP participant discontinued due to a non-treatment-related SAE, and no deaths or thromboembolic events were recorded in the phase 3 study or the LTE. Mean (95% CI) ABR for treated bleeds was reduced for OD (91.6% [88.1-94.1%]) and RP (35.2% [5.6-55.6%]) participants over the 12-month ATP and marstacimab demonstrated superiority vs OD (P&amp;lt;0.001) and non-inferiority and superiority vs RP (P=0.0376) therapy. Marstacimab was also associated with significant reductions in ABR across all breakthrough bleed categories vs OD, and numerical reductions vs RP (non-inferiority). Overall, mean ABR declined over the first 6 months of ATP, which continued to Month 12 (data not shown). Bleed rates for an additional 16 months of follow-up in the LTE were consistent with those observed during the first 12 months of treatment in the phase 3 study ( Table 2). The ABR reductions observed with marstacimab during ATP were consistent across hemophilia types and age groups for OD and were generally consistent across hemophilia types and age groups for RP, with all point estimates for a difference &amp;lt;2.5 (non-inferiority margin for the ABR of treated bleeds). HRQoL parameters demonstrated non-significant improvements vs OD therapy and non-inferiority vs RP therapy. Conclusion: Compared with previous OD or RP therapy, once weekly subcutaneous marstacimab was safe and effective for reducing bleeding events in participants with severe HA or moderately severe to severe HB without inhibitors beyond 12 months in the phase 3 study and up to an additional 16 months in the LTE study.

Robust adhesive nanocomposite sponge composed of citric acid and nano clays modified cellulose for rapid hemostasis of lethal non-compressible hemorrhage

Massive bleeding control plays the main role in saving people's lives in emergency situations. Herein, modified cellulose-based nanocomposite sponges by polydopamine (PDA) and laponite nano-clay was developed to sturdily deal with non-compressible lethal severe bleeding. PDA accomplishes supreme adhesion in the bleeding site (∼405 kPa) to form strong physical barrier and seal the position. Sponges super porous (∼70 % porosity) and super absorbent capacity (48 g blood absorbed per 1 g sponge) by concentrating the blood cells and platelets provides the requirements for primary hemostasis. Synergistically, the nanocomposite sponges' intelligent chemical structure induces hemostasis by activation of the XI, IX, X, II and FVII factors of intrinsic and extrinsic coagulation pathways. Excellent hemostatic performance of sponges in-vitro was assessed by RBC accumulation (∼100 %), blood clotting index (∼10 %), platelet aggregation/activation (∼93 %) and clotting time. The nanocomposite sponges depicted super performance in the fatal high-pressure non-compressible hemorrhage model by reducing of >2, 15 and 3 times in the bleeding amount at New Zealand rabbit's heart and liver, and rat's femoral artery bleeding models, respectively compared to commercial hemostatic agents (Pvalue˂0.001). The in-vivo host response results exhibited biosafety with no systemic and significant local inflammatory response by hematological, pathological and biochemical parameters assessments.

Bioinspired Hemostatic and Anti‐Infective Armor for Wound Healing Assisted by Metal‐Phenol‐Polyamine System

AbstractHemostatic materials facilitate rapid hemostasis and significantly mitigate the potential of fatal hemorrhage in civilian and military traumas. However, most existing hemostatic materials are limited in material‐dependent forms and fail to integrate multifunctionality, thus constraining their versatility for differing settings and wound healing capacity. Herein, a facile, versatile armor strategy is proposed to endow various biomaterials with rapid hemostasis, infection prevention, and tissue healing capabilities. The armor is fabricated on the surface of substrates first through chemical cross‐linking of adhesive catechol (phenol) and collagen (polyamine) inspired by insect sclerotization, followed by zinc ions (Zn2+) chelation based on mussel‐inspired metal‐phenol coordination chemistry, referred to “metal‐phenol‐polyamine system”. This armor facilitates clot formation by promoting platelet aggregation and activating both intrinsic and extrinsic coagulation pathways. Moreover, the integrated Zn2+ endows the armor with potent antibacterial properties against both Gram‐positive and Gram‐negative bacteria. Consequently, this strategy armors a hemostatic sponge that effectively controls bleeding in rabbit hemorrhage models and successfully facilitates the complete healing of epidermal traumas in rats within 14 days. This metal‐phenol‐polyamine system‐assisted armor provides a potential and universal strategy for efficient hemostasis and wound healing.

Precision medicine in hemostasis: a review of prothrombin complex concentrates and the role of viscoelastic tests in tailoring therapy

This review explores the role of precision medicine in the management of bleeding disorders and anticoagulation therapy, with a focus on the use of visco-elastic tests such as Thromboelastography (TEG) and Rotational Thromboelastometry (ROTEM). These tests provide real-time, dynamic insight into a patient's coagulation status, guiding the choice between three-factor prothrombin complex concentrate (PCC3) and four-factor PCC (PCC4), as well as the use of activated four-factor PCC (FEIBA). The specific ROTEM tests, INTEM and EXTEM, further enhance our understanding of the intrinsic and extrinsic coagulation pathways. Moreover, the use of tranexamic acid (TXA) and fibrinogen, guided by these visco-elastic tests, has shown promise in trauma patients. TXA has been associated with survival benefit when administered immediately or within 3 hours of injury. Fibrinogen, a key factor in clot formation, can be monitored and supplemented as needed to optimize hemostasis. In conclusion, the practice of precision medicine, with the aid of TEG and ROTEM, offers the potential to enhance the safety and efficacy of PCC therapy, TXA administration, and fibrinogen supplementation. These tools are invaluable in tailoring therapy to the specific needs of each patient, potentially optimizing patient outcomes and minimizing the risk of adverse events.

Evolution of Antidrug Antibody Assays During the Development of Anti-Tissue Factor Pathway Inhibitor Monoclonal Antibody Marstacimab.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic coagulation pathway. In patients with hemophilia A or B, inhibition of TFPI is an alternative therapeutic approach that augments the extrinsic coagulation pathway. Marstacimab is an investigational fully human monoclonal antibody that binds and neutralizes TFPI and is being evaluated as a prophylactic treatment to prevent or reduce the frequency of bleeding episodes in patients with severe hemophilia A or B, with or without inhibitors (antibodies against coagulation factors). However, the efficacy, safety, and pharmacokinetics of marstacimab may be affected by the induction of antidrug antibody (ADA) responses. Here, we describe the evolution and validation of three quasi-quantitative electrochemiluminescence-based methods to detect marstacimab ADAs, starting from their use in a first-in-human phase 1 study to their use in phase 2 and 3 clinical studies of patients with severe hemophilia. For all three methods, validation criteria evaluated the performance of the assays in screening and confirmatory cut points, precision, selectivity, drug tolerance, target interference, and stability. Additional criteria for validation were dilution linearity (Methods 1 and 2) and low positive control concentration, prozone effect, plate homogeneity, and robustness (Method 3). The three methods met validation criteria and are a potentially valuable tool in detecting the induction of marstacimab ADAs during treatment in patients with hemophilia.

Naringin binds to protein disulfide isomerase to inhibit its activity and modulate the blood coagulation rates: Implications in controlling thrombosis

Currently used antithrombotic drugs are beset with several drawbacks which necessitates the need for new and cheaper alternatives. Protein disulfide isomerase (PDI) is secreted in the blood plasma in cellular stress conditions and initiates the thrombus formation. A screening of library of natural compounds revealed that naringin had a high binding affinity for the PDI (−8.2 kcal/mol). Recombinant PDI was purified using the affinity chromatography. Incubation of purified PDI (3 μM) with naringin (0–100 μM, pH 7.4, 25 °C) partially modulated its conformation. Consequently, the fluorescence emission spectra of the PDI binding to naringin were assessed using the Stern-Volmer equation, which indicated an association constant of 2.78 × 104 M−1 suggesting an appreciable affinity for the naringin, with a unique binding site. An insulin turbidity assay showed that PDI activity is decreased in the presence of naringin indicating inhibition. Molecular dynamic simulation studies showed the changes in the PDI structure on binding to the naringin. Incubation of naringin (80 μM) in fresh human plasma along with exogenous PDI (175 nM) showed a significant delay in the intrinsic and extrinsic coagulation pathways. We show that naringin is able to modulate the PDI conformation and activity resulting in altered blood coagulation rates.

Tissue factor is a critical regulator of radiation therapy-induced glioblastoma remodeling.

Radiation therapy (RT) provides therapeutic benefits for patients with glioblastoma (GBM), but inevitably induces poorly understood global changes in GBM and its microenvironment (TME) that promote radio-resistance and recurrence. Through a cell surface marker screen, we identified that CD142 (tissue factor or F3) is robustly induced in the senescence-associated β-galactosidase (SA-βGal)-positive GBM cells after irradiation. F3 promotes clonal expansion of irradiated SA-βGal+ GBM cells and orchestrates oncogenic TME remodeling by activating both tumor-autonomous signaling and extrinsic coagulation pathways. Intratumoral F3 signaling induces a mesenchymal-like cell state transition and elevated chemokine secretion. Simultaneously, F3-mediated focal hypercoagulation states lead to activation of tumor-associated macrophages (TAMs) and extracellular matrix (ECM) remodeling. A newly developed F3-targeting agent potently inhibits the aforementioned oncogenic events and impedes tumor relapse invivo. These findings support F3 as a critical regulator for therapeutic resistance and oncogenic senescence in GBM, opening potential therapeutic avenues.

A retrospective validation of ROTEM algorithms for detecting hyperfibrinolysis demonstrates poor agreement for prediction of in-hospital mortality and transfusion requirement in a general, non-cardiac, surgical population

IntroductionHyperfibrinolysis diagnosed on Rotational Thromboelastography (ROTEM) is associated with increased transfusion requirements and mortality in trauma. The diagnosis and significance of hyperfibrinolysis in a mixed, non-cardiac, general surgical population has not been investigated. We aimed to measure agreement between four ROTEM algorithms for diagnosing hyperfibrinolysis and transfusion requirements and mortality in general surgical patients. These algorithms mostly incorporate measures of early or late clot amplitude reduction on the Extrinsic Clotting Pathway Test with Tissue Factor (EXTEM) channel. MethodFour hospital administrative data sets were linked from 2019 to 2022. Adults >18 years were included if a ROTEM was performed during their surgery (intraoperative period) or within 24-h of the surgery completion (postoperative period). The four hyperfibrinolysis criteria were applied to the ROTEM data and assessed for their agreement, intraoperative and postoperative transfusion requirements and in-patient mortality. ResultsWe linked 933 ROTEMs to 558 patient-procedures. One algorithm identified hyperfibrinolysis on only three patients so was excluded. Agreement between the remaining three was slight (Cohens Kappa 0.18 (p < 0.001)) with hyperfibrinolysis diagnosed between 22 and 69 % of the procedures. The association between hyperfibrinolysis diagnosis and intraoperative or postoperative transfusion requirement was inconsistent between the criteria. However, an algorithm put forward by Goerling et al. was more often associated with transfusion requirement and inpatient mortality. DiscussionThe poor agreement between criteria suggests that some ROTEM criteria may not transfer directly to general surgical patients. Future research should focus on optimising hyperfibrinolysis cut-off values to update algorithms for bleeding general surgical patients.

Levels of some coagulation parameters among apparently healthy Medical Laboratory Science undergraduate students of Rivers State University, Port Harcourt

Introduction: Prothrombin time (PT), International normalized ratio (INR) and activated partial thromboplastin time (APTT) are screening tests used to evaluate the overall integrity of the intrinsic, extrinsic and common coagulation pathway as well as monitoring and management of coagulation disorders. This study was aimed at assessing the PT, INR and APTT among apparently healthy undergraduate Medical Laboratory Science students of Rivers State University, Port Harcourt. Materials and Methods: A total of 100 apparently healthy undergraduate Medical Laboratory Science students of Rivers State University, Port Harcourt comprising of 44 males and 56 females within the age range of 17-40 years were recruited for this study. Five milliliters (5ml) of venous blood was collected from each participant using a standard venipuncture technique into a vacutainer bottle containing 3.2% tri-sodium citrate anticoagulant in a ratio of 1:9 of the anticoagulant and the blood and mixed thoroughly. Prothrombin time and activated partial thromboplastin time were determined by clotting method using the Helena C4 semi-automated, photo-optical coagulation analyzer and the Agappe thromboplastin test kit manufactured by Agappe Diagnostics Switzerland while INR was calculated by using the ratio of the study subjects’ PT to the mean of control PT raised to the power of the reagent international sensitivity index (ISI). The data obtained was analyzed using Graphpad Prism Software version 6.00. Data was presented as means, median, range and standard deviation. Results: The mean ± SD of the PT, INR and APTT were 12.9± 1.624s, 1.01±0.1281 and 37.99±8.898s in the same order. The reference intervals obtained for PT, INR and APTT were 9.7-16.2s, 0.8-1.3 and APTT 20.2-55.8s in the same order. The lower limits were within the ranges established by previous researchers while the upper limits were higher. The age range 21-25 had the highest frequency count (54), which was followed by &lt;20 (26), 26-30 (17) and then &lt;30 (3). The overall mean values for the age groups were within the established normal values and as such no statistically significant difference was observed between the various groups. Conclusion: This study has established the mean values and reference intervals of PT, INR and APTT for apparently healthy Medical Laboratory Science undergraduate students of Rivers State University, Port Harcourt as well as established that age does not have any significant effect on PT, INR and APTT. Due to geography, lifestyle, and genetic diversity, it is recommended that each laboratory establishes geography-specific reference intervals for PT, INR and APTT.

CAR T cells as micropharmacies against solid cancers: Combining effector T-cell mediated cell death with vascular targeting in a one-step engineering process

To enhance the potency of chimeric antigen receptor (CAR) engineered T cells in solid cancers, we designed a novel cell-based combination strategy with an additional therapeutic mode of action. CAR T cells are used as micropharmacies to produce a targeted pro-coagulatory fusion protein, truncated tissue factor (tTF)-NGR, which exerts pro-coagulatory activity and hypoxia upon relocalization to the vascular endothelial cells that invade tumor tissues. Delivery by CAR T cells aimed to induce locoregional tumor vascular infarction for combined immune-mediated and hypoxic tumor cell death. Human T cells that were one-vector gene-modified to express a GD2-specific CAR along with CAR-inducible tTF-NGR exerted potent GD2-specific effector functions while secreting tTF-NGR that activates the extrinsic coagulation pathway in a strictly GD2-dependent manner. In murine models, the CAR T cells infiltrated GD2-positive tumor xenografts, secreted tTF-NGR into the tumor microenvironment and showed a trend towards superior therapeutic activity compared with control cells producing functionally inactive tTF-NGR. In vitro evidence supports a mechanism of hypoxia-mediated enhancement of T cell cytolytic activity. We conclude that combined CAR T cell targeting with an additional mechanism of antitumor action in a one-vector engineering strategy is a promising approach to be further developed for targeted treatment of solid cancers.

Unusual Surface Coagulation Activation Patterns of Crystalline and Amorphous Silicate-Based Biominerals.

Activation of coagulation cascades, especially FX and prothrombin, prevents blood loss and reduces mortality from hemorrhagic shock. Inorganic salts are efficient but cannot stop bleeding completely in hemorrhagic events, and rebleeding carries a significant mortality risk. The coagulation mechanism of biominerals has been oversimplified in the past two decades, limiting the creation of novel hemostats. Here, at the interface, the affinity of proteins, the protease activity, fibrinolysis, hydration shell, and dynamic microenvironment are monitored at the protein level. Proteomic analysis reveals that fibrinogen and antithrombin III's affinity for kaolin's interface causes a weak thrombus and rebleeding during hemostasis. Inspiringly, amorphous bioactive glass (BG) with a transient-dynamic ion microenvironment breaches the hydration layer barrier and selectively and slightly captures procoagulant components of kiniogen-1, plasma kallikrein, FXII, and FXI proteins on its interface, concurrently generating a continuous biocatalytic interface to rapidly activate both intrinsic and extrinsic coagulation pathways. Thus, prothrombin complexes are successfully hydrolyzed to thrombin without platelet membrane involvement, speeding production of high-strength clots. This study investigates how the interface of inorganic salts assists in coagulation cascades from a more comprehensive micro-perspective that may help elucidate the clinical application issues of kaolin-gauze and pave the way to new materials for managing hemorrhage.

The role of tissue factor pathway inhibitor 2 in the coagulation and fibrinolysis system.

Tissue factor (TF), the primary initiator of the extrinsic coagulation pathway, contributes to the generation of a hypercoagulable and prothrombotic state in cancer patients. TF pathway inhibitor (TFPI) is a major inhibitor of TF-mediated coagulation pathway. The two proteins, TFPI1 and TFPI2, are encoded by separate genes. Indeed, various cancer patients with venous thromboembolism (VTE) had significantly lower TFPI1 levels than those without VTE. In contrast, serum TFPI2 level was found to increase in ovarian cancer patients with VTE. It remains unclear why TFPI2, unlike TFPI1, is elevated in ovarian cancer patients with VTE. The aim of this review is to explore the pathophysiological role of TFPI2 on the coagulation and fibrinolysis system. A literature search was performed from inception to April 30, 2022 in the PubMed and Google Scholar databases. TFPI1 and TFPI2 are homologs with different protease inhibitory activities in the coagulation and fibrinolysis system. TFPI1 inhibits TF/factor VIIa (FVIIa) catalyzed factor X (FX) activation. On the other hand, TFPI2 is unlikely to affect TF-initiated thrombin generation, but it has strong inhibitory activity against plasmin. Plasmin is involved in fibrin degradation, clot lysis, and inactivation of several coagulation factors (such as FV, FVIII, FIX, and FX). TFPI2 may increase the risk of VTE by inhibiting plasmin-dependent fibrinolysis. TFPI1 and TFPI2 may have different key functions in regulating the coagulation and fibrinolytic systems.

Tissue Factor Pathway Inhibitors as Potential Targets for Understanding the Pathophysiology of Preeclampsia.

Preeclampsia is a hypertensive disorder of pregnancy that causes maternal and perinatal morbidity and mortality worldwide. Preeclampsia is associated with complex abnormalities of the coagulation and fibrinolytic system. Tissue factor (TF) is involved in the hemostatic system during pregnancy, while the Tissue Factor Pathway Inhibitor (TFPI) is a major physiological inhibitor of the TF-initiated coagulation cascade. The imbalance in hemostatic mechanisms may lead to a hypercoagulable state, but prior research has not comprehensively investigated the roles of TFPI1 and TFPI2 in preeclamptic patients. In this review, we summarize our current understanding of the biological functions of TFPI1 and TFPI2 and discuss future directions in preeclampsia research. A literature search was performed from inception to 30 June 2022 in the PubMed and Google Scholar databases. TFPI1 and TFPI2 are homologues with different protease inhibitory activities in the coagulation and fibrinolysis system. TFPI1 is an essential physiological inhibitor of the TF-initiated extrinsic pathway of coagulation. On the other hand, TFPI2 inhibits plasmin-mediated fibrinolysis and exerts antifibrinolytic activity. It also inhibits plasmin-mediated inactivation of clotting factors and maintains a hypercoagulable state. Furthermore, in contrast to TFPI1, TFPI2 suppresses trophoblast cell proliferation and invasion and promotes cell apoptosis. TFPI1 and TFPI2 may play important roles in regulating the coagulation and fibrinolytic system and trophoblast invasion to establish and maintain successful pregnancies. Concentrations of TF, TFPI1, and TFPI2 in maternal blood and placental tissue are significantly altered in preeclamptic women compared to normal pregnancies. TFPI protein family may affect both the anticoagulant (i.e., TFPI1) and antifibrinolytic/procoagulant (i.e., TFPI2) systems. TFPI1 and TFPI2 may function as new predictive biomarkers for preeclampsia and navigate precision therapy.